New insight into mitochondrial changes in vascular endothelial cells irradiated by gamma ray.

PubMed

Hu, Shunying; Gao, Yajing; Zhou, Hao; Kong, Fanxuan; Xiao, Fengjun; Zhou, Pingkun; Chen, Yundai

2017-05-01

To investigate alterations of mitochondria in irradiated endothelial cells to further elucidate the mechanism underlying radiation-induced heart disease. Experiments were performed using human umbilical vein endothelial cells (HUVECs). HUVECs were irradiated with single gamma ray dose of 0, 5, 10 and 20 Gy, respectively. Apoptosis was assessed by flow cytometry at 24, 48 and 72 h post-irradiation, respectively. The intracellular reactive oxygen species (ROS) was measured with 2',7'-dichlorofluorescein-diacetate (DCFH-DA) at 24 h post-irradiation. Mitochondrial membrane potential (ΔΨm) by JC-1 and the opening of mitochondrial permeability transition pore (mPTP) by a calcein-cobalt quenching method were detected at 24 h post-irradiation in order to measure changes of mitochondria induced by gamma ray irradiation. Gamma ray irradiation increased HUVECs apoptosis in a dose-dependent and time-dependent manner. Irradiation also promoted ROS production in HUVECs in a dose-dependent manner. At 24 h post-irradiation, the results showed that irradiation decreases ΔΨm, however, paradoxically, flow cytometry showed green fluorescence instensity higher in irradiated HUVECs than in control HUVECs in an irradiation dose-dependent manner which indicated gamma ray irradiation inhibited mPTP opening in HUVECs. Gamma ray irradiation induces apoptosis and ROS production of endothelial cells, and decreases ΔΨm meanwhile contradictorily inhibiting the opening of mPTP.

Cannabinoids inhibit angiogenic capacities of endothelial cells via release of tissue inhibitor of matrix metalloproteinases-1 from lung cancer cells.

PubMed

Ramer, Robert; Fischer, Sascha; Haustein, Maria; Manda, Katrin; Hinz, Burkhard

2014-09-15

Cannabinoids inhibit tumor neovascularization as part of their tumorregressive action. However, the underlying mechanism is still under debate. In the present study the impact of cannabinoids on potential tumor-to-endothelial cell communication conferring anti-angiogenesis was studied. Cellular behavior of human umbilical vein endothelial cells (HUVEC) associated with angiogenesis was evaluated by Boyden chamber, two-dimensional tube formation and fibrin bead assay, with the latter assessing three-dimensional sprout formation. Viability was quantified by the WST-1 test. Conditioned media (CM) from A549 lung cancer cells treated with cannabidiol, Δ(9)-tetrahydrocannabinol, R(+)-methanandamide or the CB2 agonist JWH-133 elicited decreased migration as well as tube and sprout formation of HUVEC as compared to CM of vehicle-treated cancer cells. Inhibition of sprout formation was further confirmed for cannabinoid-treated A549 cells co-cultured with HUVEC. Using antagonists to cannabinoid-activated receptors the antimigratory action was shown to be mediated via cannabinoid receptors or transient receptor potential vanilloid 1. SiRNA approaches revealed a cannabinoid-induced expression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) as well as its upstream trigger, the intercellular adhesion molecule-1, to be causally linked to the observed decrease of HUVEC migration. Comparable anti-angiogenic effects were not detected following direct exposure of HUVEC to cannabinoids, but occurred after addition of recombinant TIMP-1 to HUVEC. Finally, antimigratory effects were confirmed for CM of two other cannabinoid-treated lung cancer cell lines (H460 and H358). Collectively, our data suggest a pivotal role of the anti-angiogenic factor TIMP-1 in intercellular tumor-endothelial cell communication resulting in anti-angiogenic features of endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

The endothelial cell secretome as a novel treatment to prime adipose-derived stem cells for improved wound healing in diabetes.

PubMed

Fromer, Marc W; Chang, Shaohua; Hagaman, Ashleigh L R; Koko, Kiavash R; Nolan, Ryan S; Zhang, Ping; Brown, Spencer A; Carpenter, Jeffrey P; Caputo, Francis J

2017-07-28

Chronic wounds are a common surgical problem exacerbated by diabetes and ischemia. Although adipose-derived stem cells (ASCs) have shown promise as a wound healing therapy, their function and proliferation are hindered in diabetes. This study examines the ability of the human umbilical vein endothelial cell (HUVEC) secretome to reverse the deleterious effects of high glucose concentrations on ASCs through priming, thereby enhancing their ability to participate in angiogenesis and wound healing. Institutional review board-approved human ASCs were cultured in M199 medium with or without glucose (30 mmol/L). HUVEC were grown in 30 mmol/L glucose-containing M199 medium; the resulting conditioned medium (HUVEC-CM) was collected every 3 days and used to prime ASCs. An aliquot of HUVEC-CM was heated (85°C for 30 minutes) to produce thermal denaturation of protein. Viability, proliferation, and endothelial differentiation were measured by MTT assays, growth curves, and quantitative polymerase chain reaction, respectively. A Matrigel assay was used to assess the ability of primed ASCs to participate in capillary-like tube formation. An Institutional Animal Care and Use Committee-approved in vivo murine model of diabetic and ischemic hindlimbs was used to evaluate the angiogenic potential of primed stem cells. Human ASCs were cultured with either control M199 or HUVEC-CM. Mice were randomized to a control group, an unprimed ASC group, or a HUVEC-primed ASC group. Cellular therapies were injected into the ischemic muscle. Thirty days later, slides were made. Microvessels were counted by three blinded observers. MTT assays revealed that HUVEC-priming induced a 1.5 times increase in cell viability over diabetic controls. This promoting effect was lost with heated HUVEC-CM (P < .001), indicating that the active molecules are of protein origin. After 9 days, ASCs cultured in 30 mmol/L glucose solution showed a 14% reduction in growth from nondiabetic controls (P = .013) and exhibited atrophic morphology. Conversely, diabetic HUVEC-primed stem cells demonstrated a nearly four-fold increase in proliferation (P < .05) and took on a fusiform, endothelial-like phenotype. Polymerase chain reaction demonstrated enhanced expression of CD31 messenger RNA by 4.7-fold after 14 days in the HUVEC-primed group, and endothelial nitric oxide synthase messenger RNA messenger RNA was increased 20.1-fold from controls. Unlike unprimed controls, HUVEC-primed ASCs readily formed capillary-like tube networks on Matrigel. Diabetic mice that were injected with HUVEC-primed ASCs demonstrated greater vessel density than both controls (2.1-fold) and unprimed stem cell treatments (P < .001). HUVECs secrete protein factors that significantly increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC-primed ASCs leads to enhanced angiogenesis. Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Activation of GPR4 by Acidosis Increases Endothelial Cell Adhesion through the cAMP/Epac Pathway

PubMed Central

Leffler, Nancy R.; Asch, Adam S.; Witte, Owen N.; Yang, Li V.

2011-01-01

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2′,5′-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a Gi signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the Gs/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the Gs/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells. PMID:22110680

Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1β Pathway in Systemic Lupus Erythematosus.

PubMed

Nhek, Sokha; Clancy, Robert; Lee, Kristen A; Allen, Nicole M; Barrett, Tessa J; Marcantoni, Emanuela; Nwaukoni, Janet; Rasmussen, Sara; Rubin, Maya; Newman, Jonathan D; Buyon, Jill P; Berger, Jeffrey S

2017-04-01

Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet-endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1±12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1±3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and intercellular adhesion molecule 1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1β-dependent pathway. Incubation of SLE-activated platelets with an interleukin-1β-neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. Platelet activity measurements and subsequent interleukin-1β-dependent activation of the endothelium are increased in subjects with SLE. Platelet-endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE. © 2017 American Heart Association, Inc.

Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1β Pathway in Systemic Lupus Erythematosus

PubMed Central

Nhek, Sokha; Clancy, Robert; Lee, Kristen A.; Allen, Nicole M.; Barrett, Tessa J.; Marcantoni, Emanuela; Nwaukoni, Janet; Rasmussen, Sara; Rubin, Maya; Newman, Jonathan D.; Buyon, Jill P.; Berger, Jeffrey S.

2017-01-01

Objective Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet–endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. Approach and Results Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1±12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1±3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte–platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and intercellular adhesion molecule 1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1β–dependent pathway. Incubation of SLE-activated platelets with an interleukin-1β–neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. Conclusions Platelet activity measurements and subsequent interleukin-1β–dependent activation of the endothelium are increased in subjects with SLE. Platelet–endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE. PMID:28153882

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

PubMed

Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

Oxidized-LDL induce morphological changes and increase stiffness of endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chouinard, Julie A.; Research Centre on Aging, Sherbrooke Geriatric University Institute, Sherbrooke, Quebec; Grenier, Guillaume

There is increasing evidence suggesting that oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury contributing to the age-related physio-pathological process of atherosclerosis. In this study, the effects of native LDL and ox-LDL on the mechanical properties of living human umbilical vein endothelial cells (HUVEC) were investigated by atomic force microscopy (AFM) force measurements. The contribution of filamentous actin (F-actin) and vimentin on cytoskeletal network organization were also examined by fluorescence microscopy. Our results revealed that ox-LDL had an impact on the HUVEC shape by interfering with F-actin and vimentin while native LDL showed no effect. AFM colloidalmore » force measurements on living individual HUVEC were successfully used to measure stiffness of cells exposed to native and ox-LDL. AFM results demonstrated that the cell body became significantly stiffer when cells were exposed for 24 h to ox-LDL while cells exposed for 24 h to native LDL displayed similar rigidity to that of the control cells. Young's moduli of LDL-exposed HUVEC were calculated using two models. This study thus provides quantitative evidence on biomechanical mechanisms related to endothelial cell dysfunction and may give new insight on strategies aiming to protect endothelial function in atherosclerosis.« less

In vitro effects of ATG-Fresenius on immune cell adhesion.

PubMed

Kanzler, I; Seitz-Merwald, I; Schleger, S; Kaczmarek, I; Kur, F; Beiras-Fernandez, A

2013-06-01

ATG-Fresenius, a purified rabbit polyclonal anti-human T-lymphocyte immunoglobulin is used for induction immunosuppression as well as prevention and treatment of acute rejection episodes among patients receiving solid organ transplants. The aim of this study was to investigate the in vitro activity of ATG-Fresenius upon immune cell adhesion, which may explain its activity to mitigate ischemia-reperfusion injury. Human vascular endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMCs) isolated from umbilical vein or peripheral blood were incubated 20 to 24 hours before analysis. HUVEC were incubated with 10 and 100 μg/mL ATG-Fresenius or reference polyclonal rabbit immunoglobulin G. Analysis of immune cell adhesion to endothelial cells was studied in cocultures of PBMCs and adherent HUVEC. Endothelial cell expression of adhesion molecules CD62E and CD54 was determined by flow cytometry. The numbers of T-, B- and natural killer cells attached to HUVEC were also determined by flow cytometry. Groups were compared using one-way analysis of variance. We showed that ATG-Fresenius binds to endothelial cells particularly activated ones expressing increased levels of E-selectin and ICAM-1. The increased binding of ATG-Fresenius to activated endothelial cells was consistent with its known binding to Intercellular Adhesion Molecule 1 (ICAM-1) and selectins. We also showed that ATG-Fresenius inhibited adhesion of prestimulated immune cells to activated endothelium. We demonstrated dose-dependent binding of ATG-Fresenius to activated endothelial cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1

NASA Technical Reports Server (NTRS)

Ding, Ke-Hong; Zhong, Qing; Isales, Carlos M.; Iscules, C. M. (Principal Investigator)

2003-01-01

We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

Influence of homeobox B2 antisense oligodeoxynucleotides on the biological characteristics of in vitro cultured primary human umbilical vein endothelial cells.

PubMed

Liu, X S; Zhang, X Q; Tian, T; Liu, L; Ming, J

2008-01-01

This study aims to explore the influence of homeobox B2 (HOXB2) antisense oligodeoxynucleotides (asodn) on the biological characteristics of in vitro cultured primary human umbilical vein endothelial cells (HUVECs). The distribution of HOXB2 asodn in the HUVECs was observed by fluorescent labelling, and the influence of different concentrations of HOXB2 asodn on the DNA synthesis of HUVECs was assessed. Flow cytometry and a reverse transcriptase-polymerase chain reaction (RT- PCR) method were employed to observe the influence of HOXB2 asodn on HOXB2 expression and the HUVEC cell cycle. After the induction of liposome, the nuclear fluorescent staining of HOXB2 asodn was weaker 15 min after transfection and the staining reached the strongest level at 4-8 h but then weakened and disappeared by 16 h after transfection. This indicated that endothelial DNA synthesis could be inhibited by HOXB2 asodn in a dose-dependent manner. Furthermore, the HUVECs could be delayed in their passage from G1 to S. Simultaneously, expression of HOXB2 mRNA had decreased significantly by 24-48 h after transfection. Clearly, HOXB2 plays important roles in the proliferation of endothelial cells and also affects the cell cycle.

Spaceflight of HUVEC: An Integrated eXperiment- SPHINX Onboard the ISS

NASA Astrophysics Data System (ADS)

Versari, S.; Maier, J. A. M.; Norfini, A.; Zolesi, V.; Bradamante, S.

2013-02-01

The spaceflight orthostatic challenge can promote in astronauts inadequate cardiovascular responses defined as cardiovascular deconditioning. In particular, disturbance of endothelial functions are known to lead to altered vascular performances, being the endothelial cells crucial in the maintenance of the functional integrity of the vascular wall. In order to evaluate whether weightlessness affects endothelial functions, we designed, developed, and performed the experiment SPHINX - SPaceflight of HUVEC: an INtegrated eXperiment - where HUVEC (Human Umbilical Vein Endothelial Cells) were selected as a macrovascular cell model system. SPHINX arrived at the International Space Station (ISS) onboard Progress 40P, and was processed inside Kubik 6 incubator for 7 days. At the end, all of the samples were suitably fixed and preserved at 6°C until return on Earth on Soyuz 23S.

Angiogenic effects of apigenin on endothelial cells after hypoxia-reoxygenation via the caveolin-1 pathway

PubMed Central

Tu, Fengxia; Pang, Qiongyi; Chen, Xiang; Huang, Tingting; Liu, Meixia; Zhai, Qiongxiang

2017-01-01

In the present study, we aimed to elucidate whether apigenin contributes to the induction of angiogenesis and the related mechanisms in cell hypoxia-reoxygenation injury. The role of apigenin was examined in human umbilical vein endothelial cell (HUVEC) viability, migration and tube formation in vitro. To investigate the related mechanisms, we used caveolin-1 short interfering RNA. The viability of HUVECs was measured using Cell Counting Kit-8 assays, HUVEC migration was analyzed by crystal violet staining, and a tube formation assay was performed using the branch point method. Expression of caveolin-1, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) in HUVECs was examined by polymerase chain reaction and western blotting. Our data revealed that apigenin induced angiogenesis in vitro by increasing the tube formation ability of HUVECs, which was counteracted by caveolin-1 silencing. Compared to the NC group, Caveolin-1 and eNOS expression was upregulated by apigenin, whereas compared to the NC group, eNOS expression was increased upon caveolin-1 silencing. The expression of VEGF was increased by treatment with apigenin; however, compared to the NC group, caveolin-1 silencing did not affect VEGF expression, and apigenin did not increase VEGF expression in HUVECs after caveolin-1 silencing. These data suggest that apigenin may be a candidate therapeutic target for stroke recovery by promoting angiogenesis via the caveolin-1 signaling pathway. PMID:29039442

Enhancement of proinflammatory and procoagulant responses to silica particles by monocyte-endothelial cell interactions

PubMed Central

2012-01-01

Background Inorganic particles, such as drug carriers or contrast agents, are often introduced into the vascular system. Many key components of the in vivo vascular environment include monocyte-endothelial cell interactions, which are important in the initiation of cardiovascular disease. To better understand the effect of particles on vascular function, the present study explored the direct biological effects of particles on human umbilical vein endothelial cells (HUVECs) and monocytes (THP-1 cells). In addition, the integrated effects and possible mechanism of particle-mediated monocyte-endothelial cell interactions were investigated using a coculture model of HUVECs and THP-1 cells. Fe3O4 and SiO2 particles were chosen as the test materials in the present study. Results The cell viability data from an MTS assay showed that exposure to Fe3O4 or SiO2 particles at concentrations of 200 μg/mL and above significantly decreased the cell viability of HUVECs, but no significant loss in viability was observed in the THP-1 cells. TEM images indicated that with the accumulation of SiO2 particles in the cells, the size, structure and morphology of the lysosomes significantly changed in HUVECs, whereas the lysosomes of THP-1 cells were not altered. Our results showed that reactive oxygen species (ROS) generation; the production of interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor (TNF)-α and IL-1β; and the expression of CD106, CD62E and tissue factor in HUVECs and monocytes were significantly enhanced to a greater degree in the SiO2-particle-activated cocultures compared with the individual cell types alone. In contrast, exposure to Fe3O4 particles had no impact on the activation of monocytes or endothelial cells in monoculture or coculture. Moreover, using treatment with the supernatants of SiO2-particle-stimulated monocytes or HUVECs, we found that the enhancement of proinflammatory response by SiO2 particles was not mediated by soluble factors but was dependent on the direct contact between monocytes and HUVECs. Furthermore, flow cytometry analysis showed that SiO2 particles could markedly increase CD40L expression in HUVECs. Our data also demonstrated that the stimulation of cocultures with SiO2 particles strongly enhanced c-Jun NH2-terminal kinase (JNK) phosphorylation and NF-κB activation in both HUVECs and THP-1 cells, whereas the phosphorylation of p38 was not affected. Conclusions Our data demonstrate that SiO2 particles can significantly augment proinflammatory and procoagulant responses through CD40–CD40L-mediated monocyte-endothelial cell interactions via the JNK/NF-κB pathway, which suggests that cooperative interactions between particles, endothelial cells, and monocytes may trigger or exacerbate cardiovascular dysfunction and disease, such as atherosclerosis and thrombosis. These findings also indicate that the monocyte-endothelial cocultures represent a sensitive in vitro model system to assess the potential toxicity of particles and provide useful information that may help guide the future design and use of inorganic particles in biomedical applications. PMID:22985792

Piper sarmentosum increases nitric oxide production in oxidative stress: a study on human umbilical vein endothelial cells.

PubMed

Ugusman, Azizah; Zakaria, Zaiton; Hui, Chua Kien; Nordin, Nor Anita Megat Mohd

2010-07-01

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs). HUVECS WERE DIVIDED INTO FOUR GROUPS: control, treatment with 180 microM hydrogen peroxide (H(2)O(2)), treatment with 150 microg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H(2)O(2) for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction. Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs. Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.

Plasma from preeclamptic women activates endothelial cells via monocyte activation in vitro.

PubMed

Faas, Marijke M; van Pampus, Maria G; Anninga, Zwanine A; Salomons, Jet; Westra, Inge M; Donker, Rogier B; Aarnoudse, Jan G; de Vos, Paul

2010-12-01

In this study we tested whether plasma from preeclamptic women contains factors that can activate endothelial cells in the presence of monocytes in vitro. Plasma from preeclamptic women (n=6), healthy pregnant women (n=6) and nonpregnant women (n=6) was incubated with mono-cultures and co-cultures of human umbilical vein endothelial cells (HUVEC) and monomac-6 monocytes. Reactive oxygen species (ROS) production and ICAM-1 expression were measured using flow cytometry. Whether scavenging of ROS by superoxide dismutase and catalase inhibited HUVEC ICAM-1 expression was also investigated. We found that in HUVEC co-cultured with monomac-6 cells but not in HUVEC cultured alone, ICAM-1 was upregulated after incubation with plasma from preeclamptic women but not plasma from non-pregnant women. Also in co-cultures, monomac-6 ICAM-1 was upregulated by plasma from preeclamptic women, while in both mono- and co-cultures monomac-6 ROS production was upregulated by plasma from pregnant and preeclamptic women, compared with plasma from non-pregnant women. Scavenging of ROS by superoxide dismutase and catalase resulted in a further upregulation of HUVEC ICAM-1 after incubation with plasma from preeclamptic women, compared with incubation without superoxide dismutase and catalase. These results show that endothelial cells in vitro are activated by plasma of preeclamptic women only if they are co-cultured with monocytes. This upregulation appeared not to be due to extracellular ROS production by monocytes or HUVEC, pointing to involvement of other mechanisms. Our data suggest that plasma of preeclamptic women activates monocytes, and that these monocytes subsequently activate endothelial cells. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Acrylamide induces accelerated endothelial aging in a human cell model.

PubMed

Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

2015-09-01

Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interaction of Poly(l-lysine)/Polysaccharide Complex Nanoparticles with Human Vascular Endothelial Cells.

PubMed

Weber, Dominik; Torger, Bernhard; Richter, Karsten; Nessling, Michelle; Momburg, Frank; Woltmann, Beatrice; Müller, Martin; Schwartz-Albiez, Reinhard

2018-05-23

Angiogenesis plays an important role in both soft and hard tissue regeneration, which can be modulated by therapeutic drugs. If nanoparticles (NP) are used as vectors for drug delivery, they have to encounter endothelial cells (EC) lining the vascular lumen, if applied intravenously. Herein the interaction of unloaded polyelectrolyte complex nanoparticles (PECNP) composed of cationic poly(l-lysine) (PLL) and various anionic polysaccharides with human vascular endothelial cells (HUVEC) was analyzed. In particular PECNP were tested for their cell adhesive properties, their cellular uptake and intracellular localization considering composition and net charge. PECNP may form a platform for both cell coating and drug delivery. PECNP, composed of PLL in combination with the polysaccharides dextran sulfate (DS), cellulose sulfate (CS) or heparin (HEP), either unlabeled or labeled with fluorescein isothiocyanate (FITC) and either with positive or negative net charge were prepared. PECNP were applied to human umbilical cord vein endothelial cells (HUVEC) in both, the volume phase and immobilized phase at model substrates like tissue culture dishes. The attachment of PECNP to the cell surface, their intracellular uptake, and effects on cell proliferation and growth behavior were determined. Immobilized PECNP reduced attachment of HUVEC, most prominently the systems PLL/HEP and PLL/DS. A small percentage of immobilized PECNP was taken up by cells during adhesion. PECNP in the volume phase showed no effect of the net charge sign and only minor effects of the composition on the binding and uptake of PECNP at HUVEC. PECNP were stored in endosomal vesicles in a cumulative manner without apparent further processing. During mitosis, internalized PECNP were almost equally distributed among the dividing cells. Both, in the volume phase and immobilized at the surface, PECNP composed of PLL/HEP and PLL/DS clearly reduced cell proliferation of HUVEC, however without an apparent cytotoxic effect, while PLL/CS composition showed minor impairment. PECNP have an anti-adhesive effect on HUVEC and are taken up by endothelial cells which may negatively influence the proliferation rate of HUVEC. The negative effects were less obvious with the composition PLL/CS. Since uptake and binding for PLL/HEP was more efficient than for PLL/DS, PECNP of PLL/HEP may be used to deliver growth factors to endothelial cells during vascularization of bone reconstitution material, whereas those of PLL/CS may have an advantage for substituting biomimetic bone scaffold material.

Shear stress reduces protease activated receptor-1 expression in human endothelial cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Induction of cysteine-rich motor neuron 1 mRNA expression in vascular endothelial cells.

PubMed

Nakashima, Yukiko; Takahashi, Satoru

2014-08-22

Cysteine-rich motor neuron 1 (CRIM1) is expressed in vascular endothelial cells and plays a crucial role in angiogenesis. In this study, we investigated the expression of CRIM1 mRNA in human umbilical vein endothelial cells (HUVECs). CRIM1 mRNA levels were not altered in vascular endothelial growth factor (VEGF)-stimulated monolayer HUVECs or in cells in collagen gels without VEGF. In contrast, the expression of CRIM1 mRNA was elevated in VEGF-stimulated cells in collagen gels. The increase in CRIM1 mRNA expression was observed even at 2h when HUVECs did not form tubular structures in collagen gels. Extracellular signal-regulated kinase (Erk) 1/2, Akt and focal adhesion kinase (FAK) were activated by VEGF in HUVECs. The VEGF-induced expression of CRIM1 mRNA was significantly abrogated by PD98059 or PF562271, but was not affected by LY294002. These results demonstrate that CRIM1 is an early response gene in the presence of both angiogenic stimulation (VEGF) and environmental (extracellular matrix) factors, and Erk and FAK might be involved in the upregulation of CRIM1 mRNA expression in vascular endothelial cells. Copyright © 2014 Elsevier Inc. All rights reserved.

[Molecular mechanism involved in adhesion of monocytes to endothelial cells induced by nicotine and Porphyromonas gingivalis-LPS].

PubMed

Wang, Yi-xiang; An, Na; Ouyang, Xiang-ying

2015-10-18

To investigate molecular mechanism involved in nicotine in combination with Porphyromonas gingivalis (P.g) caused monocyte-endothelial cell adhesion. The effect of nicotine, P.g-lipopolysaccharide (P.g-LPS) and their combination on the proliferation of U937 cells was determined by CCK-8 method. Interleukin-6 (IL-6) expression was investigated by real-time PCR after U937 cells were treated with nicotine, P.g-LPS and their combination. In human umbilical vein endothelial cells (HUVECs), the expressions of monocyte chemoattractant protein CCL-8 and adhesion molecules including vascular cell adhesion molecule 1 (Vcam-1), very late antigen 4 alpha (VLA4α), tumor necrosis factor receptor superfamily member 4 (OX40) and OX40 ligand (OX40L) were detected by real-time PCR or Western blotting assays after HUVEC cells were treated with nicotine, P.g-LPS and their combination. Adhesion of monocytes to endothelial cells was detected after the HUVECs and U937 cells were stimulated with nicotine, P.g-LPS and their combination, respectively. P.g-LPS did not affect the proliferative ability of nicotine in U937 cells. However, the ability of P.g-LPS induced IL-6 expression was inhibited by 100 μmol/L nicotine in U937 cells. In HUVECs, the expressions of CCL-8, Vcam-1, VLA4α, OX40 and OX40L were significantly up-regulated by nicotine and P.g-LPS combination compared with nicotine alone, P.g-LPS alone and the untreated control. Adhesion of monocytes to HUVECs results showed that the two types of cells treated with nicotine in combination with P.g-LPS could markedly increase the adhesion ability of monocytes to HUVECs. P.g-LPS in combination with nicotine could recruit monocytes to endothelial lesion through up-regulation of CCL-8, and promote adhesion of monocytes to endothelial cells through enhancement of Vcam-1/VLA4α and OX40/OX40L interactions, which could be involved in the initiation and development of atherosclerosis.

Grape seed proanthocyanidin extract protects human umbilical vein endothelial cells from indoxyl sulfate-induced injury via ameliorating mitochondrial dysfunction.

PubMed

Lu, Zhaoyu; Lu, Fuhua; Zheng, Yanqun; Zeng, Yuqun; Zou, Chuan; Liu, Xusheng

2016-01-01

To investigate the effects of grape seed proanthocyanidin extract (GSPE) on indoxyl sulfate-induced Human Umbilical Vein Endothelial Cells (HUVECs) injury in vitro and study its mechanism. HUVECs were incubated with indoxyl sulfate at concentrations in the range found in uremic patients. Then we determined the effect of indoxyl sulfate on endothelial phenotype, endothelial function, ROS (reactive oxygen species), cell apoptosis and mitochondrial function. In addition, we detected whether GSPE can suppress the injury of HUVECs induced by indoxyl sulfate and probe the mechanism underlying the protective effects of GSPE by analyzing mitochondrial dysfunction. GSPE treatment significantly attenuated indoxyl sulfate-induced HVUECs injury in a dose- and time-dependent manner. GSPE-enhanced eNOS and VE-cadherin expression, inhibited intracellular ROS level and cell apoptosis, adjust mitochondrial membrane potential and reduced 8-hydroxy-desoxyguanosine (8-OHdG) level induced by indoxyl sulfate. These results suggest that GSPE prevents HUVECs from indoxyl sulfate-induced injury by ameliorating mitochondrial dysfunction and may be a promising agent for treating uremia toxin-induced injury.

Protection of vascular endothelial cells from high glucose-induced cytotoxicity by emodin.

PubMed

Gao, Yun; Zhang, Jun; Li, Guilin; Xu, Hong; Yi, Yun; Wu, Qin; Song, Miaomiao; Bee, Yong Mong; Huang, Liping; Tan, Mengxia; Liang, Shangdong; Li, GuoDong

2015-03-01

Induction of endothelial cytotoxicity by hyperglycemia in diabetes has been widely accepted. Emodin is a natural anthraquinone in rhubarb used for treatment of diabetes, but its mechanism of action is not fully understood. This study aimed to examine the potential beneficial effects of emodin on endothelial cytotoxicity caused by high glucose milieu. Culture of human umbilical vein endothelial cells (HUVECs) with high concentrations of glucose resulted in damage to the cells, leading to decreased formazan products by 14-27%, reduced DNA contents by 12-19%, and increased hypodiploid apoptosis by 40-109%. These adverse effects of high glucose could be prevented to a large extent by co-culture with 3 μM of emodin which per se did not affect HUVECs viability. In addition, CCL5 expression of HUVECs cultured in high glucose medium was significantly elevated at both mRNA and protein levels, an effect abolished after treatment with emodin. Moreover, the enhanced adhesion of monocytes to HUVECs (2.1-2.2 fold over control) and elevated chemotaxis activities (2.3-2.4 fold over control) in HUVECs cultured in high glucose medium were completely reversed by emodin. Emodin also suppressed activation of p38 MAPK and ERK1/2 due to high glucose. Our data demonstrated that endothelial cytotoxicity occurred clearly when HUVECs were exposed to high glucose milieu and emodin was able to alleviate the impairments. The protective effects of emodin might be related to the inhibition of CCL5 expression and subsequent cell stress/inflammatory events possibly mediated by activation of MAPK signaling pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

[Effect of Leonurus Heterophyllus Sweet on tissue factor transcription and expression in human umbilical vein endothelial cells in vitro].

PubMed

Zheng, Lian; Fang, Chi-hua

2007-06-01

To investigate the effect of Leonurus Heterophyllus Sweet, (LHS) on tissue factor (TF) transcription and expression induced by thrombin in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with different concentrations of LHS and the TF mRNA expression was detected by reverse transcript-polymerase chain reaction (RT-PCR). LHS treatment of HUVECs at different concentrations and for different times resulted in significant differences in TF expression (Plt;0.01). The transcription of TF in LHS-treated cells was significantly different from that of the blank control group (Plt;0.01). LHS can decrease the expression of TF and intervene with TF transcription in HUVECs in vitro.

Co-Seeding Human Endothelial Cells with Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells on Calcium Phosphate Scaffold Enhances Osteogenesis and Vascularization in Rats.

PubMed

Liu, Xian; Chen, Wenchuan; Zhang, Chi; Thein-Han, Wahwah; Hu, Kevin; Reynolds, Mark A; Bao, Chongyun; Wang, Ping; Zhao, Liang; Xu, Hockin H K

2017-06-01

A major challenge in repairing large bone defects with tissue-engineered constructs is the poor vascularization in the defect. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of seeded cells. To achieve favorable regenerative effects, prevascularization of tissue-engineered constructs by co-culturing of endothelial cells and bone cells is a promising strategy. The aim of this study was to investigate the effects of human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) co-cultured with human umbilical vein endothelial cells (HUVECs) for prevascularization of calcium phosphate cement (CPC) scaffold on bone regeneration in vivo for the first time. HUVECs co-cultured with hiPSC-MSCs formed microcapillary-like structures in vitro. HUVECs promoted mineralization of hiPSC-MSCs on CPC scaffolds. Four groups were tested in a cranial bone defect model in nude rats: (1) CPC scaffold alone (CPC control); (2) HUVEC-seeded CPC (CPC-HUVEC); (3) hiPSC-MSC-seeded CPC (CPC-hiPSC-MSC); and (4) HUVECs co-cultured with hiPSC-MSCs on CPC scaffolds (co-culture group). After 12 weeks, the co-culture group achieved the greatest new bone area percentage of 46.38% ± 3.8% among all groups (p < 0.05), which was more than four folds of the 10.61% ± 1.43% of CPC control. In conclusion, HUVECs co-cultured with hiPSC-MSCs substantially promoted bone regeneration. The novel construct of HUVECs co-cultured with hiPSC-MSCs delivered via CPC scaffolds is promising to enhance bone and vascular regeneration in orthopedic applications.

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells.

PubMed

Oliveira, Jessica Silva Santos de; Santos, Gabriela da Silva; Moraes, João Alfredo; Saliba, Alessandra Mattos; Barja-Fidalgo, Thereza Christina; Mattos-Guaraldi, Ana Luíza; Nagao, Prescilla Emy

2018-01-01

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Evodiamine attenuates TGF-β1-induced fibroblast activation and endothelial to mesenchymal transition.

PubMed

Wu, Qing-Qing; Xiao, Yang; Jiang, Xiao-Han; Yuan, Yuan; Yang, Zheng; Chang, Wei; Bian, Zhou-Yan; Tang, Qi-Zhu

2017-06-01

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.

Recombinant Treponema pallidum Protein Tp0965 Activates Endothelial Cells and Increases the Permeability of Endothelial Cell Monolayer

PubMed Central

Zhang, Rui-Li; Zhang, Jing-Ping; Wang, Qian-Qiu

2014-01-01

The recombinant Treponema pallidum protein Tp0965 (rTp0965), one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs) with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells) to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis. PMID:25514584

Role of tumour necrosis factor receptor-1 and nuclear factor-κB in production of TNF-α-induced pro-inflammatory microparticles in endothelial cells.

PubMed

Lee, S K; Yang, S-H; Kwon, I; Lee, O-H; Heo, J H

2014-09-02

Tumour necrosis factor-α (TNF-α) is upregulated in many inflammatory diseases and is also a potent agent for microparticle (MP) generation. Here, we describe an essential role of TNF-α in the production of endothelial cell-derived microparticles (EMPs) in vivo and the function of TNF-α-induced EMPs in endothelial cells. We found that TNF-α rapidly increased blood levels of EMPs in mice. Treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α also induced EMP formation in a time-dependent manner. Silencing of TNF receptor (TNFR)-1 or inhibition of the nuclear factor-κB (NF-κB) in HUVECs impaired the production of TNF-α-induced EMP. Incubation of HUVECs with PKH-67-stained EMPs showed that endothelial cells readily engulfed EMPs, and the engulfed TNF-α-induced EMPs promoted the expression of pro-apoptotic molecules and upregulated intercellular adhesion molecule-1 level on the cell surface, which led to monocyte adhesion. Collectively, our findings indicate that the generation of TNF-α-induced EMPs was mediated by TNFR1 or NF-κB and that EMPs can contribute to apoptosis and inflammation of endothelial cells.

Vitreous Cryopreservation of Human Umbilical Vein Endothelial Cells with Low Concentration of Cryoprotective Agents for Vascular Tissue Engineering

PubMed Central

Zheng, Yuanyuan; Panhwar, Fazil

2016-01-01

Cryopreservation of human umbilical vein endothelial cells (HUVECs) is important to tissue engineering applications and the study of the role of endothelial cells in cardiovascular and cerebrovascular diseases. The traditional methods for cryopreservation by vitrification (cooling samples to a cryogenic temperature without apparent freezing) using high concentration of cryoprotective agents (CPAs) and slow freezing are suboptimal due to the severe toxicity of high concentration of CPAs and ice formation-induced cryoinjuries, respectively. In this study, we developed a method to cryopreserve HUVECs by vitrification with low concentration of CPAs. This is achieved by optimizing the CPAs and using highly thermally conductive quartz capillary (QC) to contain samples for vitrification. The latter minimizes the thermal mass to create ultra-fast cooling/warming rates. Our data demonstrate that HUVECs can be vitrified in the QC using 1.4 mol/L ethylene glycol and 1.1 mol/L dimethyl sulfoxide with more than 90% viability. Moreover, this method significantly improves the attachment efficiency of the cryopreserved HUVECs. The attached cells post-cryopreservation proliferate similarly to fresh cells. Therefore, this study may provide an effective vitrification technique to bank HUVECs for vascular tissue engineering and other applications. PMID:27673413

Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis.

PubMed

Huet, Olivier; Cherreau, Christaine; Nicco, Carole; Dupic, Laurent; Conti, Marc; Borderie, Didier; Pene, Frédéric; Vicaut, Eric; Benhamou, Dan; Mira, Jean-Paul; Duranteau, Jacques; Batteux, Frédéric

2008-08-01

Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. Prospective, observational study. Medical intensive care unit in a university hospital. Twenty-five patients with septic shock and 10 healthy volunteers. Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2',7'-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. On admission, the septic shock population's mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.

Kisspeptin-10 induces endothelial cellular senescence and impaired endothelial cell growth.

PubMed

Usui, Sayaka; Iso, Yoshitaka; Sasai, Masahiro; Mizukami, Takuya; Mori, Hiroyoshi; Watanabe, Takuya; Shioda, Seiji; Suzuki, Hiroshi

2014-07-01

The KPs (kisspeptins) are a family of multifunctional peptides with established roles in cancer metastasis, puberty and vasoconstriction. The effects of KPs on endothelial cells have yet to be determined. The aim of the present study was to investigate the effects of KP-10 on endothelial cell growth and the mechanisms underlying those effects. The administration of recombinant KP-10 into the hindlimbs of rats with ischaemia significantly impaired blood flow recovery, as shown by laser Doppler, and capillary growth, as shown using histology, compared with the controls. HUVECs (human umbilical vein endothelial cells) express the KP receptor and were treated with KP-10 in culture studies. KP-10 inhibited endothelial cell tube formation and proliferation in a significant and dose-dependent manner. The HUVECs treated with KP exhibited the senescent phenotype, as determined using a senescence-associated β-galactosidase assay, cell morphology analysis, and decreased Sirt1 (sirtuin 1) expression and increased p53 expression shown by Western blot analysis. Intriguingly, a pharmacological Rho kinase inhibitor, Y-27632, was found to increase the proliferation of HUVECs and to reduce the number of senescent phenotype cells affected by KP-10. In conclusion, KP-10 suppressed endothelial cells growth both in vivo and in vitro in the present study. The adverse effect of KP on endothelial cells was attributable, at least in part, to the induction of cellular senescence.

Angiogenic effects of apigenin on endothelial cells after hypoxia-reoxygenation via the caveolin-1 pathway.

PubMed

Tu, Fengxia; Pang, Qiongyi; Chen, Xiang; Huang, Tingting; Liu, Meixia; Zhai, Qiongxiang

2017-12-01

In the present study, we aimed to elucidate whether apigenin contributes to the induction of angiogenesis and the related mechanisms in cell hypoxia-reoxygenation injury. The role of apigenin was examined in human umbilical vein endothelial cell (HUVEC) viability, migration and tube formation in vitro. To investigate the related mechanisms, we used caveolin-1 short interfering RNA. The viability of HUVECs was measured using Cell Counting Kit-8 assays, HUVEC migration was analyzed by crystal violet staining, and a tube formation assay was performed using the branch point method. Expression of caveolin-1, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) in HUVECs was examined by polymerase chain reaction and western blotting. Our data revealed that apigenin induced angiogenesis in vitro by increasing the tube formation ability of HUVECs, which was counteracted by caveolin-1 silencing. Compared to the NC group, Caveolin-1 and eNOS expression was upregulated by apigenin, whereas compared to the NC group, eNOS expression was increased upon caveolin-1 silencing. The expression of VEGF was increased by treatment with apigenin; however, compared to the NC group, caveolin-1 silencing did not affect VEGF expression, and apigenin did not increase VEGF expression in HUVECs after caveolin-1 silencing. These data suggest that apigenin may be a candidate therapeutic target for stroke recovery by promoting angiogenesis via the caveolin-1 signaling pathway.

Discovering the infectome of human endothelial cells challenged with Aspergillus fumigatus applying a mass spectrometry label-free approach.

PubMed

Curty, N; Kubitschek-Barreira, P H; Neves, G W; Gomes, D; Pizzatti, L; Abdelhay, E; Souza, G H M F; Lopes-Bezerra, L M

2014-01-31

Blood vessel invasion is a key feature of invasive aspergillosis. This angioinvasion process contributes to tissue thrombosis, which can impair the access of leukocytes and antifungal drugs to the site of infection. It has been demonstrated that human umbilical vein endothelial cells (HUVECs) are activated and assume a prothrombotic phenotype following contact with Aspergillus fumigatus hyphae or germlings, a process that is independent of fungus viability. However, the molecular mechanisms by which this pathogen can activate endothelial cells, together with the endothelial pathways that are involved in this process, remain unknown. Using a label-free approach by High Definition Mass Spectrometry (HDMS(E)), differentially expressed proteins were identified during HUVEC-A. fumigatus interaction. Among these, 89 proteins were determined to be up- or down-regulated, and another 409 proteins were exclusive to one experimental condition: the HUVEC control or HUVEC:AF interaction. The in silico predictions provided a general view of which biological processes and/or pathways were regulated during HUVEC:AF interaction, and they mainly included cell signaling, immune response and hemostasis pathways. This work describes the first global proteomic analysis of HUVECs following interaction with A. fumigatus germlings, the fungus morphotype that represents the first step of invasion and dissemination within the host. A. fumigatus causes the main opportunistic invasive fungal infection related to neutropenic hematologic patients. One of the key steps during the establishment of invasive aspergillosis is angioinvasion but the mechanism associated with the interaction of A. fumigatus with the vascular endothelium remains unknown. The identification of up- and down-regulated proteins expressed by human endothelial cells in response to the fungus infection can contribute to reveal the mechanism of endothelial response and, to understand the physiopathology of this high mortality disease. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 Elsevier B.V. All rights reserved.

Glycolipids from spinach suppress LPS-induced vascular inflammation through eNOS and NK-κB signaling.

PubMed

Ishii, Masakazu; Nakahara, Tatsuo; Araho, Daisuke; Murakami, Juri; Nishimura, Masahiro

2017-07-01

Glycolipids are the major constituent of the thylakoid membrane of higher plants and have a variety of biological and pharmacological activities. However, anti-inflammatory effects of glycolipids on vascular endothelial cells have not been elucidated. Here, we investigated the effect of glycolipids extracted from spinach on lipopolysaccharides (LPS)-induced endothelial inflammation and evaluated the underlying molecular mechanisms. Treatment with glycolipids from spinach had no cytotoxic effects on cultured human umbilical vein endothelial cells (HUVECs) and significantly blocked the expression of LPS-induced interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and intracellular adhesion molecule-1 (ICAM-1) in them. Glycolipids treatment also effectively suppressed monocyte adhesion to HUVECs. Treatment with glycolipids inhibited LPS-induced NF-κB phosphorylation and nuclear translocation. In addition, glycolipids treatment significantly promoted endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production in HUVECs. Furthermore, glycolipids treatment blocked LPS-induced inducible NOS (iNOS) expression in HUVECs. Pretreatment with a NOS inhibitor attenuated glycolipids-induced suppression of NF-κB activation and adhesion molecule expression, and abolished the glycolipids-mediated suppression of monocyte adhesion to HUVECs. These results indicate that glycolipids suppress LPS-induced vascular inflammation through attenuation of the NF-κB pathway by increasing NO production in endothelial cells. These findings suggest that glycolipids from spinach may have a potential therapeutic use for inflammatory vascular diseases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Investigation of antiviral state mediated by interferon-inducible transmembrane protein 1 induced by H9N2 virus and inactivated viral particle in human endothelial cells.

PubMed

Feng, Bo; Zhao, Lihong; Wang, Wei; Wang, Jianfang; Wang, Hongyan; Duan, Huiqin; Zhang, Jianjun; Qiao, Jian

2017-11-03

Endothelial cells are believed to play an important role in response to virus infection. Our previous microarray analysis showed that H9N2 virus infection and inactivated viral particle inoculation increased the expression of interferon-inducible transmembrane protein 1 (IFITM1) in human umbilical vein endothelial cells (HUVECs). In present study, we deeply investigated the expression patterns of IFITM1 and IFITM1-mediated antiviral response induced by H9N2 virus infection and inactivated viral particle inoculation in HUVECs. Epithelial cells that are considered target cells of the influenza virus were selected as a reference control. First, we quantified the expression levels of IFITM1 in HUVECs induced by H9N2 virus infection or viral particle inoculation using quantitative real-time PCR and western blot. Second, we observed whether hemagglutinin or neuraminidase affected IFITM1 expression in HUVECs. Finally, we investigated the effect of induced-IFITM1 on the antiviral state in HUVECs by siRNA and activation plasmid transfection. Both H9N2 virus infection and viral particle inoculation increased the expression of IFITM1 without elevating the levels of interferon-ɑ/β in HUVECs. HA or NA protein binding alone is not sufficient to increase the levels of IFITM1 and interferon-ɑ/β in HUVECs. IFITM1 induced by viral particle inoculation significantly decreased the virus titers in culture supernatants of HUVECs. Our results showed that inactivated viral particle inoculation increased the expression of IFITM1 at mRNA and protein levels. Moreover, the induction of IFITM1 expression mediated the antiviral state in HUVECs.

Persistent Infection of Human Fetal Endothelial Cells with Rubella Virus

PubMed Central

Perelygina, Ludmila; Zheng, Qi; Metcalfe, Maureen; Icenogle, Joseph

2013-01-01

Cardiovascular abnormalities are the leading cause of neonatal death among patients with congenital rubella syndrome (CRS). Although persistence of rubella virus (RV) in fetal endothelium has been repeatedly suggested as a possible cause of cardiovascular birth defects, evidence of the permissiveness of fetal endothelial cells to RV is lacking. In this study we evaluated the ability of RV to infect and persist in primary fetal endothelial cells derived from human umbilical vein (HUVEC). We found that wild type (wt) low passage clinical RV productively infected HUVEC cultures without producing cytopathology or ultrastructural changes. RV did not inhibit host cell protein synthesis, cell proliferation, or interfere with the cell cycle. Persistently infected cultures were easily established at low and high multiplicities of infection (MOI) with both laboratory and wt clinical RV strains. However, synchronous infections of entire HUVEC monolayers were only observed with clinical RV strains. The release of infectious virions into media remained at consistently high levels for several subcultures of infected HUVEC. The results indicate that macrovascular fetal endothelial cells are highly permissive to RV and allow slow persistent RV replication. The findings provide more evidence for the suggestion that vascular pathologies in CRS are triggered by persistent rubella virus infection of the endothelium. PMID:23940821

High-density lipoproteins protect endothelial cells from tumor necrosis factor-alpha-induced apoptosis.

PubMed

Sugano, M; Tsuchida, K; Makino, N

2000-06-16

High-density lipoproteins (HDL) levels have been shown to be inversely correlated with coronary heart disease, but the mechanisms of the direct protective effect of HDL on endothelial cells are not fully understood. The apoptosis of endothelial cells induced by cytokines and/or oxidized low-density lipoproteins, etc. may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis. Here we report that HDL prevent the apoptosis of human umbilical venous endothelial cells (HUVECs) induced by tumor necrosis factor-alpha (TNF-alpha) via an inhibition of CPP32-like protease activity. The incubation of HUVECs with TNF-alpha significantly increased the CPP32-like protease activity, and induced apoptosis. Preincubation of HUVECs with HDL before incubation with TNF-alpha significantly suppressed the increase in the CPP32-like protease activity, preventing apoptosis in a concentration-dependent manner. These results suggest that HDL prevent the suicide pathway leading to apoptosis of endothelial cells by decreasing the CPP32-like protease activity and that HDL thus play a protective role against the "response-to-injury" hypothesis of atherogenesis. Copyright 2000 Academic Press.

A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells.

PubMed

Kawasaki, Kengo; Muroyama, Koutarou; Yamamoto, Norio; Murosaki, Shinji

2015-01-01

The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.

Dehydrodiconiferyl alcohol suppresses monocyte adhesion to endothelial cells by attenuation of JNK signaling pathway.

PubMed

Tsuneyoshi, Tadamitsu; Kanamori, Yuta; Matsutomo, Toshiaki; Morihara, Naoaki

2015-09-25

Several clinical studies have shown that the intake of aged garlic extract improves endothelial dysfunction. Lignan compounds, (+)-(2S,3R)-dehydrodiconiferyl alcohol (DDC) and (-)-(2R,3S)-dihydrodehydrodiconiferyl alcohol (DDDC), have been isolated as antioxidants in aged garlic extract. There is evidence showing the importance of oxidative stress in endothelial dysfunction. In the present study, we examined whether DDC and DDDC enhance endothelial cell function in vitro. Cell adhesion assay was performed using THP-1 monocyte and human umbilical vein endothelial cells (HUVECs) which were activated by lipopolysaccharide (LPS) or advanced glycation end products (AGEs)-BSA. Cellular ELISA method was used for the evaluation of vascular cell adhesion molecule 1 (VCAM-1) expression on HUVECs. DDC and DDDC suppressed the adhesion of THP-1 to HUVECs which was activated by LPS or AGEs-BSA. DDC and DDDC also inhibited VCAM-1 expression induced by LPS or AGEs-BSA, but DDDC was less effective than DDC. In addition, the inhibitory effect of DDC on VCAM-1 expression involved suppressing JNK/c-Jun pathway rather than NF-κB pathway. DDC has an inhibitory effect on VCAM-1 expression via JNK pathway in endothelial cells and therefore may serve as a novel pharmacological agent to improve endothelial dysfunction. Copyright © 2015 Elsevier Inc. All rights reserved.

Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells.

PubMed Central

Savage, C. O.; Pottinger, B. E.; Gaskin, G.; Pusey, C. D.; Pearson, J. D.

1992-01-01

The ability of vasculitis-associated anti-neutrophil cytoplasm antibodies (ANCA) to activate neutrophils and mediate release of radiolabel from 111Indium-labeled cultured human umbilical vein endothelial cells (HUVEC) was determined as a measure of the potential cytotoxicity of ANCA-activated neutrophils against vascular endothelium. Priming of neutrophils with low doses of phorbol 12-myristate 13-acetate (PMA) (1 ng/ml) and ionomycin (0.1 mumol/1) was required, together with pretreatment of endothelial cells with BCNU (1,3-bis-[2-chloroethyl]-1-nitrosourea; 0.26 mmol/l). Under these conditions and using a 4-hour serum-free assay system, mouse monoclonal antibodies (MAb) to the target autoantigens proteinase-3 (Pr-3) and myeloperoxidase (MPO) mediated enhanced release of 111Indium from HUVEC compared with control MAb. Human IgG Fab2 C-ANCA (recognizing Pr-3) and P-ANCA (recognizing MPO) did likewise. Preactivation of HUVEC with TNF (50 U/ml, 4 hr) enhanced the release of 111Indium from HUVEC generated by neutrophils activated with anti-Pr-3 and anti-MPO MAb. These data support the suggestion that activation of neutrophils by ANCA within the vascular lumen may contribute to endothelial cell injury. PMID:1323218

Prevention of Osmotic Injury to Human Umbilical Vein Endothelial Cells for Biopreservation: A First Step Toward Biobanking of Endothelial Cells for Vascular Tissue Engineering.

PubMed

Niu, Dan; Zhao, Gang; Liu, Xiaoli; Zhou, Ping; Cao, Yunxia

2016-03-01

High-survival-rate cryopreservation of endothelial cells plays a critical role in vascular tissue engineering, while optimization of osmotic injuries is the first step toward successful cryopreservation. We designed a low-cost, easy-to-use, microfluidics-based microperfusion chamber to investigate the osmotic responses of human umbilical vein endothelial cells (HUVECs) at different temperatures, and then optimized the protocols for using cryoprotective agents (CPAs) to minimize osmotic injuries and improve processes before freezing and after thawing. The fundamental cryobiological parameters were measured using the microperfusion chamber, and then, the optimized protocols using these parameters were confirmed by survival evaluation and cell proliferation experiments. It was revealed for the first time that HUVECs have an unusually small permeability coefficient for Me2SO. Even at the concentrations well established for slow freezing of cells (1.5 M), one-step removal of CPAs for HUVECs might result in inevitable osmotic injuries, indicating that multiple-step removal is essential. Further experiments revealed that multistep removal of 1.5 M Me2SO at 25°C was the best protocol investigated, in good agreement with theory. These results should prove invaluable for optimization of cryopreservation protocols of HUVECs.

[The influence of HOXB2 anti-sense oligodeoxynucleotides on the proliferation and expression of human umbilical vein endothelial cells].

PubMed

Zhang, X; Liu, X; Liu, L

2001-12-01

To explore the effects of HOXB2 anti-sense oligodeoxynucleotides (asodn) on the proliferation and the expression of human umbilical vein endothelial cells (HUVECs). Various concentrations of HOXB2 ASODN modified by thiophosphate were transfected into HUVECs by liposome mediation. MTT and RT-PCR methods were employed to determine the influence of different concentrations of ASODN on endothelial proliferation and the expression level of HOXB2 mRNA. After the transfection of HOXB2 ASODN, the endothelial proliferation was inhibited in dose-dependent manner. Simultaneously, the expression level of HOXB2 mRNA decreased significantly. HOXB2 might play important roles in the proliferation of endothelial cells.

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF- R75, and by receptor type-specific agonists, binding exclusively to TNF- R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha- dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF- R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences. PMID:8386742

Serum podocalyxin is significantly increased in early-onset preeclampsia and may represent a novel marker of maternal endothelial cell dysfunction.

PubMed

Chen, Qi; Wang, Yao; Li, Ying; Zhao, Min; Nie, Guiying

2017-11-01

Podocalyxin is a glomerular podocyte protein and increased in urine of preeclampsia. However, podocalyxin is also expressed in endothelial cells of other organs. Here we investigated whether podocalyxin is detectable in pregnant serum and whether the levels are altered in preeclampsia. Podocalyxin was determined by ELISA in sera collected from normal pregnancy across gestation (n = 44) and from preeclamptic pregnancies at diagnosis (n = 34) with gestation-age-matched controls (n = 68). Immunohistochemistry examined podocalyxin in placentas and in 32 human tissues on a tissue array. Human umbilical vein endothelial cells (HUVECs) were treated with interleukin (IL)-6 and podocalyxin was analysed by ELISA and western blotting. Podocalyxin was detected in serum of normal pregnancy, with levels increasing progressively with advancing gestation. Podocalyxin serum levels were significantly elevated in preeclampsia, especially the early-onset subtype. Within the placenta, blood vessels but not trophoblasts expressed podocalyxin, and preeclampsia didn't differ from controls. Endothelial cells in all 32 human organs examined, as well as HUVECs, expressed podocalyxin. Its levels increased in the conditioned media but decreased in the lysates when HUVECs were treated with IL-6. Podocalyxin likely derived from maternal endothelial cells is present in pregnant serum and significantly increased in early-onset preeclampsia. Podocalyxin release was stimulated by IL-6 in HUVECs.

Hydrogen sulfide facilities production of nitric oxide via the Akt/endothelial nitric oxide synthases signaling pathway to protect human umbilical vein endothelial cells from injury by angiotensin II.

PubMed

Cui, Jiasen; Zhuang, Shunjiu; Qi, Shaohong; Li, Li; Zhou, Junwen; Zhang, Wan; Zhao, Yun; Qi, Ning; Yin, Yangjun; Huang, Lu

2017-11-01

Angiotensin II (Ang II) has been reported as key in inducing endothelial cell injury, and endothelial cells may produce nitric oxide (NO) to protect themselves. However, the underlying mechanism remains elusive. Human umbilical vein endothelial cells (HUVECs) were divided into five treatment groups as follows: Normal control, Ang II, Ang II + sodium hydrosulfide [NaHS; hydrogen sulfide (H2S) donor], Ang II + Akt inhibitors + NaHS, and Ang II + endothelial nitric oxide synthases (eNOS) inhibitors + NaHS. Subsequently, cell viability, apoptosis, migration, proliferation and adhesion ability were determined. In addition, tubular structure formation was observed, and the NO and phosphorylation levels of Akt and eNOS were evaluated. Compared with the normal control group, Ang II treatment reduced the viability of HUVECs and increased the level of cell apoptosis (P<0.05). Furthermore, Ang II treatment inhibited the phosphorylation level of eNOS and Akt, as well as the generation of NO (P<0.05). H2S reversed the above‑mentioned effects significantly and increased cell proliferation, adhesion ability and promoted tubular structure formation (P<0.05); however, H2S did not reverse the impact of eNOS and Akt phosphorylation levels after being processed with Akt and eNOS inhibitors, which indicates that H2S is capable of protecting HUVECs via the eNOS/Akt signaling pathway (P<0.05). Thus, H2S stimulates the production of NO and protects HUVECs via inducing the Akt/eNOS signaling pathway.

Cell-Responsive Hydrogel for Encapsulation of Vascular Cells

PubMed Central

Kraehenbuehl, Thomas P.; Ferreira, Lino S.; Zammaretti, Prisca; Hubbell, Jeffrey A.; Langer, Robert

2014-01-01

The in vitro potential of a synthetic matrix metalloproteinase (MMP)-responsive polyethylene glycol) (PEG)-based hydrogel as a bioactive co-encapsulation system for vascular cells and a small bioactive peptide, thymosin β4 (Tp4), was examined. We show that the physical incorporation of Tβ4 in this bioactive matrix creates a three-dimensional (3D) environment conducive for human umbilical vein endothelial cell (HUVEC) adhesion, survival, migration and organization. Gels with entrapped Tβ4 increased the survival of HUVEC compared to gels without Tp4, and significantly up-regulated the endothelial genes vascular endothelial-cadherin and angiopoietin-2, whereas von Willebrand factor was significantly down-regulated. Incorporation of Tβ4 significantly increased MMP-2 and MMP-9 secretion of encapsulated HUVEC. The gel acts as a controlled Tβ4-release system, as MMP-2 and MMP-9 enzymes trigger the release. In addition, Tβ4 facilitated HUVEC attachment and induced vascular-like network formation upon the PEG-hydrogels. These MMP-responsive PEG-hydrogels may thus serve as controlled co-encapsulation system of vascular cells and bioactive factors for in situ regeneration of ischemic tissues. PMID:19500842

Vitamin D protects endothelial cells from irradiation-induced senescence and apoptosis by modulating MAPK/SirT1 axis.

PubMed

Marampon, F; Gravina, G L; Festuccia, C; Popov, V M; Colapietro, E A; Sanità, P; Musio, D; De Felice, F; Lenzi, A; Jannini, E A; Di Cesare, E; Tombolini, V

2016-04-01

Radiotherapy toxicity is related to oxidative stress-mediated endothelial dysfunction. Here, we investigated on radioprotective properties of Vitamin D (Vit.D) on human endothelial cells (HUVEC). HUVEC, pre-treated with Vit.D, were exposed to ionizing radiation (IR): ROS production, cellular viability, apoptosis, senescence and western blot for protein detection were performed. The role of MAPKs pathway was investigated by using U0126 (10 μM) MEKs/ERKs-, SB203580 (2.5 μM) p38-inhibitor or by over/expressing MKK6 p38-upstream activator. Vit.D reduced IR-induced ROS production protecting proliferating and quiescent HUVEC from cellular apoptosis or senescence, respectively, by regulating MAPKs pathways. In proliferating HUVEC, Vit.D prevented IR-induced apoptosis by activating ERKs while in quiescent HUVEC counteracted IR-induced senescence by inhibiting the p38-IR-induced activation. MEKs&ERKs inhibition in proliferating or MKK6/mediated p38 activation in quiescent HUVEC, respectively, reverted anti-apoptotic or anti-senescent Vit.D properties. SirT1 protein expression levels were up-regulated by Vit.D. ERKs inhibition blocked Vit.D-induced SirT1 protein up-regulation in proliferating cells. In quiescent HUVEC cells, p38 inhibition counteracted the IR-induced SirT1 protein down-regulation, while MKK6 transfection abrogated the Vit.D positive effects on SirT1 protein levels after irradiation. SirT1 inhibition by sirtinol blocked the Vit.D radioprotective effects. Vit.D protects HUVEC from IR induced/oxidative stress by positively regulating the MAPKs/SirT1 axis.

Circular RNA hsa_circ_0003575 regulates oxLDL induced vascular endothelial cells proliferation and angiogenesis.

PubMed

Li, Chen-Ye; Ma, Lan; Yu, Bo

2017-11-01

Circular RNAs (circRNAs) are a novel class of RNAs generated from back-splicing and characterized by covalently closed continuous loops. Recently, circRNAs have recently shown large regulation on cardiovascular system, including atherosclerosis. The present study aims to investigate the circRNA expression profile and identify their roles on vascular endothelial cells induced by oxLDL. Human circRNA microarray analysis revealed that total 943 differently expressed circRNAs were screened with 2 fold change. Hsa_circ_0003575 was validated to be significantly up-regulated in oxLDL induced HUVECs. Loss-of-function experiments indicated that hsa_circ_0003575 silencing promoted the proliferation and angiogenesis ability of HUVECs. Bioinformatics online programs predicted the potential circRNA-miRNA-mRNA network for hsa_circ_0003575. In summary, circRNA microarray analysis reveals the expression profiles of HUVECs and verifies the role of hsa_circ_0003575 on HUVECs, providing a therapeutic strategy for vascular endothelial cell injury of atherosclerosis. Copyright © 2017. Published by Elsevier Masson SAS.

Effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells.

PubMed

Cai, Guoping; Lai, Binbin; Hong, Huaxing; Lin, Peng; Chen, Weifu; Zhu, Zhong; Chen, Haixiao

2017-07-01

Cryopreservation is widely used in regenerative medicine for tissue preservation. In the present study, the effects of cryopreservation on excretory function, cellular adhesion molecules and vessel lumen formation in human umbilical vein endothelial cells (HUVECs) were investigated. After 0, 4, 8, 12 or 24 weeks of cryopreservation in liquid nitrogen, the HUVECs were thawed. The excretory functions markers (endothelin‑1, prostaglandin E1, von Willebrand factor and nitric oxide) of HUVECs were measured by ELISA assay. The expression of intercellular adhesion molecule‑1 (ICAM‑1) in HUVECs was analyzed using flow cytometry. An angiogenesis assay was used to determine the angiogeneic capabilities of the thawed HUVECs. The results demonstrated that cryopreserved/thawed and recultivated HUVECs were unsuitable for tissue‑engineered microvascular construction. Specifically, the excretory function of the cells was significantly decreased in the post‑cryopreserved HUVECs at 24 weeks. In addition, the level of ICAM‑1 in HUVECs was significantly upregulated from the fourth week of cryopreservation. Furthermore, the tube‑like structure‑forming potential was weakened with increasing cryopreservation duration, and the numbers of lumen and the length of the pipeline were decreased in the thawed HUVECs, in a time‑dependent manner. In conclusion, the results of the present study revealed that prolonged cryopreservation may lead to HUVEC dysfunction and did not create stable cell lines for tissue‑engineered microvascular construction.

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

PubMed

Lukashevich, I S; Maryankova, R; Vladyko, A S; Nashkevich, N; Koleda, S; Djavani, M; Horejsh, D; Voitenok, N N; Salvato, M S

1999-12-01

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF. Copyright 1999 Wiley-Liss, Inc.

Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

PubMed

Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

2015-01-01

The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the supernatant increased significantly after 1.5 min to 466 ± 543 pg·mL-1 (p <  0.001) and after 5 min to 408 ± 458 pg·mL-1 (p <  0.001), while in control cells the prostacyclin concentration did not change remaining in the range of 50 ± 48.9 pg·mL-1. This study revealed that the exchange of the cell culture medium led to a rapid disturbance of the HUVEC with stress fiber formation, disconnection of cell-cell contacts and an altered prostacyclin secretion, which had regressed nearly completely after 12 hours. Therefore, the evaluation of HUVEC on body foreign materials should be performed not earlier than 12 hours after cell culture medium exchange to avoid a misinterpretation of the endothelial cell morphological state. This procedure minimizes the risk of a misinterpretation of the endothelial cell morphology - caused by the culture medium exchange and not by the interaction between biomaterials and HUVEC.

Far-infrared protects vascular endothelial cells from advanced glycation end products-induced injury via PLZF-mediated autophagy in diabetic mice

PubMed Central

Chen, Cheng-Hsien; Chen, Tso-Hsiao; Wu, Mei-Yi; Chou, Tz-Chong; Chen, Jia-Rung; Wei, Meng-Jun; Lee, San-Liang; Hong, Li-Yu; Zheng, Cai-Mei; Chiu, I-Jen; Lin, Yuh-Feng; Hsu, Ching-Min; Hsu, Yung-Ho

2017-01-01

The accumulation of advanced glycation end products (AGEs) in diabetic patients induces vascular endothelial injury. Promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor that can be activated by low-temperature far-infrared (FIR) irradiation to exert beneficial effects on the vascular endothelium. In the present study, we investigated the influence of FIR-induced PLZF activation on AGE-induced endothelial injury both in vitro and in vivo. FIR irradiation inhibited AGE-induced apoptosis in human umbilical vein endothelial cells (HUVECs). PLZF activation increased the expression of phosphatidylinositol-3 kinases (PI3K), which are important kinases in the autophagic signaling pathway. FIR-induced PLZF activation led to autophagy in HUVEC, which was mediated through the upregulation of PI3K. Immunofluorescence staining showed that AGEs were engulfed by HUVECs and localized to lysosomes. FIR-induced autophagy promoted AGEs degradation in HUVECs. In nicotinamide/streptozotocin-induced diabetic mice, FIR therapy reduced serum AGEs and AGEs deposition at the vascular endothelium. FIR therapy also reduced diabetes-induced inflammatory markers in the vascular endothelium and improved vascular endothelial function. These protective effects of FIR therapy were not found in PLZF-knockout mice. Our data suggest that FIR-induced PLZF activation in vascular endothelial cells protects the vascular endothelium in diabetic mice from AGE-induced injury. PMID:28071754

[Protective effect and mechanism of compound Ginkgo biloba granules on oxidative stress injury of HUVEC].

PubMed

Li, Qi; Chen, Xi; Kan, Xiao-Xi; Li, Yu-Jie; Yang, Qing; Wang, Ya-Jie; Chen, Ying; Weng, Xiao-Gang; Cai, Wei-Yan; Huang, He-Fei; Zhu, Xiao-Xin

2016-02-01

To reveal the protective and anti-apoptosis effect of compound Ginkgo biloba granules on oxidative stress injury of human umbilical vein endothelial cells (HUVEC). Negative control group, H2O2 model group and 4 drug pretreatment groups (80, 160, 320, 640 mg• L⁻¹) were established. The cell proliferation, morphological changes in each group after oxidative stress injury was detected by MTT assay and through microscope observation respectively. The content of LDH, MDA, SOD and NO and SOD activity in supernatant were detected to judge the protection effect of the drugs on endothelial cells. The protective effect on HUVEC apoptosis was analyzed by Caspase-3 activity test and Annexin V-FITC/PI staining. Western blot was used to observe the expression of apoptosis-related proteins Bcl-2 and Bax. Results showed that 1 200 μmol• L⁻¹ H2O2 can induce oxidative stress injury in endothelial cells and reduce the cell survival rate; cell proliferation inhibition degree is positively correlated with the effect time of H2O2. Besides, 80, 160, 320 640 mg•L⁻¹ compound Ginkgo biloba granules can protect HUVEC from oxidative stress injury, recover the normal proliferation level of cells, improve their state, prohibit cell apoptosis, and can up-regulate and down-regulate the expression level of Bcl-2 and Bax respectively. In conclusion, compound G. biloba granules can protect HUVEC from the oxidative stress injury induced by H2O2, its mechanism may be correlated with inhibition of the mitochondrial apoptotic pathway in HUVEC. Copyright© by the Chinese Pharmaceutical Association.

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin.

PubMed

Giraux, J L; Matou, S; Bros, A; Tapon-Bretaudière, J; Letourneur, D; Fischer, A M

1998-12-01

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.

Radiation therapy affects the mechanical behavior of human umbilical vein endothelial cells.

PubMed

Mohammadkarim, Alireza; Tabatabaei, Mohammad; Parandakh, Azim; Mokhtari-Dizaji, Manijhe; Tafazzoli-Shadpour, Mohammad; Khani, Mohammad-Mehdi

2018-06-06

Radiation therapy has been widely utilized as an effective method to eliminate malignant tumors and cancerous cells. However, subjection of healthy tissues and the related networks of blood vessels adjacent to the tumor area to irradiation is inevitable. The aim of this study was to investigate the consequent effects of fractionation radiotherapy on the mechanical characteristics of human umbilical vein endothelial cells (HUVECs) through alterations in cytoskeleton organization and cell and nucleus morphology. In order to simulate the clinical condition of radiotherapy, the HUVECs were exposed to the specific dose of 2 Gy for 1-4 times among four groups with incremental total dose from 2 Gy up to 8 Gy. Fluorescence staining was performed to label F-actin filaments and nuclei. Micropipette aspiration and standard linear solid model were employed to evaluate the elastic and viscoelastic characteristics of the HUVECs. Radiotherapy significantly increased cell elastic moduli. Due to irradiation, instantaneous and equilibrium Young's modulus were also increased. Radiotherapy diminished HUVECs viscoelastic behavior and shifted their creep compliance curves downward. Furthermore, gamma irradiation elevated the nuclei sizes and to a lesser extent the cells sizes resulting in the accumulation of F-actin filaments within the rest of cell body. Endothelial stiffening correlates with endothelial dysfunction, hence the results may be helpful when the consequent effects of radiotherapy are the focus of concern. Copyright © 2018. Published by Elsevier Ltd.

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

PubMed Central

de Oliveira, Jessica Silva Santos; Santos, Gabriela da Silva; Moraes, João Alfredo; Saliba, Alessandra Mattos; Barja-Fidalgo, Thereza Christina; Mattos-Guaraldi, Ana Luíza; Nagao, Prescilla Emy

2018-01-01

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis. PMID:29641644

Advanced glycation end product Nε-carboxymethyllysine induces endothelial cell injury: the involvement of SHP-1-regulated VEGFR-2 dephosphorylation.

PubMed

Liu, Shing Hwa; Sheu, Wayne Huey Herng; Lee, Maw Rong; Lee, Wen Jane; Yi, Yu Chiao; Yang, Tzung Jie; Jen, Jen Fon; Pan, Hung Chuan; Shen, Chin Chang; Chen, Wen Bao; Tien, Hsing Ru; Sheu, Meei Ling

2013-06-01

N(ε)-carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes-induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML-related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)-mediated SH2 domain-containing tyrosine phosphatase-1 (SHP-1) activation by CML inhibits the VEGF receptor-2 (VEGFR-2, KDR/Flk-1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR-2 activation in parallel with the increased SHP-1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP-1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP-1 with VEGFR-2. Consistently, SHP-1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [(3)H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML-increased SHP-1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP-1 and CML was increased, but phospho-VEGFR-2 staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Agglucetin, a tetrameric C-type lectin-like venom protein, regulates endothelial cell survival and promotes angiogenesis by activating integrin {alpha}v{beta}3 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, W.-J.

2008-05-02

Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-{alpha}v{beta}3 monoclonal antibody (mAb), but not antibodies against {alpha}2 and {alpha}5, toward HUVEC and purified {alpha}v{beta}3 also bound to immobilizedmore » agglucetin-{beta} in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering {alpha}v{beta}3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.« less

Human endothelial cells hollow fiber membrane bioreactor as a model of the blood vessel for in vitro studies.

PubMed

Ciechanowska, Anna; Ladyzynski, Piotr; Hoser, Grazyna; Sabalinska, Stanislawa; Kawiak, Jerzy; Foltynski, Piotr; Wojciechowski, Cezary; Chwojnowski, Andrzej

2016-09-01

Human endothelial cells are used in experimental models for studying in vitro pathophysiological mechanisms of different diseases. We developed an original bioreactor, which can simulate human blood vessel, with capillary polysulfone membranes covered with the human umbilical vein endothelial cells (HUVECs) and we characterized its properties. The elaborated cell seeding and culturing procedures ensured formation of a confluent cell monolayer on the inside surface of capillaries within 24 h of culturing under the shear stress of 6.6 dyn/cm(2). The optimal density of cells to be seeded was 60,000 cells/cm(2). Labeling HUVECs with carboxyfluorescein succinimidyl ester (CFSE) did not influence cells' metabolism. Flow cytometry-based analysis of HUVECs stained with CFSE demonstrated that in a presence of the shear stress cells' proliferation was much inhibited (after 72 h proliferation index was equal to 1.9 and 6.2 for cultures with and without shear stress, respectively) and the monolayer was formed mainly due to migration and spreading of cells that were physiologically elongated in a direction of the flow. Monitoring of cells' metabolism showed that HUVECs cultured in a presence of the shear stress preferred anaerobic metabolism and they consumed 1.5 times more glucose and produced 2.3 times more lactate than the cells cultured under static conditions. Daily von Willebrand factor production by HUVECs was near 2 times higher in a presence of the shear stress. The developed model can be used for at least 3 days in target studies under conditions mimicking the in vivo state more closely than the static HUVEC cultures.

Calcium influx through the TRPV1 channel of endothelial cells (ECs) correlates with a stronger adhesion between monocytes and ECs.

PubMed

Himi, N; Hamaguchi, A; Hashimoto, K; Koga, T; Narita, K; Miyamoto, O

2012-01-01

Atherosclerosis is thought to be initiated by the transendothelial migration of monocytes. In the early stage of this process, the adhesion of monocytes to endothelial cells is supported by an increase in the intracellular concentration of calcium ion ([Ca(2+)]i) in endothelial cells. However, the main source of Ca(2+) has been unclear. In this study, the changes in ionic transmittance and [Ca(2+)]i due to the adhesion of monocytes were continuously measured by an electrophysiological technique and fluorescent imaging. Especially, we focused on transient receptor potential vanilloid channel 1 (TRPV1) as a Ca(2+) channel that could influence the adhesion of monocytes. Whole-cell current was continuously recorded in human umbilical vein endothelial cells (HUVECs) by a patch electrode. The adhesion of monocytes (THP-1) induced a transient inward current in HUVECs, as well as an elevation of [Ca(2+)]i. This inward element was abolished by the application of 100 nM SB366,791, a selective antagonist of TRPV1 channel. Furthermore, SB366,791 significantly decreased the number of THP-1 cells that adhered to HUVECs (control: 231 ± 38, SB366,791: 96 ± 16 cells/mm2). These results suggest that an inward calcium current via the TRPV1 channels of endothelial cells correlates with a stronger adhesion between monocytes and endothelial cells.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

PubMed

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

Omentin inhibits TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via ERK/NF-{kappa}B pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Xia, E-mail: zhongxia1977@126.com; Li, Xiaonan; Liu, Fuli

2012-08-24

Highlights: Black-Right-Pointing-Pointer Omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Black-Right-Pointing-Pointer Omentin reduces expression of ICAM-1 and VCAM-1 induced by TNF-{alpha} in HUVECs. Black-Right-Pointing-Pointer Omentin inhibits TNF-{alpha}-induced ERK and NF-{kappa}B activation in HUVECs. Black-Right-Pointing-Pointer Omentin supreeses TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 via ERK/NF-{kappa}B pathway. -- Abstract: In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha}) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibitedmore » TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-{alpha}-activated signal pathway of nuclear factor-{kappa}B (NF-{kappa}B) by preventing NF-{kappa}B inhibitory protein (I{kappa}B{alpha}) degradation and NF-{kappa}B/DNA binding activity. Omentin pretreatment significantly inhibited TNF-{alpha}-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-{alpha}-induced NF-{kappa}B activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-{alpha}. These results suggest that omentin may inhibit TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-{kappa}B pathway.« less

Low Concentration of S100A8/9 Promotes Angiogenesis-Related Activity of Vascular Endothelial Cells: Bridges among Inflammation, Angiogenesis, and Tumorigenesis?

PubMed Central

Li, Changyou; Li, Siyuan; Jia, Changkai; Yang, Lingling; Song, Zicheng; Wang, Yiqiang

2012-01-01

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 μg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 μg/mL S100A8 revealed that ribosome pathway, pathogenic Escherichia coli infection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis. PMID:22685372

Effect of Granulocyte-Colony Stimulating Factor on Endothelial Cells and Osteoblasts

PubMed Central

Liu, Xi Ling; Hu, Xiang; Cai, Wei Xin; Lu, Weijia William; Zheng, Li Wu

2016-01-01

Objectives. Some animal studies showed that granulocyte-colony stimulating factor (G-CSF) provides beneficial environment for bone healing. It has been well documented that endothelial cells and osteoblasts play critical roles in multiple phases of bone healing. However, the biological effects of G-CSF on these cells remain controversial. This study aimed to investigate the influence of G-CSF at various concentrations on endothelial cells and osteoblasts. Materials and Methods. Human umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) were treated with G-CSF at 1000, 100, 10, and 0 ng/mL, respectively. The capacity of cell proliferation, migration, and tube formation of HUVECs was evaluated at 72, 8, and 6 hours after treatment, respectively. The capacity of proliferation, differentiation, and mineralization of hOBs was evaluated at 24 hours, 72 hours, and 21 days after treatment, respectively. Results. HUVECs treated with 100 and 1000 ng/mL G-CSF showed a significantly higher value comparing with controls in migration assay (p < 0.001, p < 0.01, resp.); the group treated with 1000 ng/mL G-CSF showed a significantly lower value on tube formation. No significant difference was detected in groups of hOBs. Conclusions. G-CSF showed favorable effects only on the migration of HUVECs, and no direct influence was found on hOBs. PMID:27006951

Differential ability of formononetin to stimulate proliferation of endothelial cells and breast cancer cells via a feedback loop involving MicroRNA-375, RASD1, and ERα.

PubMed

Chen, Jian; Zhang, Xing; Wang, Yong; Ye, Yu; Huang, Zhaoquan

2018-05-02

For postmenopausal cardiovascular disease, long-term estrogen therapy may increase the risk of breast cancer. To reduce this risk, estrogen may be replaced with the phytoestrogen formononetin, but how formononetin acts on vascular endothelial cells (ECs) and breast cancer cells is unclear. Here, we show that low concentrations of formononetin induced proliferation and inhibited apoptosis more strongly in cultured human umbilical vein endothelial cells (HUVECs) than in breast cancer cells expressing estrogen receptor α (ERα) (MCF-7, BT474) or not (MDA-MB-231), and that this differential stimulation was associated with miR-375 up-regulation in HUVECs. For the first time, we demonstrate the presence of a feedback loop involving miR-375, ras dexamethasone-induced 1 (RASD1), and ERα in normal HUVECs, and we show that formononetin stimulated this feedback loop in HUVECs but not in MCF-7 or BT474 cells. In all three cell lines, formononetin increased Akt phosphorylation and Bcl-2 expression. Inhibiting miR-375 blocked these changes and increased proliferation in HUVECs, but not in MCF-7 or BT474 cells. In ovariectomized rats, formononetin increased uterine weight and caused similar changes in levels of miR-375, RASD1, ERα, and Bcl-2 in aortic ECs as in cultured HUVECs. In mice bearing MCF-7 xenografts, tumor growth was stimulated by 17β-estradiol but not by formononetin. These results suggest selective action of formononetin in ECs (proliferation stimulation and apoptosis inhibition) relative to breast cancer cells, possibly via a feedback loop involving miR-375, RASD1, and ERα. This differential effect may explain why formononetin may not increase the risk of postmenopausal breast cancer. © 2018 Wiley Periodicals, Inc.

Ezetimibe inhibits platelet activation and uPAR expression on endothelial cells.

PubMed

Becher, Tobias; Schulze, Torsten J; Schmitt, Melanie; Trinkmann, Frederik; El-Battrawy, Ibrahim; Akin, Ibrahim; Kälsch, Thorsten; Borggrefe, Martin; Stach, Ksenija

2017-01-15

Lipid lowering therapy constitutes the basis of cardiovascular disease therapy. The purpose of this study was to investigate effects of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on platelets and endothelial cells in an in vitro endothelial cell model. After a 24h incubation period with ezetimibe (concentrations 1, 50, 100 and 1000ng/ml), human umbilical vein endothelial cells (HUVEC) were stimulated for 1h with lipopolysaccharide (LPS) and were then incubated in direct contact with activated platelets. Following this, the expression of CD40L and CD62P on platelets, and the expression of ICAM-1, VCAM-1, uPAR, and MT1-MMP on endothelial cells were measured by flow cytometry. Supernatants were analysed by enzyme linked immunosorbent assay for soluble MCP-1, IL-6 and MMP-1. The increased expression of uPAR on endothelial cells by proinflammatory stimulation with LPS and by direct endothelial contact with activated platelets was significantly reduced through pre-incubation with 100ng/ml and 1000ng/ml ezetimibe (p<0.05). Platelets directly incubated with ezetimibe but without endothelial cell contact showed significantly reduced CD62P and CD40L surface expression (p<0.05). Ezetimibe had no significant effects on HUVEC expression of MT1-MMP, ICAM-1 and VCAM-1 and on CD40L expression on platelets in direct contact with endothelial cells. Levels of soluble IL-6 in HUVEC supernatants were significantly lower after pre-incubation with ezetimibe. In this in vitro analysis, ezetimibe directly attenuates platelet activation and has significant endothelial cell mediated effects on selected markers of atherosclerosis. Copyright © 2016. Published by Elsevier Ireland Ltd.

Neuropilin2 expressed in gastric cancer endothelial cells increases the proliferation and migration of endothelial cells in response to VEGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Woo Ho; Lee, Sun Hee; Jung, Myung Hwan

2009-08-01

The structure and characteristics of the tumor vasculature are known to be different from those of normal vessels. Neuropilin2 (Nrp2), which is expressed in non-endothelial cell types, such as neuronal or cancer cells, functions as a receptor for both semaphorin and vascular endothelial growth factor (VEGF). After isolating tumor and normal endothelial cells from advanced gastric cancer tissue and normal gastric mucosa tissues, respectively, we identified genes that were differentially expressed in gastric tumor endothelial (TEC) and normal endothelial cells (NEC) using DNA oligomer chips. Using reverse transcriptase-PCR, we confirmed the chip results by showing that Nrp2 gene expression ismore » significantly up-regulated in TEC. Genes that were found to be up-regulated in TEC were also observed to be up-regulated in human umbilical vein endothelial cells (HUVECs) that were co-cultured with gastric cancer cells. In addition, HUVECs co-cultured with gastric cancer cells showed an increased reactivity to VEGF-induced proliferation and migration. Moreover, overexpression of Nrp2 in HUVECs significantly enhanced the proliferation and migration induced by VEGF. Observation of an immunohistochemical analysis of various human tumor tissue arrays revealed that Nrp2 is highly expressed in the tumor vessel lining and to a lesser extent in normal tissue microvessels. From these results, we suggest that Nrp2 may function to increase the response to VEGF, which is more significant in TEC than in NEC given the differential expression, leading to gastric TEC with aggressive angiogenesis phenotypes.« less

Trimethylamine N-oxide in atherogenesis: impairing endothelial self-repair capacity and enhancing monocyte adhesion.

PubMed

Ma, GuoHua; Pan, Bing; Chen, Yue; Guo, CaiXia; Zhao, MingMing; Zheng, LeMin; Chen, BuXing

2017-04-30

Several studies have reported a strong association between high plasma level of trimethylamine N-oxide (TMAO) and atherosclerosis development. However, the exact mechanism underlying this correlation is unknown. In the present study, we try to explore the impact of TMAO on endothelial dysfunction. After TMAO treatment, human umbilical vein endothelial cells (HUVECs) showed significant impairment in cellular proliferation and HUVECs-extracellular matrix (ECM) adhesion compared with control. Likewise, TMAO markedly suppressed HUVECs migration in transwell migration assay and wound healing assay. In addition, we found TMAO up-regulated vascular cell adhesion molecule-1 (VCAM-1) expression, promoted monocyte adherence, activated protein kinase C (PKC) and p-NF-κB. Interestingly, TMAO-stimulated VCAM-1 expression and monocyte adherence were diminished by PKC inhibitor. These results demonstrate that TMAO promotes early pathological process of atherosclerosis by accelerating endothelial dysfunction, including decreasing endothelial self-repair and increasing monocyte adhesion. Furthermore, TMAO-induced monocyte adhesion is partly attributable to activation of PKC/NF-κB/VCAM-1. © 2017 The Author(s).

ITE Suppresses Angiogenic Responses in Human Artery and Vein Endothelial Cells: Differential Roles of AhR.

PubMed

Li, Yan; Wang, Kai; Zou, Qing-Yun; Jiang, Yi-Zhou; Zhou, Chi; Zheng, Jing

2017-12-01

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor is involved in regulation of many essential biological processes including vascular development and angiogenesis. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an AhR ligand, which regulates immune responses and cancer cell growth. However, the roles of the ITE/AhR pathway in mediating placental angiogenesis remains elusive. Here, we determined if ITE affected placental angiogenic responses via AhR in human umbilical vein (HUVECs) and artery endothelial (HUAECs) cells in vitro. We observed that ITE dose- and time-dependently inhibited proliferation and viability of HUAECs and HUVECs, whereas it inhibited migration of HUAECs, but not HUVECs. While AhR siRNA significantly suppressed AhR protein expression in HUVECs and HUAECs, it attenuated the ITE-inhibited angiogenic responses of HUAECs, but not HUVECs. Collectively, ITE suppressed angiogenic responses of HUAECs and HUVECs, dependent and independent of AhR, respectively. These data suggest that ITE may regulate placental angiogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Taspine isolated from Radix et Rhizoma Leonticis inhibits growth of human umbilical vein endothelial cell (HUVEC) by inducing its apoptosis.

PubMed

Zhang, Yanmin; He, Langchong; Zhou, Yali

2008-01-01

The present study was to evaluate the effects of taspine isolated from Radix et Rhizoma Leonticsi on the growth and apoptosis of human umbilical vein endothelial cell (HUVEC) line by MTT and flow cytometer, respectively. At the same time, a series of changes were observed in HUVEC treated by taspine, including microstructure, protein expression of bax, bcl-2 and VEGF. The change of microstructure was observed by transmission electron microscope (TEM). The protein expression of bax and bcl-2 was detected by immunohistochemistry (IHC), and VEGF protein secreted was determined by enzyme-linked immunosorbent assay (ELISA). The results showed taspine could inhibit growth and induce apoptosis of HUVEC in a dose-dependent manner. Cell cycle was significantly stopped at the S phase. Under electronic microscope, the morphology of HUVEC treated with taspine showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body. Bcl-2 and VEGF expressions were decreased and bax expression was increased. All these results demonstrate that taspine has an inhibitory effect on growth of HUVEC and can induce its apoptosis.

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

PubMed Central

Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay

2014-01-01

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis. PMID:24615331

Adipose-derived stem cell-derived microvesicle-released miR-210 promoted proliferation, migration and invasion of endothelial cells by regulating RUNX3.

PubMed

Zheng, Zeqi; Liu, Lijuan; Zhan, Yuliang; Yu, Songping; Kang, Ting

2018-06-18

To explore the potential mechanism of miRNA released from adipose-derived stem cell (ADSC)-derived micro vesicle (MV) on the modulation of proliferation, migration and invasion of endothelial cells. miR-210 level was detected by qT-PCR. Alix, VEGF and RUNX3 expressions were detected by Western blot. The proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) were observed by MTT assay and Transwell assay. Luciferase reporter gene assay was conducted to validate the targeting activity of MVs-released miR-210 on RUNX3. Hypoxia significantly increased the expression of MVs-released miR-210. MVs released from ADSCs in hypoxic group significantly promoted the proliferation, migration and invasion of HUVECs. Overexpression of miR-210 significantly upregulated VEGF expression, and promoted the proliferation, migration and invasion of HUVECs. Besides, RUNX3 was identified as the direct of miR-210 in HUVECs. Overexpression of miR-210 decreased RUNX3 expression and promoted the proliferation, migration and invasion of HUVECs, while overexpression of RUNX3 inhibited these promotion effects. In vivo experiment showed that MVs derived from ADSCs under hypoxia increased miR-210 level and capillary density, and inhibition of miR-210 decreased capillary density. We also found MVs downregulated RUNX3 expression, and inhibition of miR-210 upregulated RUNX3 expression. miR-210 released from ADSCs-derived MVs promoted proliferation, migration and invasion of HUVECs by targeting RUNX3, which revealed one of the mechanisms of ADSCs-derived MVs on the promotion of proliferation, migration and invasion of HUVECs.

Expansion and cryopreservation of porcine and human corneal endothelial cells.

PubMed

Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

2017-08-01

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Association of Plasmodium falciparum with Human Endothelial Cells in vitro

PubMed Central

Utter, Christopher; Serrano, Adelfa E.; Glod, John W.; Leibowitz, Michael J.

2017-01-01

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria. PMID:28656007

Interleukin-6 and intercellular cell adhesion molecule-1 expression remains elevated in revived live endothelial cells following spaceflight.

PubMed

Muid, S; Froemming, G R A; Ali, A M; Nawawi, H

2013-12-01

The effects of spaceflight on cardiovascular health are not necessarily seen immediately after astronauts have returned but can be delayed. It is important to investigate the long term effects of spaceflight on protein and gene expression of inflammation and endothelial activation as a predictor for the development of atherosclerosis and potential cardiovascular problems. The objectives of this study were to investigate the (a) protein and gene expression of inflammation and endothelial activation, (b) expression of nuclear factor kappa B (NFκB), signal transducer and activator of transcription-3 (STAT-3) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) 3 months post-space flight travel compared to ground controls. HUVEC cultured on microcarriers in fluid processing apparatus were flown to the International Space Station (ISS) by the Soyuz TMA-11 rocket. After landing, the cells were detached from microcarriers and recultured in T-25 cm(2) culture flasks (Revived HUVEC). Soluble protein expression of IL-6, TNF-α, ICAM-1, VCAM-1 and e-selectin were measured by ELISA. Gene expression of these markers and in addition NFκB, STAT-3 and eNOS were measured. Spaceflight induced IL-6 and ICAM-1 remain elevated even after 3 months post spaceflight travel and this is mediated via STAT-3 pathway. The downregulation of eNOS expression in revived HUVEC cells suggests a reduced protection of the cells and the surrounding vessels against future insults that may lead to atherosclerosis. It would be crucial to explore preventive measures, in relation to atherosclerosis and its related complications.

Prostaglandin E2 induces expression of P-selectin (CD62P) on cultured human umbilical vein endothelial cells and enhances endothelial binding of CD4-T-cells.

PubMed

Hailer, N P; Oppermann, E; Leckel, K; Cinatl, J; Markus, B H; Blaheta, R A

2000-07-15

Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.

EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways.

PubMed

Liu, Shumin; Sun, Zhengwu; Chu, Peng; Li, Hailong; Ahsan, Anil; Zhou, Ziru; Zhang, Zonghui; Sun, Bin; Wu, Jingjun; Xi, Yalin; Han, Guozhu; Lin, Yuan; Peng, Jinyong; Tang, Zeyao

2017-05-01

Homocysteine (Hcy) induced vascular endothelial injury leads to the progression of endothelial dysfunction in atherosclerosis. Epigallocatechin gallate (EGCG), a natural dietary antioxidant, has been applied to protect against atherosclerosis. However, the underlying protective mechanism of EGCG has not been clarified. The present study investigated the mechanism of EGCG protected against Hcy-induced human umbilical vein endothelial cells (HUVECs) apoptosis. Methyl thiazolyl tetrazolium assay (MTT), transmission electron microscope, fluorescent staining, flow cytometry, western blot were used in this study. The study has demonstrated that EGCG suppressed Hcy-induced endothelial cell morphological changes and reactive oxygen species (ROS) generation. Moreover, EGCG dose-dependently prevented Hcy-induced HUVECs cytotoxicity and apoptotic biochemical changes such as reducing mitochondrial membrane potential (MMP), decreasing Bcl-2/Bax protein ratio and activating caspase-9 and 3. In addition, EGCG enhanced the protein ratio of p-Akt/Akt, endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) formation in injured cells. In conclusion, the present study shows that EGCG prevents Hcy-induced HUVECs apoptosis via modulating mitochondrial apoptotic and PI3K/Akt/eNOS signaling pathways. Furthermore, the results indicate that EGCG is likely to represent a potential therapeutic strategy for atherosclerosis associated with Hyperhomocysteinemia (HHcy).

Asiatic Acid Inhibits Pro-Angiogenic Effects of VEGF and Human Gliomas in Endothelial Cell Culture Models

PubMed Central

Kavitha, Chandagirikoppal V.; Agarwal, Chapla; Agarwal, Rajesh; Deep, Gagan

2011-01-01

Malignant gliomas are one of the most devastating and incurable tumors. Sustained excessive angiogenesis by glioma cells is the major reason for their uncontrolled growth and resistance toward conventional therapies resulting in high mortality. Therefore, targeting angiogenesis should be a logical strategy to prevent or control glioma cell growth. Earlier studies have shown that Asiatic Acid (AsA), a pentacyclic triterpenoid, is effective against glioma and other cancer cells; however, its efficacy against angiogenesis remains unknown. In the present study, we examined the anti-angiogenic efficacy of AsA using human umbilical vein endothelial cells (HUVEC) and human brain microvascular endothelial cells (HBMEC). Our results showed that AsA (5–20 µM) inhibits HUVEC growth and induces apoptotic cell death by activating caspases (3 and 9) and modulating the expression of apoptosis regulators Bad, survivin and pAkt-ser473. Further, AsA showed a dose-dependent inhibition of HUVEC migration, invasion and capillary tube formation, and disintegrated preformed capillary network. AsA also inhibited the VEGF-stimulated growth and capillary tube formation by HUVEC and HBMEC. Next, we analyzed the angiogenic potential of conditioned media collected from human glioma LN18 and U87-MG cells treated with either DMSO (control conditioned media, CCM) or AsA 20 µM (AsA20 conditioned media, AsA20CM). CCM from glioma cells significantly enhanced the capillary tube formation in both HUVEC and HBMEC, while capillary tube formation in both endothelial cell lines was greatly compromised in the presence of AsA20CM. Consistent with these results, VEGF expression was lesser in AsA20CM compared to CCM, and indeed AsA strongly inhibited VEGF level (both cellular and secreted) in glioma cells. AsA also showed dose-dependent anti-angiogenic efficacy in Matrigel plug assay, and inhibited the glioma cells potential to attract HUVEC/HBMEC. Overall, the present study clearly showed the strong anti-angiogenic potential of AsA and suggests its usefulness against malignant gliomas. PMID:21826202

Effect of Calcium-Infiltrated Hydroxyapatite Scaffolds on the Hematopoietic Fate of Human Umbilical Vein Endothelial Cells.

PubMed

Zhang, Qinghao; Gerlach, Jörg C; Schmelzer, Eva; Nettleship, Ian

2017-01-01

Foamed hydroxyapatite offers a three-dimensional scaffold for the development of bone constructs, mimicking perfectly the in vivo bone structure. In vivo, calcium release at the surface is assumed to provide a locally increased gradient supporting the maintenance of the hematopoietic stem cells niche. We fabricated hydroxyapatite scaffolds with high surface calcium concentration by infiltration, and used human umbilical vein endothelial cells (HUVECs) as a model to study the effects on hematopoietic lineage direction. HUVECs are umbilical vein-derived and thus possess progenitor characteristics, with a prospective potential to give rise to hematopoietic lineages. HUVECs were cultured for long term on three-dimensional porous hydroxyapatite scaffolds, which were either infiltrated biphasic foams or untreated. Controls were cultured in two-dimensional dishes. The release of calcium into culture medium was determined, and cells were analyzed for typical hematopoietic and endothelial gene expressions, surface markers by flow cytometry, and hematopoietic potential using colony-forming unit assays. Our results indicate that the biphasic foams promoted a hematopoietic lineage direction of HUVECs, suggesting an improved in vivo-like scaffold for hematopoietic bone tissue engineering. © 2017 S. Karger AG, Basel.

Turkish propolis protects human endothelial cells in vitro from homocysteine-induced apoptosis.

PubMed

Darendelioglu, Ekrem; Aykutoglu, Gurkan; Tartik, Musa; Baydas, Giyasettin

2016-05-01

Chronic cardiovascular and neurodegenerative complications induced by hyperhomocysteinemia have been most relatively associated with endothelial cell injury. Elevated homocysteine (Hcy) generates reactive oxygen species (ROS) accompanying with oxidative stress which is hallmarks of the molecular mechanisms responsible for cardiovascular disease. Propolis is a natural product, obtained by honeybee from various oils, pollens, special resins and wax materials, conventionally used with the purpose of treatment by folks Propolis has various biological activities and powerful antioxidant capacity. The flavonoids and phenolic acids, most bioactive components of propolis, have superior antioxidant ability to defend cell from free radicals. This study was designed to examine the protective effects of Turkish propolis (from east of country) on Hcy induced ROS production and apoptosis in human vascular endothelial cells (HUVECs). According to results, co-treatment of HUVECs with propolis decreased Hcy-induced ROS overproduction and lipid peroxidation (LPO) levels. Furthermore, overproductions of Bax, caspase-9 and caspase-3 protein, elevation of cytochrome c release in Hcy-treated HUVECs were significantly reduced by propolis. It was concluded that propolis has cytoprotective ability against cytotoxic effects of high Hcy in HUVECs. Copyright © 2016 Elsevier GmbH. All rights reserved.

Expression of human olfactory receptor 10J5 in heart aorta, coronary artery, and endothelial cells and its functional role in angiogenesis.

PubMed

Kim, Sung-Hee; Yoon, Yeo Cho; Lee, Ae Sin; Kang, NaNa; Koo, JaeHyung; Rhyu, Mee-Ra; Park, Jae-Ho

2015-05-01

ORs are ectopically expressed in non-chemosensory tissues including muscle, kidney, and keratinocytes; however, their physiological roles are largely unknown. We found that human olfactory receptor 10J5 (OR10J5) is expressed in the human aorta, coronary artery, and umbilical vein endothelial cells (HUVEC). Lyral induces Ca(2+) and phosphorylation of AKT in HUVEC. A knockdown study showed the inhibition of the lyral-induced Ca(2+) and the phosphorylation AKT and implied that these processes are mediated by OR10J5. In addition, lyral enhanced migration of HUVEC, which were also inhibited by RNAi in a migration assay. In addition, matrigel plug assay showed that lyral enhanced angiogenesis in vivo. Together these data demonstrate the physiological role of OR10J5 in angiogenesis and represent roles of ORs in HUVEC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

MCPIP1-induced autophagy mediates ischemia/reperfusion injury in endothelial cells via HMGB1 and CaSR.

PubMed

Xie, Xiaolong; Zhu, Tiebing; Chen, Lulu; Ding, Shuang; Chu, Han; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-29

Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) plays a important role in ischemia/reperfusion (I/R) injury. Autophagy is involved in activating endothelial cells in response to I/R. However, researchers have not clearly determined whether MCPIP1 mediates I/R injury in endothelial cells via autophagy, and its downstream mechanism remains unclear. Western blotting analyses and immunocytochemistry were applied to detect protein levels were detected in HUVECs. An in vitro scratch assay was used to detect cell migration. Cells were transfected with siRNAs to knockdown MCPIP1 and high mobility group box 1 (HMGB1) expression. The pharmacological activator of autophagy rapamycin and the specific calcium-sensing receptor (CaSR) inhibitor NPS-2143 were used to confirm the roles of autophagy and CaSR in I/R injury. I/R induced HMGB1 and CaSR expression, which subsequently upreguated the migration and apoptosis of HUVECs and coincided with the increase of autophagy. HMGB1 was involved in cell migration, whereas CaSR specifically participated in I/R-induced HUVEC apoptosis. Based on these findings, I/R-induced MCPIP1 expression regulates the migration and apoptosis of HUVECs via HMGB1 and CaSR, respectively, suggesting a new therapeutic targetof I/R injury.

Epicatechin and catechin modulate endothelial activation induced by platelets of patients with peripheral artery disease.

PubMed

Carnevale, R; Loffredo, L; Nocella, C; Bartimoccia, S; Bucci, T; De Falco, E; Peruzzi, M; Chimenti, I; Biondi-Zoccai, G; Pignatelli, P; Violi, F; Frati, G

2014-01-01

Platelet activation contributes to the alteration of endothelial function, a critical initial step in atherogenesis through the production and release of prooxidant mediators. There is uncertainty about the precise role of polyphenols in interaction between platelets and endothelial cells (ECs). We aimed to investigate whether polyphenols are able to reduce endothelial activation induced by activated platelets. First, we compared platelet activation and flow-mediated dilation (FMD) in 10 healthy subjects (HS) and 10 patients with peripheral artery disease (PAD). Then, we evaluated the effect of epicatechin plus catechin on platelet-HUVEC interaction by measuring soluble cell adhesion molecules (CAMs), NOx production, and eNOS phosphorylation (p-eNOS) in HUVEC. Compared to HS, PAD patients had enhanced platelet activation. Conversely, PAD patients had lower FMD than HS. Supernatant of activated platelets from PAD patients induced an increase of sCAMs release and a decrease of p-eNOS and nitric oxide (NO) bioavailability compared to unstimulated HUVEC. Coincubation of HUVEC, with supernatant of PAD platelets patients, pretreated with a scalar dose of the polyphenols, resulted in a decrease of sCAMs release and in an increase of p-eNOS and NO bioavailability. This study demonstrates that epicatechin plus catechin reduces endothelial activation induced by activated platelets.

Plant proteolytic enzyme papain abrogates angiogenic activation of human umbilical vein endothelial cells (HUVEC) in vitro

PubMed Central

2013-01-01

Background Vascular endothelial growth factor (VEGF) is a key regulator of physiologic and pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. It is known that cysteine proteases from plants, like bromelain and papain are capable to suppress inflammatory activation. Recent studies have demonstrated that they may interfere with angiogenesis related pathways as well. The aim of this study was to investigate the anti-angiogenic effects of papain on human umbilical vein endothelial cells (HUVEC) in vitro. Methods Cell viability after prolonged treatment with papain was investigated by life cell staining and lactate dehydrogenase release assay. Angiogenic activation was assessed by ELISA against phosphorylated proteins AKT, MEK1/2, ERK1/2, SAPK/JNK and p38-MAPK. Growth inhibition was determined by means of an MTT-assay and cell migration by means of a scratch assay. Capability to form a capillary network was investigated using a tube formation assay. Results Papain did not induce proteolysis or cell detachment of HUVEC in a concentration range between 0 and 25 μg/mL. Four hours treatment with 10 μg/mL papain resulted in a reduced susceptibility of endothelial cells to activation by VEGF as determined by phosphorylation levels of Akt, MEK1/2, SAPK/JNK. Papain exerted a distinct inhibitory effect on cell growth, cell migration and tube formation with inhibition of tube formation detectable at concentrations as low as 1 μg/mL. Bromelain and ficin displayed similar effects with regard to cell growth and tube formation. Conclusion Papain showed a strong anti-angiogenic effect in VEGF activated HUVEC. This effect may be due to interference with AKT, MEK1/2 and SAPK/JNK phosphorylation. Two other plant derived cysteine proteases displayed similar inhibition of HUVEC cell growth and tube formation. These findings indicate that plant proteolytic enzymes may have potential as preventive and therapeutic agents against angiogenesis related human diseases. PMID:24053149

Alteration of pancreatic carcinoma and promyeloblastic cell adhesion in liver microvasculature by co-culture of hepatocytes, hepatic stellate cells and endothelial cells in a physiologically-relevant model.

PubMed

Danoy, Mathieu; Shinohara, Marie; Rizki-Safitri, Astia; Collard, Dominique; Senez, Vincent; Sakai, Yasuyuki

2017-04-18

In vitro models of the liver microvasculature, especially with respect to cancer cell extravasation, should include not only endothelial and cancer cells but also surrounding cells to mimic the physiological situation. To this end, in the present study, we established a physiologically-relevant hierarchical co-culture model by stacking layers of primary rat hepatocytes (Hep), hepatic stellate cells embedded in collagen gel (LX-2) and endothelial cells (HUVECs) on a specially designed oxygen-permeable polydimethylsiloxane PDMS bottom plate. The model was used to investigate the role and contribution of each of the three cell types in pancreatic cancer and promyeloblast cell adhesion. In particular, we showed an increase in albumin production by the primary hepatocytes and in the consumption of the produced vascular endothelial growth factors (VEGFs). Furthermore, in co-culture, the HUVECs exhibited a mature vascular endothelial and non-inflamed phenotype, as evidenced by Stabilin-1, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), intercellular adhesion molecule (ICAM-1), and vascular adhesion protein-1 (VAP-1) expression. The HUVECs were also successfully activated with an inflammatory cytokine and their ICAM-1 response was found to be higher in monoculture compared to co-culture. Additionally, the adhesion of MiaPaCa-2 pancreatic cancer cells and HL60 promyeloblasts was tested in both cases (i.e.: activation or not by an inflammatory cytokine). It has been found that their adhesion was always reduced in the co-culture model. These results highlight the importance of integrating hepatic stellate cells in the design of biomimetic models of the hepatic endothelial barrier.

Coenzyme Q10 Protects Human Endothelial Cells from β-Amyloid Uptake and Oxidative Stress-Induced Injury

PubMed Central

Durán-Prado, Mario; Frontiñán, Javier; Santiago-Mora, Raquel; Peinado, Juan Ramón; Parrado-Fernández, Cristina; Gómez-Almagro, María Victoria; Moreno, María; López-Domínguez, José Alberto; Villalba, José Manuel; Alcaín, Francisco J.

2014-01-01

Neuropathological symptoms of Alzheimer's disease appear in advances stages, once neuronal damage arises. Nevertheless, recent studies demonstrate that in early asymptomatic stages, ß-amyloid peptide damages the cerebral microvasculature through mechanisms that involve an increase in reactive oxygen species and calcium, which induces necrosis and apoptosis of endothelial cells, leading to cerebrovascular dysfunction. The goal of our work is to study the potential preventive effect of the lipophilic antioxidant coenzyme Q (CoQ) against ß-amyloid-induced damage on human endothelial cells. We analyzed the protective effect of CoQ against Aβ-induced injury in human umbilical vein endothelial cells (HUVECs) using fluorescence and confocal microscopy, biochemical techniques and RMN-based metabolomics. Our results show that CoQ pretreatment of HUVECs delayed Aβ incorporation into the plasma membrane and mitochondria. Moreover, CoQ reduced the influx of extracellular Ca2+, and Ca2+ release from mitochondria due to opening the mitochondrial transition pore after β-amyloid administration, in addition to decreasing O2 .− and H2O2 levels. Pretreatment with CoQ also prevented ß-amyloid-induced HUVECs necrosis and apoptosis, restored their ability to proliferate, migrate and form tube-like structures in vitro, which is mirrored by a restoration of the cell metabolic profile to control levels. CoQ protected endothelial cells from Aβ-induced injury at physiological concentrations in human plasma after oral CoQ supplementation and thus could be a promising molecule to protect endothelial cells against amyloid angiopathy. PMID:25272163

Interaction of hydroxyapatite nanoparticles with endothelial cells: internalization and inhibition of angiogenesis in vitro through the PI3K/Akt pathway

PubMed Central

Shi, Xingxing; Zhou, Kai; Huang, Fei; Wang, Chen

2017-01-01

Nano-hydroxyapatite (nano-HAP) has been proposed as a better candidate for bone tissue engineering; however, the interactions of nano-HAP with endothelial cells are currently unclear. In this study, HAP nanoparticles (HANPs; 20 nm np20 and 80 nm np80) and micro-sized HAP particles (m-HAP; 12 μm) were employed to explore and characterize cellular internalization, subcellular distribution, effects of HANPs on endothelial cell function and underlying mechanisms using human umbilical vein endothelial cells (HUVECs) as an in vitro model. It was found that HANPs were able to accumulate in the cytoplasm, and both adhesion and uptake of the HANPs followed a function of time; compared to np80, more np20 had been uptaken at the end of the observation period. HANPs were mainly uptaken via clathrin- and caveolin-mediated endocytosis, while macropinocytosis was the main pathway for m-HAP uptake. Unexpectedly, exposure to HANPs suppressed the angiogenic ability of HUVECs in terms of cell viability, cell cycle, apoptosis response, migration and capillary-like tube formation. Strikingly, HANPs reduced the synthesis of nitric oxide (NO) in HUVECs, which was associated with the inhibition of phosphatidylinositol 3-kinase (PI3K) and phosphorylation of eNOS. These findings provide additional insights into specific biological responses as HANPs interface with endothelial cells. PMID:28848353

Streptococcus pyogenes Phospholipase A2 Induces the Expression of Adhesion Molecules on Human Umbilical Vein Endothelial Cells and Aorta of Mice.

PubMed

Oda, Masataka; Domon, Hisanori; Kurosawa, Mie; Isono, Toshihito; Maekawa, Tomoki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

2017-01-01

The Streptococcus pyogenes phospholipase A 2 (SlaA) gene is highly conserved in the M3 serotype of group A S. pyogenes , which often involves hypervirulent clones. However, the role of SlaA in S. pyogenes pathogenesis is unclear. Herein, we report that SlaA induces the expression of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) via the arachidonic acid signaling cascade. Notably, recombinant SlaA induced ICAM1 and VCAM1 expression in human umbilical vein endothelial cells (HUVECs), resulting in enhanced adhesion of human monocytic leukemia (THP-1) cells. However, C134A, a variant enzyme with no enzymatic activity, did not induce such events. In addition, culture supernatants from S. pyogenes SSI-1 enhanced the adhesion of THP-1 cells to HUVECs, but culture supernatants from the Δ slaA isogenic mutant strain had limited effects. Aspirin, a cyclooxygenase 2 inhibitor, prevented the adhesion of THP-1 cells to HUVECs and did not induce ICAM1 and VCAM1 expression in HUVECs treated with SlaA. However, zileuton, a 5-lipoxygenase inhibitor, did not exhibit such effects. Furthermore, pre-administration of aspirin in mice intravenously injected with SlaA attenuated the transcriptional abundance of ICAM1 and VCAM1 in the aorta. These results suggested that SlaA from S. pyogenes stimulates the expression of adhesion molecules in vascular endothelial cells. Thus, SlaA contributes to the inflammation of vascular endothelial cells upon S. pyogenes infection.

Melatonin prevents human pancreatic carcinoma cell PANC-1-induced human umbilical vein endothelial cell proliferation and migration by inhibiting vascular endothelial growth factor expression.

PubMed

Cui, Peilin; Yu, Minghua; Peng, Xingchun; Dong, Lv; Yang, Zhaoxu

2012-03-01

Melatonin is an important natural oncostatic agent, and our previous studies have found its inhibitory action on tumor angiogenesis, but the mechanism remains unclear. It is well known that vascular endothelial growth factor (VEGF) plays key roles in tumor angiogenesis and has become an important target for antitumor therapy. Pancreatic cancer is a representative of the most highly vascularized and angiogenic solid tumors, which responds poorly to chemotherapy and radiation. Thus, seeking new treatment strategies targeting which have anti-angiogenic capability is urgent in clinical practice. In this study, a co-culture system between human umbilical vein endothelial cells (HUVECs) and pancreatic carcinoma cells (PANC-1) was used to investigate the direct effect of melatonin on the tumor angiogenesis and its possible action on VEGF expression. We found HUVECs exhibited an increased cell proliferation and cell migration when co-cultured with PANC-1 cells, but the process was prevented when melatonin added to the incubation medium. Melatonin at concentrations of 1 μm and 1 mm inhibited the cell proliferation and migration of HUVECs and also decreased both the VEGF protein secreted to the cultured medium and the protein produced by the PANC-1 cells. In addition, the VEGF mRNA expression was also down-regulated by melatonin. Taken together, our present study shows that melatonin at pharmacological concentrations inhibited the elevated cell proliferation and cell migration of HUVECs stimulated by co-culturing them with PANC-1 cells; this was associated with a suppression of VEGF expression in PANC-1 cells. © 2011 John Wiley & Sons A/S.

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

PubMed Central

Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

2013-01-01

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value. PMID:23613998

Melanoma upregulates ICAM-1 expression on endothelial cells through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway

PubMed Central

Zhang, Pu; Goodrich, Chris; Fu, Changliang; Dong, Cheng

2014-01-01

Cancer metastasis involves multistep adhesive interactions between tumor cells (TCs) and endothelial cells (ECs), but the molecular mechanisms of intercellular communication in the tumor microenvironment remain elusive. Using static and flow coculture systems in conjunction with flow cytometry, we discovered that certain receptors on the ECs are upregulated on melanoma cell adhesion. Direct contact but not separate coculture between human umbilical endothelial cells (HUVECs) and a human melanoma cell line (Lu1205) increased intercellular adhesion molecule 1 (ICAM-1) and E-selectin expression on HUVECs by 3- and 1.5-fold, respectively, compared with HUVECs alone. The nonmetastatic cell line WM35 failed to promote ICAM-1 expression changes in HUVECs on contact. Enzyme-linked immunosorbent assay (ELISA) revealed that EC–TC contact has a synergistic effect on the expression of the cytokines interleukin (IL)-8, IL-6, and growth-related oncogene α (Gro-α). By using E-selectin cross-linking and beads coated with CD44 immunopurified from Lu1205 cells, we showed that CD44/selectin ligation was responsible for the ICAM-1 up-regulation on HUVECs. Protein kinase Cα (PKC-α) activation was found to be the downstream target of the CD44/selectin-initiated signaling, as ICAM-1 elevation was inhibited by siRNA targeting PKCα or a dominant negative form of PKCα (PKCα DN). Western blot analysis and electrophoretic mobility shift assays (EMSAs) showed that TC–EC contact mediated p38 phosphorylation and binding of the transcription factor SP-1 to its regulation site. In conclusion, CD44/selectin binding signals ICAM-1 up-regulation on the EC surface through a PKCα–p38–SP-1 pathway, which further enhances melanoma cell adhesion to ECs during metastasis.—Zhang, P., Goodrich, C., Fu, C., Dong, C. Melanoma upregulates ICAM-1 expression on ECs through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38–SP-1 pathway. PMID:25138157

Effects of phytoestrogens derived from soy bean on expression of adhesion molecules on HUVEC.

PubMed

Andrade, C M de; Sá, M F Silva de; Toloi, M R Torqueti

2012-04-01

The risks of hormone replacement therapy have led to a search for new alternatives such as phytoestrogens, plant compounds with estrogen-like biological activity. Isoflavones are the phytoestrogens most extensively studied and can be found in soybean, red clover and other plants. Due to this estrogen-like activity, phytoestrogens can have some effect on atherosclerosis. Human umbilical vein endothelial cells (HUVEC) have been extensively used to study the biology and pathobiology of human endothelial cells and most of the knowledge acquired is due to experiments with cultures of these cells. To evaluate the effects of the phytoestrogen extracts from Glycine max soy bean, genistein, formononetin, biochanin A and daidzein, as well as a mixture of these extracts (Mix), on expression of adhesion molecules, VCAM-1, ICAM-1 and E-selectin, by endothelial cell HUVEC, stimulated with lipopolysaccharide. HUVEC were cultured in medium EBM(2), pretreated with isoflavones for 24 and 48 h and then stimulated with lipopolysaccharide; in addition, isoflavones were added, after stimulation by lipopolysaccharide, to HUVEC. We evaluated the production of VCAM-1, ICAM-1 and E-selectin on cell surface, by cell-based enzyme immunoassay, and of sVCAM-1, sICAM-1 and sE-selectin in culture supernatant, by ELISA. Genistein, formononetin, biochanin A and daidzein, as well as the Mix were able to reduce VCAM-1, ICAM-1 and E-selectin on cell surface and in culture supernatant. Conclusion Isoflavones extracted from Glycine max soy bean, in vitro, presented antiatherogenic effects, reducing the expression of adhesion molecules and acting as preventive agents as well as therapeutic agents.

Effect of low level laser therapy and high intensity laser therapy on endothelial cell proliferation in vitro: preliminary communication

NASA Astrophysics Data System (ADS)

Lukowicz, Malgorzata; Szymanska, Justyna; Goralczyk, Krzysztof; Zajac, Andrzej; Rość, Danuta

2013-01-01

Background: The main purpose of this study was to analyze the influence of power intensity and wavelength of Low Level Laser Therapy (LLLT) and HILT (High Intensity Laser Therapy) on endothelial cell proliferation. Material and methods: The tests were done on human umbilical vein endothelial cells (HUVEC). Cultures were exposed to laser irradiation of 660 nm and 670 nm at different dosages, power output was 10 - 40 mW as well as 820 nm with power 100 mW and 808 nm with power 1500 mW. Energy density was from 0.28 to 11,43 J/cm2. Cell proliferation of a control and tested culture was evaluated with a colorimetric device to detect live cells. The tests were repeated 8 times. Results: We observed good effects of LLLT on live isolated ECs and no effects in experiments on previous deep-frozen cultures. Also HILT stimulated the proliferation of HUVEC. Conclusion: Endothelial cells play a key role in vascular homeostasis in humans. We observed the stimulatory effect of LLLT and HILT on proliferation of HUVEC. Many factors influence the proliferation of EC, so is it necessary to continue the experiment with different doses, intensity and cell concentration.

Ganoderma atrum polysaccharide ameliorates anoxia/reoxygenation-mediated oxidative stress and apoptosis in human umbilical vein endothelial cells.

PubMed

Zhang, Yan-Song; Li, Wen-Juan; Zhang, Xian-Yi; Yan, Yu-Xin; Nie, Shao-Ping; Gong, De-Ming; Tang, Xiao-Fang; He, Ming; Xie, Ming-Yong

2017-05-01

Ganoderma atrum polysaccharide (PSG-1), a main polysaccharide from Ganoderma atrum, possesses potent antioxidant capacity and cardiovascular benefits. The aim of this study was to investigate the role of PSG-1 in oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs) under anoxia/reoxygenation (A/R) injury conditions. The results showed that exposure of HUVECs to A/R triggered cell death and apoptosis. Administration of PSG-1 significantly inhibited A/R-induced cell death and apoptosis in HUVECs. PSG-1-reduced A/R injury was mediated via mitochondrial apoptotic pathway, as evidenced by elevation of mitochondrial Bcl-2 protein and mitochondrial membrane potential, and attenuation of Bax translocation, cytochrome c release and caspases activation. Furthermore, PSG-1 enhanced the activities of superoxide dismutase, catalase and glutathione peroxidase and glutathione content, and concomitantly attenuated reactive oxygen species generation, lipid peroxidation and glutathione disulfide content. The antioxidant, N-acetyl-l-cysteine, significantly ameliorated all of these endothelial injuries caused by A/R, suggesting that antioxidant activities might play a key role in PSG-1-induced endothelial protection. Taken together, these findings suggested that PSG-1 could be as a promising adjuvant against endothelial dysfunction through ameliorating oxidative stress and apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Bisphenol A Disrupts Transcription and Decreases Viability in Aging Vascular Endothelial Cells

PubMed Central

Ribeiro-Varandas, Edna; Pereira, H. Sofia; Monteiro, Sara; Neves, Elsa; Brito, Luísa; Boavida Ferreira, Ricardo; Viegas, Wanda; Delgado, Margarida

2014-01-01

Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis. PMID:25207595

Chitosan microsphere scaffold tethered with RGD-conjugated poly(methacrylic acid) brushes as effective carriers for the endothelial cells.

PubMed

Yang, Zhenyi; Yuan, Shaojun; Liang, Bin; Liu, Yang; Choong, Cleo; Pehkonen, Simo O

2014-09-01

Endothelial cell-matrix interactions play a vital role in promoting vascularization of engineered tissues. The current study reports a facile and controllable method to develop a RGD peptide-functionalized chitosan microsphere scaffolds for rapid cell expansion of human umbilical vein endothelial cells (HUVECs). Functional poly(methacrylic acid) (PMAA) brushes are grafted from the chitosan microsphere surfaces via surface-initiated ATRP. Subsequent conjugation of RGD peptides on the pendent carboxyl groups of PMAA side chain is accomplished by carbodiimide chemistry to facilitate biocompatibility of the 3D CS scaffolding system. In vitro cell-loading assay of HUVECs exhibits a significant improvment of cell adhesion, spreading, and proliferation on the RGD peptide-immobilized CS microsphere surfaces. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Influence de revetements bioactifs sur les cellules endotheliales: Vers des protheses vasculaires non thrombotiques

NASA Astrophysics Data System (ADS)

Fadlallah, Hicham

Developing vascular prostheses of small diameter to replace vessels closed by atherosclerosis remains a challenge because of the risk of thrombosis. Seeding of endothelial cells, which have antithrombogenic properties, is a promising solution, but we must create surfaces which promote their adhesion and retention to resist shear stresses created by the blood flow on the surface. This master's project aimed at investigating the effect of a plasma polymerized coating rich in primary amines (called LP), with or without elements of the extracellular matrix (Fibronectin (FN) or chondroitin sulphate (CS)) on the endothelial cells and hemocompatibility of polyethylene terephthalate (PET). The adhesion, growth and retention of human umbilical vein endothelial cells (HUVECs) on PET and LP, in the presence and absence of FN or CS, have been studied. In addition, platelet adhesion on different surfaces was evaluated by a perfusion test with whole blood, platelets being previously labeled with rhodamine. Finally a double fluorescent labeling (using Cellview Maroon to mark the HUVECs and CD61 antibody for platelets) was developed to study the retention of endothelial cells, under blood flow and verify their non thrombogenic character. The results obtained show that both the LP coating and the adsorbed FN, strongly increase both the cellular adhesion and growth on PET; however they have no additional effect when the two are combined. They also augment cellular retention to surface, but this remains incomplete. Moreover we observed that the plasma coating (LP) greatly increases the thrombogenicity of the surface, with strong platelet adhesion and activation. This thrombogenicity is extremely reduced when endothelial cells cover the surface, but cell loss under the effect of shear produced by the perfusion creates significant areas of platelet adhesion. The grafting of CS on LP also permits good HUVECs adhesion, growth and retention under shear stress on HUVEC, with no difference from the LP alone. In addition, the CS sharply decreases the platelet adhesion, which is found below the value observed for PET. The double cell labeling also showed that cells adhered on LP+CS has an anti-thrombotic phenotype and can resist blood flow. These studies suggest that a coating of CS is a promising strategy for vascular prosthesis given the combination of the good adhesion of endothelial cells and the low thrombogenicity of the underlying surface. Keywords: vascular prostheses, polymers, bioactive coating, thrombogenicity, HUVECs.

Mechanical property quantification of endothelial cells using scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Shelke, A.; Brand, S.; Kundu, T.; Bereiter-Hahn, J.; Blase, C.

2012-04-01

The mechanical properties of cells reflect dynamic changes of cellular organization which occur during physiologic activities like cell movement, cell volume regulation or cell division. Thus the study of cell mechanical properties can yield important information for understanding these physiologic activities. Endothelial cells form the thin inner lining of blood vessels in the cardiovascular system and are thus exposed to shear stress as well as tensile stress caused by the pulsatile blood flow. Endothelial dysfunction might occur due to reduced resistance to mechanical stress and is an initial step in the development of cardiovascular disease like, e.g., atherosclerosis. Therefore we investigated the mechanical properties of primary human endothelial cells (HUVEC) of different age using scanning acoustic microscopy at 1.2 GHz. The HUVECs are classified as young (tD < 90 h) and old (tD > 90 h) cells depending upon the generation time for the population doubling of the culture (tD). Longitudinal sound velocity and geometrical properties of cells (thickness) were determined using the material signature curve V(z) method for variable culture condition along spatial coordinates. The plane wave technique with normal incidence is assumed to solve two-dimensional wave equation. The size of the cells is modeled using multilayered (solid-fluid) system. The propagation of transversal wave and surface acoustic wave are neglected in soft matter analysis. The biomechanical properties of HUVEC cells are quantified in an age dependent manner.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation.

PubMed

Lekowski, R; Collard, C D; Reenstra, W R; Stahl, G L

2001-02-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

PubMed Central

Lekowski, Robert; Collard, Charles D.; Reenstra, Wende R.; Stahl, Gregory L.

2001-01-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O2, 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 ± 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (≤ 100 μmol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC50 = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC50 ≈ 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress. PMID:11266613

Aqueous extracts of Tribulus terrestris protects against oxidized low-density lipoprotein-induced endothelial dysfunction.

PubMed

Jiang, Yue-hua; Yang, Chuan-hua; Li, Wei; Wu, Sai; Meng, Xian-qing; Li, Dong-na

2016-03-01

To investigate the role of aqueous extracts of Tribulus terrestris (TT) against oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) dysfunction in vitro. HUVECs were pre-incubated for 60 min with TT (30 and 3 μg/mL respectively) or 10(-5) mol/L valsartan (as positive controls) and then the injured endothelium model was established by applying 100 μg/mL ox-LDL for 24 h. Cell viability of HUVECs was observed by real-time cell electronic sensing assay and apoptosis rate by Annexin V/PI staining. The cell migration assay was performed with a transwell insert system. Cytoskeleton remodeling was observed by immunofluorescence assay. The content of endothelial nitric oxide synthase (eNOS) was measured by enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) generation was assessed by immunofluorescence and flow cytometer. Key genes associated with the metabolism of ox-LDL were chosen for quantitative real-time polymerase chain reaction to explore the possible mechanism of TT against oxidized LDL-induced endothelial dysfunction. TT suppressed ox-LDL-induced HUVEC proliferation and apoptosis rates significantly (41.1% and 43.5% after treatment for 3 and 38 h, respectively; P<0.05). It also prolonged the HUVEC survival time and postponed the cell's decaying stage (from the 69th h to over 100 h). According to the immunofluorescence and transwell insert system assay, TT improved the endothelial cytoskeletal network, and vinculin expression and increased cell migration. Additionally, TT regulated of the synthesis of endothelial nitric oxide synthase and generation of intracellular reactive oxygen species (P<0.05). Both 30 and 3 μg/mL TT demonstrated similar efficacy to valsartan. TT normalized the increased mRNA expression of PI3Kα and Socs3. It also decreased mRNA expression of Akt1, AMPKα1, JAK2, LepR and STAT3 induced by ox-LDL. The most notable changes were JAK2, LepR, PI3Kα, Socs3 and STAT3. TT demonstrated potential lowering lipid benefits, anti-hypertension and endothelial protective effects. It also suggested that the JAK2/STAT3 and/or PI3K/AKT pathway might be a very important pathway which was involved in the pharmacological mechanism of TT as the vascular protective agent.

Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells

PubMed Central

Yi, Wei; Yang, Yang; Zhao, Dajun; Yang, Honggang; Geng, Ting; Xing, Jianzhou; Zhang, Yu; Tan, Songtao; Yi, Dinghua

2014-01-01

Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine (sICAM1, sE-selectin, MCP-1 and IL-8) production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK). EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin (THA, an ERS inducer) attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS. PMID:25470819

Efficacy for lung metastasis induced by the allogeneic bEnd3 vaccine in mice.

PubMed

Zhao, Jun; Lu, Jing; Zhou, Lurong; Zhao, Jimin; Dong, Ziming

2018-05-04

The mouse brain microvascular endothelial cell line bEnd.3 was used to develop a vaccine and its anti-tumor effect on lung metastases was observed in immunized mice. Mouse bEnd.3 cells cultured in-vitro and then fixed with glutaraldehyde was used to immunize mice; mice were challenged with the metastatic cancer cell line U14, and changes in metastatic cancer tissues were observed through hematoxylin and eosin staining. Carboxyfluorescein succinimidyl amino ester (CSFE) and propidium iodide (PI) were used to detect cytotoxic activity of spleen T lymphocytes; the ratio of CD3 + and CD8 + T-cell sub-sets was determined by flow cytometry. Enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and immunoblot were used to examine the specific response of the antisera of immunized mice. The number of metastatic nodules in bEnd.3 and human umbilical vein endothelial cell (HUVEC) vaccine groups was less than NIH3T3 vaccine group and phosphate buffered saline (PBS) control group. The bEnd.3-induced and HUVEC-induced cytotoxic T-lymphocytes (CTLs) showed significant lytic activity against bEnd.3 and HUVEC target cells, while the antisera of mice in bEnd.3 and HUVEC vaccine groups showed specific immune responses to membrane proteins and inhibited target cell proliferation in-vitro. Immunoblot results showed specific bands at 180KD and 220KD in bEnd.3 and at 130 kD and 220 kD in HUVEC lysates. Allogeneic bEnd.3 vaccine induced an active and specific immune response to tumor vascular endothelial cells that resulted in production of antibodies against the proliferation antigens VEGF-R II, integrin, Endog etc. Immunization with this vaccine inhibited lung metastasis of cervical cancer U14 cells and prolonged the survival of these mice.

Angiotensin II type 1 receptor blockers prevent tumor necrosis factor-alpha-mediated endothelial nitric oxide synthase reduction and superoxide production in human umbilical vein endothelial cells.

PubMed

Kataoka, Hiroki; Murakami, Ryuichiro; Numaguchi, Yasushi; Okumura, Kenji; Murohara, Toyoaki

2010-06-25

Decrease in endothelial nitric oxide synthase (eNOS) expression is one of the adverse outcomes of endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha) is known to decrease eNOS expression and is an important mediator of endothelial dysfunction. We hypothesized that an angiotensin II type 1 (AT1) receptor blocker would improve endothelial function via not only inhibition of the angiotensin II signaling but also inhibition of the TNF-alpha-mediated signaling. Therefore we investigated whether an AT1 receptor blocker would restore the TNF-alpha-induced decrease in eNOS expression in cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with an antioxidant (superoxide dismutase, alpha-tocopherol) or AT1 receptor blockers (olmesartan or candesartan) restored the TNF-alpha-dependent reduction of eNOS. The AT1 receptor blocker decreased the TNF-alpha-dependent increase of 8-isoprostane. The superoxide dismutase activities in HUVEC were stable during AT1 receptor blocker treatment, and the AT1 receptor blocker did not scavenge superoxide directly. The AT1 receptor blocker also decreased TNF-alpha-induced phosphorylation of I kappaB alpha and cell death. These results suggest that AT1 receptor blockers are able to ameliorate TNF-alpha-dependent eNOS reduction or cell injury by inhibiting superoxide production or nuclear factor-kappaB activation. (c) 2010 Elsevier B.V. All rights reserved.

Formononetin accelerates wound repair by the regulation of early growth response factor-1 transcription factor through the phosphorylation of the ERK and p38 MAPK pathways.

PubMed

Huh, Jeong-Eun; Nam, Dong-Woo; Baek, Young-Hyun; Kang, Jung Won; Park, Dong-Suk; Choi, Do-Young; Lee, Jae-Dong

2011-01-01

Formononetin, a phytoestrogen from the root of Astragalus membranaceus, is used as a blood enhancer and to improve blood microcirculation in complementary and alternative medicine. The present study investigated the influence of formononetin on the expression of early growth response factor-1 (Egr-1) and growth factors contributing to wound healing. Formononetin significantly increased growth factors such as transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) in human umbilical vein endothelial cells (HUVECs). Formononetin also increased the expression of Egr-1 transcription factor by 3.2- and 10.5-fold, compared with recombinant VEGF(125) in HUVECs. The formononetin-mediated 12%-43% increase induced endothelial cell proliferation and recovered the migration of wounded HUVECs. In an ex vivo angiogenesis assay, formononetin produced a larger capillary sprouting area than produced using recombinant VEGF(125). Cell proliferation and migration of HUVECs were also greater in the presence of formonectin than VEGF(125). Western blot analysis of scratch-wounded confluent HUVECs showed that formononetin induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slightly inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The formononetin-mediated sustained activation of Egr-1 was suppressed by the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PD98059 inhibited the formononetin-induced endothelial proliferation and repair in scratch-wounded HUVECs, SB203580 increased the cell proliferation and wound healing. Formononetin accelerate wound closure rate as early as day 3 after surgery and consistently observed until day 10 after in wound animal model. These data suggest that formononetin promotes endothelial repair and wound healing in a process involving the over-expression of Egr-1 transcription factor through the regulation of the ERK1/2 and p38 MAPK pathways. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Human brain microvascular endothelial cells resist elongation due to shear stress.

PubMed

Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

2015-05-01

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

Therapeutic potential of ixmyelocel-T, an expanded autologous multicellular therapy for treatment of ischemic cardiovascular diseases.

PubMed

Ledford, Kelly J; Murphy, Nikki; Zeigler, Frank; Bartel, Ronnda L; Tubo, Ross

2015-03-13

Bone marrow derived cellular therapies are an emerging approach to promoting therapeutic angiogenesis in ischemic cardiovascular disease. However, the percentage of regenerative cells in bone marrow mononuclear cells (BMMNCs) is small, and large amounts of BMMNCs are required. Ixmyelocel-T, an expanded autologous multicellular therapy, is manufactured from a small sample of bone marrow aspirate. Ixmyelocel-T contains expanded populations of mesenchymal stromal cells (MSCs) and M2-like macrophages, as well as many of the CD45+ cells found in the bone marrow. It is hypothesized that this expanded multi-cellular therapy would induce angiogenesis and endothelial repair. A rat model of hind limb ischemia was used to determine the effects of ixmyelocel-T on blood flow recovery. To further determine the effects on endothelial cells, ixmyelocel-T was co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell® inserts. Co-culture of HUVECs with ixmyelocel-T resulted secretion of a variety of pro-angiogenic factors. HUVECs stimulated by ixmyelocel-T exhibited enhanced migration, proliferation, and branch formation. Ixmyelocel-T co-culture also resulted in increased endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. In tumor necrosis factor alpha (TNFα)-stimulated HUVECs, ixmyelocel-T co-culture decreased apoptosis and reactive oxygen species generation, increased super oxide dismutase activity, and decreased nuclear factor kappa B (NFκB) activation. Treatment with ixmyelocel-T in a rat model of hind limb ischemia resulted in significantly increased blood flow perfusion and capillary density, gene expression and plasma levels of the anti-inflammatory cytokine interleukin (IL)-10, plasma nitrates, plasma platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF) expression, and significantly decreased plasma thiobarbituric acid reactive substances (TBARS). This work demonstrates that ixmyelocel-T interacts with endothelial cells in a paracrine manner, resulting in angiogenesis and endothelial protection. This data suggests that ixmyelocel-T could be useful for promoting of angiogenesis and tissue repair in ischemic cardiovascular diseases. In conclusion, ixmyelocel-T therapy may provide a new aspect of therapeutic angiogenesis in this patient population where expanded populations of regenerative cells might be required.

PTEN inhibition enhances angiogenesis in an in vitro model of ischemic injury by promoting Akt phosphorylation and subsequent hypoxia inducible factor-1α upregulation.

PubMed

Xue, Lixia; Huang, Jiankang; Zhang, Ting; Wang, Xiuzhe; Fu, Jianliang; Geng, Zhi; Zhao, Yuwu; Chen, Hao

2018-06-24

Angiogenesis is an important pathophysiological response to cerebral ischemia. PTEN is a lipid phosphatase whose loss activates PI3K/Akt signaling, which is related to HIF-1α upregulation and enhanced angiogenesis in human cancer cells. However, the specific roles of PTEN in endothelial cell functions and angiogenesis after cerebral ischemia remain unknown. Therefore, we sought to examine the potential effects of PTEN inhibition on post-ischemic angiogenesis in human blood vessel cells and to determine the underlying mechanism. In this present study, human umbilical vein endothelial cells (HUVECs) were exposed to oxygen-glucose deprivation (OGD), cell proliferation, migration and apoptosis, in vitro tube formation and expression of PTEN/Akt pathway and angiogenic factors were examined in HUVECs after treatment with PTEN inhibitor bisperoxovanadium (bpV) at different doses. The results showed that bpV significantly increased the cell proliferation and reduced cell apoptosis indicating that the drug exerts a cytoprotective effect on HUVECs with OGD exposure. bpV also enhanced cell migration and tube formation in HUVECs following OGD, and upregulated HIF-1α and VEGF expressions, but attenuated endostatin expression. Additionally, western blotting analysis demonstrated that Akt phosphorylation in HUVECs was significantly increased after bpV treatment. These findings suggest that PTEN inhibition promotes post-ischemic angiogenesis in HUVECs after exposure to OGD and this enhancing effect might be achieved through activation of the Akt signal cascade.

Effect of anti-sense oligodeoxynucleotides homeobox B2 on the proliferation and expression of primary human umbilical vein endothelial cells.

PubMed

Liu, Xusheng; Zhang, Xiaoqi

2002-02-01

To explore the effect of homeobox B2 (HOXB2) anti sense oligodeoxynucleotides (asodn) on the proliferation and expression of primary human umbilical vein endothelial cells (HUVECs). Various concentrations of HOXB2 asodn modified by thiophosphate transfected the induction of liposome into HUVECs. MTT a nd RT-PCR methods were employed to determine the effect of different conc ent rations of asodn on the endothelial proliferation and the expression level of HOXB2 mRNA. After the transfection of HOXB2 asodn, the endothelial proliferation was inhibited in a dose-dependent fashion. Simultaneously, the expression of HOXB2 mRNA decreased significantly. HOXB2 plays an important role in the proliferation of endothelia.

Diesel Exhaust Particulate Extracts Inhibit Transcription of Nuclear Respiratory Factor-1 and Cell Viability in Human Umbilical Vein Endothelial Cells

PubMed Central

Mattingly, Kathleen A.; Klinge, Carolyn M.

2011-01-01

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1 regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription and this suppression was not ablated by concomitant treatment with E2, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E2 increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. PMID:22105178

Racial differences in tumor necrosis factor-α-induced endothelial microparticles and interleukin-6 production

PubMed Central

Brown, Michael D; Feairheller, Deborah L; Thakkar, Sunny; Veerabhadrappa, Praveen; Park, Joon-Young

2011-01-01

African Americans (AA) tend to have heightened systemic inflammation and endothelial dysfunction. Endothelial microparticles (EMP) are released from activated/apoptotic endothelial cells (EC) when stimulated by inflammation. The purpose of our study was to assess EMP responses to inflammatory cytokine (TNF-α) and antioxidant (superoxide dismutase, SOD) conditions in human umbilical vein ECs (HUVECs) obtained from AA and Caucasians. EMPs were measured under four conditions: control (basal), TNF-α, SOD, and TNF-α + SOD. Culture supernatant was collected for EMP analysis by flow cytometry and IL-6 assay by ELISA. IL-6 protein expression was assessed by Western blot. AA HUVECs had greater EMP levels under the TNF-α condition compared to the Caucasian HUVECs (6.8 ± 1.1 vs 4.7% ± 0.4%, P = 0.04). The EMP level increased by 89% from basal levels in the AA HUVECs under the TNF-α condition (P = 0.01) compared to an 8% increase in the Caucasian HUVECs (P = 0.70). Compared to the EMP level under the TNF-α condition, the EMP level in the AA HUVECs was lower under the SOD only condition (2.9% ± 0.3%, P = 0.005) and under the TNF-α + SOD condition (2.1% ± 0.4%, P = 0.001). Basal IL-6 concentrations were 56.1 ± 8.8 pg/mL/μg in the AA and 30.9 ± 14.9 pg/mL/μg in the Caucasian HUVECs (P = 0.17), while basal IL-6 protein expression was significantly greater (P < 0.05) in the AA HUVECs. These preliminary observational results suggest that AA HUVECs may be more susceptible to the injurious effects of the proinflammatory cytokine, TNF-α. PMID:21966220

Diesel Exhaust Particle Exposure Causes Redistribution of Endothelial Tube VE-Cadherin

PubMed Central

Chao, Ming-Wei; Kozlosky, John; Po, Iris P.; Strickland, Pamela Ohman; Svoboda, Kathy K. H.; Cooper, Keith; Laumbach, Robert; Gordon, Marion K.

2010-01-01

Whether diesel exhaust particles (DEPs) potentially have a direct effect on capillary endothelia was examined by following the adherens junction component, vascular endothelial cell cadherin (VE-cadherin). This molecule is incorporated into endothelial adherens junctions at the cell surface, where it forms homodimeric associations with adjacent cells and contributes to the barrier function of the vasculature (Dejana et al., 2008; Venkiteswaran et al., 2002; Villasante et al., 2007). Human umbilical vein endothelial cells (HUVECs) that were pre-formed into capillary-like tube networks in vitro were exposed to DEPs for 24 hr. After exposure, the integrity of VE-cadherin in adherens junctions was assessed by immunofluorescence analysis, and demonstrated that increasing concentrations of DEPs caused increasing redistribution of VE-cadherin away from the cell-cell junctions toward intracellular locations. Since HUVEC tube networks are three-dimensional structures, whether particles entered the endothelial cells or tubular lumens was also examined. The data indicate that translocation of the particles does occur. The results, obtained in a setting that removes the confounding effects of inflammatory cells or blood components, suggest that if DEPs encounter alveolar capillaries in vivo, they may be able to directly affect the endothelial cell-cell junctions. PMID:20887764

EphrinB2 Stabilizes Vascularlike Structures Generated by Endothelial Cells and Stem Cells from Apical Papilla.

PubMed

Yuan, Changyong; Wang, Penglai; Zhu, Shaoyue; Zou, Ting; Wang, Shuai; Xu, Jianguang; Heng, Boon Chin; Diogenes, Anibal; Zhang, Chengfei

2016-09-01

This study aimed to investigate the roles of ephrinB2 in stabilizing vascularlike structures generated by stem cells from apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs). HUVECs were seeded alone or with SCAPs concurrently or 12 hours later. Angiogenesis and ephrinB2 phosphorylation were assayed at different time points. Additionally, ephrinB2 expression in SCAPs and HUVECs was silenced with small interfering RNA, and vascularlike structure formation within coculture was assessed; 1 × 10(5) HUVECs were seeded in transwell inserts, and 6 × 10(5) SCAPs were plated in lower wells with or without ephrinB2-Fc. Migratory cells were stained and counted. Delayed addition of ephrinB2-Fc to the coculture of HUVECs and SCAPs was performed to evaluate the role of ephrinB2 on the stabilization of vascularlike structures. Concurrent coculture of SCAPs and HUVECs yielded significantly longer tubule lengths at 4, 8, and 12 hours (P < .05). Delayed addition of SCAPs to coculture with HUVECs resulted in vascularlike structures persisting longer than the HUVEC monoculture. Western blot confirmed that ephrinB2 phosphorylation was initiated at 0.5 hours of coculture and peaked at 1 hour. Silencing ephrinB2 expression in SCAPs and HUVECs resulted in the absence of vascularlike structures. Enhanced migration of HUVECs by SCAPs could be inhibited by ephrinB2-Fc. When ephrinB2-Fc was added at 3 hours of coculture, the vascularlike structures were stabilized for more than 12 hours as compared with 9 hours in the control group. EphrinB2 plays an important role in the stabilization of vascularlike structures generated by HUVECs and SCAPs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Leonurine protects against tumor necrosis factor-α-mediated inflammation in human umbilical vein endothelial cells.

PubMed

Liu, Xinhua; Pan, Lilong; Wang, Xianli; Gong, Qihai; Zhu, Yi Zhun

2012-05-01

Leonurine, a bioactive alkaloid compound in Herba leonuri, has various pharmacological activities, including antioxidant and anti-apoptotic capacities. This study was conducted to test the hypothesis that leonurine was able to attenuate tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) activation and the underlying molecular mechanisms. Mitogen-activated protein kinases (MAPK) activation, nuclear factor-κB (NF-κB) activation, and inflammatory mediators expression were detected by Western blot or enzyme-liked immunosorbent assay, intracellular reactive oxygen species (ROS) and NF-κB p65 translocation were measured by immunofluorescence, endothelial cell-monocyte interaction was detected by microscope. Leonurine inhibited U937 cells adhesion to TNF-α-activated HUVEC in a concentration dependent manner. Treatment with leonurine blocked TNF-α-induced mRNA and protein expression of adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), cyclooxygenase-2, and monocyte chemoattractant protein-1 in endothelial cells. In addition, leonurine attenuated TNF-α-induced intracellular ROS production in HUVEC. Furthermore, leonurine also suppressed the TNF-α-activated p38 phosphorylation and IκBα degradation. Subsequently, reduced NF-κB p65 phosphorylation, nuclear translocation, and DNA-binding activity were also observed. Our results demonstrated for the first time that the anti-inflammatory properties of leonurine in endothelial cells, at least in part, through suppression of NF-κB activation, which may have a potential therapeutic use for inflammatory vascular diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

In vitro study of stimulation effect on endothelialization by a copper bearing cobalt alloy.

PubMed

Jin, Shujing; Qi, Xun; Wang, Tongmin; Ren, Ling; Yang, Ke; Zhong, Hongshan

2018-02-01

Endothelialization is an important process after stenting in coronary artery. Recovery of the injured site timely can reduce the neointima formation and platelet absorbance, leading to a lower risk of in-stent restenosis. Copper is known to be critical in vascular construction. Thus a combination of copper with stent materials is a meaningful attempt. A copper bearing L605-Cu cobalt alloy was prepared and its effect on human umbilical vein endothelial cells (HUVECs) was evaluated in vitro in this study. It was found that HUVECs attached and stretched better on the surface of L605-Cu compared with L605, and the apoptosis of cells was decreased simultaneously. The migration and tube formation of HUVECs were also enhanced by the extract of L605-Cu. Furthermore, L605-Cu increased the mRNA expression of VEGF in HUVECs significantly. However it had no effect on the secretion of NO or mRNA expression of eNOS. The result of blood clotting test indicated that L605-Cu had better blood compatibility. These results above have demonstrated that the L605-Cu alloy is promising to be a new stent material with function of accelerating endothelialization. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 561-569, 2018. © 2017 Wiley Periodicals, Inc.

Maggot debridement therapy promotes diabetic foot wound healing by up-regulating endothelial cell activity.

PubMed

Sun, Xinjuan; Chen, Jin'an; Zhang, Jie; Wang, Wei; Sun, Jinshan; Wang, Aiping

2016-03-01

To determine the role of maggot debridement therapy (MDT) on diabetic foot wound healing, we compared growth related factors in wounds before and after treatment. Furthermore, we utilized human umbilical vein endothelial cells (HUVECs) to explore responses to maggot excretions/secretions on markers of angiogenesis and proliferation. The results showed that there was neo-granulation and angiogenesis in diabetic foot wounds after MDT. Moreover, significant elevation in CD34 and CD68 levels was also observed in treated wounds. In vitro, ES increased HUVEC proliferation, improved tube formation, and increased expression of vascular endothelial growth factor receptor 2 in a dose dependent manner. These results demonstrate that MDT and maggot ES can promote diabetic foot wound healing by up-regulating endothelial cell activity. Copyright © 2016. Published by Elsevier Inc.

Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

PubMed Central

Schleicher, Martina; Hansmann, Jan; Elkin, Bentsian; Kluger, Petra J.; Liebscher, Simone; Huber, Agnes J. T.; Fritze, Olaf; Schille, Christine; Müller, Michaela; Schenke-Layland, Katja; Seifert, Martina; Walles, Heike; Wendel, Hans-Peter; Stock, Ulrich A.

2012-01-01

In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene)) coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2) and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P < 0.05). Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization. PMID:22481939

Human umbilical vein endothelial cells synergize osteo/odontogenic differentiation of periodontal ligament stem cells in 3D cell sheets.

PubMed

Pandula, P K C Prgeeth; Samaranayake, L P; Jin, L J; Zhang, C F

2014-06-01

To investigate the expression of osteo/odontogenic differentiation markers and vascular network formation in a 3D cell sheet with varying cell ratios of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs). Human PDLSCs were isolated and characterized by flow cytometry, and co-cultured with HUVECs for the construction of cell sheets. Both types of cells were seeded on temperature-responsive culture dishes with PDLSCs alone, HUVECs alone and various ratios of the latter cells (1 : 1, 2 : 1, 5 : 1 and 1 : 5) to obtain confluent cell sheets. The expressions of osteo/odontogenic pathway markers, including alkaline phosphatase (ALP), bone sialoprotein (BSP) and runt-related transcription factor 2 (RUNX2), were analyzed at 3 and 7 d using RT-PCR. Further ALP protein quantification was performed at 7 and 14 d using ALP assay. The calcium nodule formation was assessed qualitatively and quantitatively by alizarin red assay. Histological evaluations of three cell sheet constructs treated with different combinations (PDLSC-PDLSC-PDLSC/PDLSC-HUVEC-PDLSC/co-culture-co-culture-co-culture) were performed with hematoxylin and eosin and immunofluorescence staining. Statistical analysis was performed using t-test (p < 0.05). Significantly higher ALP gene expression was observed at 3 d in 1 : 1 (PDLSC-HUVEC) (2.52 ± 0.67) and 5 : 1 (4.05 ± 1.07) co-culture groups compared with other groups (p < 0.05); this was consistent with ALP protein quantification. However, the expression of BSP and RUNX2 genes was higher at 7 d compared to 3 d. Significant calcium mineralization was detected as quantified by alizarin red assay at 14 d in 1 : 1 (1323.55 ± 6.54 μm) and 5 : 1 (994.67 ± 4.15 μm) co-cultures as compared with monoculture cell sheets (p < 0.05). Hematoxylin and eosin and CD31 immunostaining clearly exemplified the development of a layered cell sheet structure with endothelial cell islands within the constructed PDLSC-HUVEC-PDLSC and co-culture groups. Furthermore, HUVECs invaded the layered cell sheet, suggestive of rudimentary vascular network initiation. This study suggests that the PDLSC-HUVEC co-culture, cell sheet, model exhibits significantly high levels of osteo/odontogenic markers with signs of initial vascular formation. This novel 3D cell sheet-based approach may be potentially beneficial for periodontal regenerative therapy. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of clopidogrel on inflammatory cytokines and adhesion molecules in human endothelial cells: Role of nitric oxide mediating pleiotropic effects.

PubMed

Cerda, Alvaro; Pavez, Monica; Manriquez, Victor; Luchessi, Andre Ducati; Leal, Pamela; Benavente, Felipe; Fajardo, Cristina Moreno; Salazar, Luis; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo

2017-08-01

Clopidogrel is commonly used in prevention and treatment of atherothrombosis. Some previous studies have suggested a pleiotropic effect of clopidogrel; however, when this drug causes platelet-independent effects on endothelial function remains unclear. To evaluate the influence of clopidogrel on inflammatory biomarkers and adhesion molecules in human endothelial cells and the role of nitric oxide (NO) in this process. TNF-α-induced human umbilical vein endothelial cells (HUVEC) were exposed to clopidogrel. Gene expression and protein expression of ICAM-1, P-selectin, IL-8, IL-6, and MCP-1 were evaluated by qPCR, flux cytometry, or milliplex technology. Expression of endothelial nitric oxide synthase (NOS3) and NO release were also evaluated. Influence of clopidogrel was further evaluated in NOS3 downregulated HUVEC by RNAi. Clopidogrel at 20 μmol/L induced NO release in HUVEC after 24-hours treatment. Gene expressions of inflammatory markers IL-8 and MCP1 were reduced after clopidogrel treatment (P<.05); however, only MCP-1 remained reduced at protein level. IL-6 was not modified by clopidogrel treatment. Gene expression and protein expression of ICAM-1 were diminished by 24-hours clopidogrel exposure, whereas P-selectin was not modified. NOS3 downregulated HUVEC model revealed that ICAM-1 modification by clopidogrel is dependent of this via, whereas MCP-1 is modulated in an NO-independent form. Our results support new evidence for pleiotropic effects of clopidogrel on inflammation and endothelial function. Reduction in ICAM-1 and MCP-1 in human endothelium is an important extent of the use of this drug for treatment of cardiovascular diseases, and NO has an important role in this process. © 2017 John Wiley & Sons Ltd.

Sandwiched polymer fibre in fibrin matrices for the dictation of endothelial cells undergoing angiogenesis

NASA Astrophysics Data System (ADS)

Sukmana, I.; Djuansjah, J. R. P.

2013-04-01

We present here a three-dimensional (3D) sandwich system made by poly(ethylene terephthalate) (PET) fibre and fibrin extracellular matrix (ECM) for endothelial cell dictation and angiogenesis guidance. In this three-dimensional system, Human Umbilical Vein Endothelial cells (HUVECs) were firstly cultured for 2 (two) days to cover the PET fibre before sandwiched in two layer fibrin gel containing HUVECs. After 4 (four) days of culture, cel-to-cel connection, tube-like structure and multi-cellular lumen formation were then assessed and validated. Phase contrast and fluorescence imaging using an inverted microscope were used to determine cell-to-cell and cell-ECM interactions. Laser scanning confocal microscopy and histological techniques were used to confirm the development of tube-like structure and multi-cellular lumen formation. This study shows that polymer fibres sandwiched in fibrin gel can be used to dictate endothelial cells undergoing angiogenesis with potential application in cancer and cardiovascular study and tissue engineering vascularisation.

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

NASA Astrophysics Data System (ADS)

Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

2014-04-01

To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

Edaravone attenuates monocyte adhesion to endothelial cells induced by oxidized low-density lipoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhijuan, E-mail: zjlee038@163.com; Cheng, Jianxin; Wang, Liping

2015-10-30

Oxidized low-density lipoprotein (oxLDL) plays a vital role in recruitment of monocytes to endothelial cells, which is important during early stages of atherosclerosis development. Edaravone, a potent and novel scavenger of free radicals inhibiting hydroxyl radicals, has been clinically used to reduce the neuronal damage following ischemic stroke. In the present study, Edaravone was revealed to markedly reduce oxLDL-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). The inhibitory mechanism of Edaravone was associated with suppression of the chemokine MCP-1 and adhesion molecule VCAM-1 and ICAM-1 expression. In addition, luciferase reporter assay results revealed that administration of Edaravone attenuatedmore » the increase in NF-κB transcriptional activity induced by oxLDL. Notably, it's also shown that Edaravone treatment blocked oxLDL induced p65 nuclear translocation in HUVECs. Results indicate that Edaravone negatively regulates endothelial inflammation. - Highlights: • Edaravone reduces oxLDL-induced monocyte adhesion to HUVECs. • Edaravone attenuates oxLDL-induced expression of MCP-1, VCAM-1, and ICAM-1. • Edaravone reduces NF-κB transcriptional activity and p65 nuclear translocation.« less

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Haibing; Department of Ophthalmology, Anhui Provincial Hospital, Hefei; Jia Weiping

2008-05-02

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might representmore » a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.« less

Cell death in a co-culture of hepatocellular carcinoma cells and human umbilical vascular endothelial cells in a medium lacking glucose and arginine.

PubMed

Tomizawa, Minoru; Shinozaki, Fuminobu; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Ishige, Naoki

2017-01-01

Human primary hepatocytes are able to survive in a medium without glucose and arginine that is instead supplemented with galactose and ornithine (hepatocyte selection medium; HSM). This is because the cells produce glucose and arginine by the action of galactokinase (GALK) and ornithine carbamoyltransferase (OTC), respectively. It was expected that hepatocellular carcinoma (HCC) cells do not survive in HSM. In the current study, HCC cell lines (namely HLE, HLF, PLC/PRL/5, Hep3B and HepG2) and human umbilical vascular endothelial cells (HUVECs) were cultured in HSM, and the expression levels of GALK1, GALK2 and OTC were analyzed by reverse transcription-quantitative polymerase chain reaction. HLE, HLF and PLC/PRL/5 cells died on day 11, while Hep3B, HepG2 and HUVECs died on day 7. HLF cells were further analyzed as these cells had lower expression levels of GALK1, GALK2 and OTC compared with adult liver cells, and survived until day 11. In these cells, the expression levels of GALK1, GALK2 and OTC did not change on days 3 and 7 as compared to day 0. In addition, a co-culture of HLF cells with HUVECs was established and the medium was changed to HSM. It was observed that HLF cells and HUVECs in co-culture were damaged in HSM. In summary, HCC cells and HUVECs died in a medium without glucose and arginine that was supplemented with galactose and ornithine. HCC cells and HUVECs were damaged in HSM, suggesting a potential application for treatment with the medium.

Rhynchophylla total alkaloid rescues autophagy, decreases oxidative stress and improves endothelial vasodilation in spontaneous hypertensive rats.

PubMed

Li, Chao; Jiang, Feng; Li, Yun-Lun; Jiang, Yue-Hua; Yang, Wen-Qing; Sheng, Jie; Xu, Wen-Juan; Zhu, Qing-Jun

2018-03-01

Autophagy plays an important role in alleviating oxidative stress and stabilizing atherosclerotic plaques. However, the potential role of autophagy in endothelial vasodilation function has rarely been studied. This study aimed to investigate whether rhynchophylla total alkaloid (RTA) has a positive role in enhancing autophagy through decreasing oxidative stress, and improving endothelial vasodilation. In oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs), RTA (200 mg/L) significantly suppressed ox-LDL-induced oxidative stress through rescuing autophagy, and decreased cell apoptosis. In spontaneous hypertensive rats (SHR), administration of RTA (50 mg·kg -1 ·d -1 , ip, for 6 weeks) improved endothelin-dependent vasodilation of thoracic aorta rings. Furthermore, RTA administration significantly increased the antioxidant capacity and alleviated oxidative stress through enhancing autophagy in SHR. In ox-LDL-treated HUVECs, we found that the promotion of autophagy by RTA resulted in activation of the AMP-activated protein kinase (AMPK) signaling pathway. Our results show that RTA treatment rescues the ox-LDL-induced autophagy impairment in HUVECs and improves endothelium-dependent vasodilation function in SHR.

[In vitro anti-angiogenic action of lambda-carrageenan oligosaccharides].

PubMed

Chen, Hai-Min; Yan, Xiao-Jun; Wang, Feng; Lin, Jing; Xu, Wei-Feng

2007-06-01

This study was designed to evaluate the inhibition effect of lambda-carrageenan oligosaccharides on neovascularization in vitro by chick chorioallantoic membrane (CAM) model and human umbilical vein endothelial cell ( HUVEC). lambda-Carrageenan oligosaccharides caused a dose-dependent decrease of the vascular density of CAM, and adversely affected capillary plexus formation. At a high concentration of 1 mg x mL(-1), this compound inhibited the endothelial cell proliferation, while low concentration of lambda-carrageenan oligosaccharides (< 250 microg x mL(-1)) affected the cell survival slightly (> 95%). Different cytotoxic sensitivity of lambda-carrageenan oligosaccharides in three kinds of cells was observed, of which HUVEC is the most sensitive to this oligosaccharides. The inhibitory action of lambda-carrageenan oligosaccharides on the endothelial cell invasion and migration was also observed at relatively low concentration (150 - 300 microg x mL(-1)) through down-regulation of intracellular matrix metalloproteinases-2 (MMP-2) expression on endothelial cells. Current observations demonstrated that lambda-carrageenan oligosaccharides are potential angiogenesis inhibitor with combined effects of inhibiting invasion, migration and proliferation.

Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi

2015-07-03

Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed,more » because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.« less

Rhubarb Antagonizes Matrix Metalloproteinase-9-induced Vascular Endothelial Permeability

PubMed Central

Cui, Yun-Liang; Zhang, Sheng; Tian, Zhao-Tao; Lin, Zhao-Fen; Chen, De-Chang

2016-01-01

Background: Intact endothelial structure and function are critical for maintaining microcirculatory homeostasis. Dysfunction of the latter is an underlying cause of various organ pathologies. In a previous study, we showed that rhubarb, a traditional Chinese medicine, protected intestinal mucosal microvascular endothelial cells in rats with metastasizing septicemia. In this study, we investigated the effects and mechanisms of rhubarb on matrix metalloproteinase-9 (MMP9)-induced vascular endothelial (VE) permeability. Methods: Rhubarb monomers were extracted and purified by a series of chromatography approaches. The identity of these monomers was analyzed by hydrogen-1 nuclear magnetic resonance (NMR), carbon-13 NMR, and distortionless enhancement by polarization transfer magnetic resonance spectroscopy. We established a human umbilical vein endothelial cell (HUVEC) monolayer on a Transwell insert. We measured the HUVEC permeability, proliferation, and the secretion of VE-cadherin into culture medium using fluorescein isothiocyanate-dextran assay, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, and enzyme-linked immunosorbent assay, respectively, in response to treatment with MMP9 and/or rhubarb monomers. Results: A total of 21 rhubarb monomers were extracted and identified. MMP9 significantly increased the permeability of the HUVEC monolayer, which was significantly reduced by five individual rhubarb monomer (emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein) or a combination of all five monomers (1 μmol/L for each monomer). Mechanistically, the five-monomer mixture at 1 μmol/L promoted HUVEC proliferation. In addition, MMP9 stimulated the secretion of VE-cadherin into the culture medium, which was significantly inhibited by the five-monomer mixture. Conclusions: The rhubarb mixture of emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein, at a low concentration, antagonized the MMP9-induced HUVEC monolayer permeability by promoting HUVEC proliferation and reducing extracellular VE-cadherin concentrations. PMID:27411464

[DNA microarray reveals changes in gene expression of endothelial cells under shear stress].

PubMed

Cheng, Min; Zhang, Wensheng; Chen, Huaiqing; Wu, Wenchao; Huang, Hua

2004-04-01

cDNA microarray technology is used as a powerful tool for rapid, comprehensive, and quantitative analysis of gene profiles of cultured human umbilical vein endothelial cells(HUVECs) in the normal static group and the shear stressed (4.20 dyne/cm2, 2 h) group. The total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The expression ratios reported are the average from the two separate experiments. After bioinformatics analysis, we identified a total of 108 genes (approximately 0.026%) revealing differential expression. Of these 53 genes expressions were up-regulated, the most enhanced ones being human homolog of yeast IPP isomerase, human low density lipoprotein receptor gene, Squalene epoxidase gene, 7-dehydrocholesterol reductase, and 55 were down-regulated, the most decreased ones being heat shock 70 kD protein 1, TCB gene encoding cytosolic thyroid hormone-binding protein in HUVECs exposed to low shear stress. These results indicate that the cDNA microarray technique is effective in screening the differentially expressed genes in endothelial cells induced by various experimental conditions and the data may serve as stimuli to further researches.

Inhibitory effect of indigo naturalis on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells.

PubMed

Chang, Hsin-Ning; Pang, Jong-Hwei Su; Yang, Sien-Hung; Hung, Chi-Feng; Chiang, Chi-Hsin; Lin, Tung-Yi; Lin, Yin-Ku

2010-09-14

The use of indigo naturalis to treat psoriasis has proved effective in our previous clinical studies. The present study was designed to examine the anti-inflammatory effect of indigo naturalis in primary cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of cells with indigo naturalis extract attenuated TNF-α-induced increase in Jurkat T cell adhesion to HUVECs as well as decreased the protein and messenger (m)RNA expression levels of vascular cell adhesion molecule-1 (VCAM-1) on HUVECs. Indigo naturalis extract also inhibited the protein expression of activator protein-1 (AP-1)/c-Jun, a critical transcription factor for the activation of VCAM-1 gene expression. Since the reduction of lymphocyte adhesion to vascular cells by indigo naturalis extract could subsequently reduce the inflammatory reactions caused by lymphocyte infiltration in the epidermal layer and help to improve psoriasis, this study provides a potential mechanism for the anti-inflammatory therapeutic effect of indigo naturalis extract in psoriasis.

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

PubMed Central

Pan, Qunwen; Zhao, Yuhui; Chen, Ji; Zhao, Bin; Chen, Yanfang

2013-01-01

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs. PMID:24358213

Tetrahydroxystilbene glucoside improves TNF-α-induced endothelial dysfunction: involvement of TGFβ/Smad pathway and inhibition of vimentin expression.

PubMed

Yao, Wenjuan; Gu, Chengjing; Shao, Haoran; Meng, Guoliang; Wang, Huiming; Jing, Xiang; Zhang, Wei

2015-01-01

Endothelial dysfunction plays an important role in the pathogenesis of atherogenesis. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum (PM), exhibits significant anti-atherosclerotic activity. Here, we used human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-α (TNF-α) in vitro to investigate the cytoprotective effects of TSG on TNF-α-induced endothelial injury and the related mechanisms. Pretreatment with 50 and 100 μM TSG markedly attenuated TNF-α-induced loss of cell viability and release of lactate dehydrogenase (LDH) and inhibited TNF-α-induced cell apoptosis. The inhibition of vimentin expression was involved in the cytoprotection afforded by TSG. Using inhibitors for PI3K and TGFβ or siRNA for Akt and Smad2, we found that vimentin production in HUVECs is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by TNF-α. These results suggest that TSG protects HUVECs against TNF-α-induced cell damage by inhibiting vimentin expression via the interruption of the TGFβ/Smad signaling pathway.

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth.

PubMed

Wang, Dong-an; Ji, Jian; Sun, Yong-hong; Shen, Jia-cong; Feng, Lin-xian; Elisseeff, Jennifer H

2002-01-01

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.

Mesenchymal stem cell-derived microparticles ameliorate peritubular capillary rarefaction via inhibition of endothelial-mesenchymal transition and decrease tubulointerstitial fibrosis in unilateral ureteral obstruction.

PubMed

Choi, Hoon Young; Lee, Hyun Gyu; Kim, Beom Seok; Ahn, Sun Hee; Jung, Ara; Lee, Mirae; Lee, Jung Eun; Kim, Hyung Jong; Ha, Sung Kyu; Park, Hyeong Cheon

2015-03-11

Microparticles (MPs) derived from kidney-derived mesenchymal stem cells (KMSCs) have recently been reported to ameliorate rarefaction of peritubular capillaries (PTC) in ischemic kidneys via delivery of proangiogenic effectors. This study aimed to investigate whether KMSC-derived MPs show anti-fibrotic effects by ameliorating endothelial-to-mesenchymal transition (EndoMT) in human umbilical vein endothelial cells (HUVEC) in vitro and by preserving PTC in kidneys with unilateral ureteral obstruction (UUO) in vivo. MPs isolated from the supernatants of KMSC were co-cultured with HUVEC to assess their in vitro biologic effects on endothelial cells. Mice were treated with MPs via the tail vein after UUO injury to assess their anti-fibrotic and PTC sparing effects. Renal tubulointerstitial damage and inflammatory cell infiltration were examined with Masson's trichrome, F4/80 and α-smooth muscle actin (α-SMA) staining and PTC rarefaction index was determined by CD31 staining. KMSC-derived MPs significantly ameliorated EndoMT and improved in vitro proliferation of TGF-β1 treated HUVEC. In vivo administration of KMSC-derived MPs significantly inhibited EndoMT of PTC endothelial cells and improved PTC rarefaction in UUO kidneys. Furthermore, administration of KMSC-derived MPs inhibited inflammatory cell infiltration as well as tubulointerstitial fibrosis in UUO mice as demonstrated by decreased F4/80 and α-SMA-positive cells and Masson's trichrome staining, respectively. Our results suggest that KMSC-derived MPs ameliorate PTC rarefaction via inhibition of EndoMT and protect against progression of renal damage by inhibiting tubulointerstitial fibrosis.

Low grade inflammation inhibits VEGF induced HUVECs migration in p53 dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panta, Sushil; Yamakuchi, Munekazu; Kagoshima University Hospital, Kagoshima

In the course of studying crosstalk between inflammation and angiogenesis, high doses of pro-inflammatory factors have been reported to induce apoptosis in cells. Under normal circumstances also the pro-inflammatory cytokines are being released in low doses and are actively involved in cell signaling pathways. We studied the effects of low grade inflammation in growth factor induced angiogenesis using tumor necrosis factor alfa (TNFα) and vascular endothelial growth factor A (VEGF) respectively. We found that low dose of TNFα can inhibit VEGF induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Low dose of TNFα induces mild upregulation and moreover nuclearmore » localization of tumor suppressor protein 53 (P53) which causes decrease in inhibitor of DNA binding-1 (Id1) expression and shuttling to the cytoplasm. In absence of Id1, HUVECs fail to upregulate β{sub 3}-integrin and cell migration is decreased. Connecting low dose of TNFα induced p53 to β{sub 3}-integrin through Id1, we present additional link in cross talk between inflammation and angiogenesis. - Highlights: • Low grade inflammation (low dose of TNF alfa) inhibits VEGF induced endothelial cells migration. • The low grade inflammation with VEGF treatment upregulates P53 to a nonlethal level. • P53 activation inhibits Id1 shuttling to the cytoplasm in endothelial cells. • Inhibition of Id1 resulted in downregulation of β{sub 3}-integrin which cause decrease in cell migration. • Inflammation and angiogenesis might cross-talk by P53 – Id1 – β{sub 3}-integrin pathway in endothelial cells.« less

Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

PubMed

Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

2016-07-01

To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.

Bioglass promotes wound healing by affecting gap junction connexin 43 mediated endothelial cell behavior.

PubMed

Li, Haiyan; He, Jin; Yu, Hongfei; Green, Colin R; Chang, Jiang

2016-04-01

It is well known that gap junctions play an important role in wound healing, and bioactive glass (BG) has been shown to help healing when applied as a wound dressing. However, the effects of BG on gap junctional communication between cells involved in wound healing is not well understood. We hypothesized that BG may be able to affect gap junction mediated cell behavior to enhance wound healing. Therefore, we set out to investigate the effects of BG on gap junction related behavior of endothelial cells in order to elucidate the mechanisms through which BG is operating. In in vitro studies, BG ion extracts prevented death of human umbilical vein endothelial cells (HUVEC) following hypoxia in a dose dependent manner, possibly through connexin hemichannel modulation. In addition, BG showed stimulatory effects on gap junction communication between HUVECs and upregulated connexin43 (Cx43) expression. Furthermore, BG prompted expression of vascular endothelial growth factor and basic fibroblast growth factor as well as their receptors, and vascular endothelial cadherin in HUVECs, all of which are beneficial for vascularization. In vivo wound healing results showed that the wound closure of full-thickness excisional wounds of rats was accelerated by BG with reduced inflammation during initial stages of healing and stimulated angiogenesis during the proliferation stage. Therefore, BG can stimulate wound healing through affecting gap junctions and gap junction related endothelial cell behaviors, including prevention of endothelial cell death following hypoxia, stimulation of gap junction communication and upregulation of critical vascular growth factors, which contributes to the enhancement of angiogenesis in the wound bed and finally to accelerate wound healing. Although many studies have reported that BG stimulates angiogenesis and wound healing, this work reveals the relationship between BG and gap junction connexin 43 mediated endothelial cell behavior and elucidates one of the possible mechanisms through which BG stimulates wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular Hydrogen Alleviates Cellular Senescence in Endothelial Cells.

PubMed

Hara, Fumihiko; Tatebe, Junko; Watanabe, Ippei; Yamazaki, Junichi; Ikeda, Takanori; Morita, Toshisuke

2016-08-25

Substantial evidence indicates that molecular hydrogen (H2) has beneficial vascular effects because of its antioxidant and/or anti-inflammatory effects. Thus, hydrogen-rich water may prove to be an effective anti-aging drink. This study examined the effects of H2on endothelial senescence and clarified the mechanisms involved. Hydrogen-rich medium was produced by a high-purity hydrogen gas generator. Human umbilical vein endothelial cells (HUVECs) were incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for various time periods in normal or hydrogen-rich medium. The baseline H2concentration in hydrogen-rich medium was 0.55±0.07 mmol/L. This concentration gradually decreased, and H2was almost undetectable in medium after 12 h. At 24 h after TCDD exposure, HUVECs treated with TCDD exhibited increased 8OHdG and acetyl-p53 expression, decreased nicotinamide adenine dinucleotide (NAD(+))/NADH ratio, impaired Sirt1 activity, and enhanced senescence-associated β-galactosidase. However, HUVECs incubated in hydrogen-rich medium did not exhibit these TCDD-induced changes accompanying Nrf2 activation, which was observed even after H2was undetectable in the medium. Chrysin, an inhibitor of Nrf2, abolished the protective effects of H2on HUVECs. H2has long-lasting antioxidant and anti-aging effects on vascular endothelial cells through the Nrf2 pathway, even after transient exposure to H2. Hydrogen-rich water may thus be a functional drink that increases longevity. (Circ J 2016; 80: 2037-2046).

Anti-inflammatory and anti-angiogenic effects of flavonoids isolated from Lycium barbarum Linnaeus on human umbilical vein endothelial cells.

PubMed

Wu, Wen-Bin; Hung, Dian-Kun; Chang, Fung-Wei; Ong, Eng-Thaim; Chen, Bing-Huei

2012-10-01

Anti-inflammatory and anti-angiogenic effects of flavonoids isolated from Lycium barbarum fruits, a traditional Chinese medicine, on human umbilical vein endothelial cells (HUVECs) were investigated. Initially, flavonoids were extracted with 80% ethanol and separated using a Cosmosil 140 C18-OPN column, with the acidic fraction eluted with deionized water being composed of chlorogenic acid, caffeoyl quinic acid, caffeic acid and p-coumaric acid and the neutral fraction eluted with methanol composed of quercetin-diglycoside, rutin and kaempferol-O-rutinoside. Flavonoid extract was effective in inhibiting expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) induced by TNF-α in HUVECs. The RT-PCR analysis indicated that ICAM-1 mRNA induced by TNF-α was inhibited by flavonoid extract. The flavonoid extract attenuated TNF-α-induced IκB phosphorylation as well as NF-κB, p65 and p50 translocation from cytosol to nucleus, through inhibition on TNF-α- and H(2)O(2)-induced intracellular reactive oxygen species (ROS) production. For the anti-angiogenic study, the flavonoid extract inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation and migration in HUVECs, as well as angiogenesis. However, the flavonoid extract did not inhibit VEGF signaling. Surprisingly, HUVECs adhesion to the extracellular matrix was compromised and adhesion-induced signaling was retarded by the flavonoid extract.

Edaravone attenuates monocyte adhesion to endothelial cells induced by oxidized low-density lipoprotein.

PubMed

Li, Zhijuan; Cheng, Jianxin; Wang, Liping

2015-10-30

Oxidized low-density lipoprotein (oxLDL) plays a vital role in recruitment of monocytes to endothelial cells, which is important during early stages of atherosclerosis development. Edaravone, a potent and novel scavenger of free radicals inhibiting hydroxyl radicals, has been clinically used to reduce the neuronal damage following ischemic stroke. In the present study, Edaravone was revealed to markedly reduce oxLDL-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). The inhibitory mechanism of Edaravone was associated with suppression of the chemokine MCP-1 and adhesion molecule VCAM-1 and ICAM-1 expression. In addition, luciferase reporter assay results revealed that administration of Edaravone attenuated the increase in NF-κB transcriptional activity induced by oxLDL. Notably, it's also shown that Edaravone treatment blocked oxLDL induced p65 nuclear translocation in HUVECs. Results indicate that Edaravone negatively regulates endothelial inflammation. Copyright © 2015. Published by Elsevier Inc.

A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

PubMed

Sabra, Georges; Vermette, Patrick

2013-02-01

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. Copyright © 2012 Wiley Periodicals, Inc.

Antiangiogenic Effects and Therapeutic Targets of Azadirachta indica Leaf Extract in Endothelial Cells

PubMed Central

Mahapatra, Saswati; Young, Charles Y. F.; Kohli, Manish; Karnes, R. Jeffrey; Klee, Eric W.; Holmes, Michael W.; Tindall, Donald J.; Donkena, Krishna Vanaja

2012-01-01

Azadirachta indica (common name: neem) leaves have been found to possess immunomodulatory, anti-inflammatory and anti-carcinogenic properties. The present study evaluates anti-angiogenic potential of ethanol extract of neem leaves (EENL) in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with EENL inhibited VEGF induced angiogenic response in vitro and in vivo. The in vitro proliferation, invasion and migration of HUVECs were suppressed with EENL. Nuclear fragmentation and abnormally small mitochondria with dilated cristae were observed in EENL treated HUVECs by transmission electron microscopy. Genome-wide mRNA expression profiling after treatment with EENL revealed differentially regulated genes. Expression changes of the genes were validated by quantitative real-time polymerase chain reaction. Additionally, increase in the expression of HMOX1, ATF3 and EGR1 proteins were determined by immunoblotting. Analysis of the compounds in the EENL by mass spectrometry suggests the presence of nimbolide, 2′,3′-dehydrosalannol, 6-desacetyl nimbinene and nimolinone. We further confirmed antiproliferative activity of nimbolide and 2′,3′-dehydrosalannol in HUVECs. Our results suggest that EENL by regulating the genes involved in cellular development and cell death functions could control cell proliferation, attenuate the stimulatory effects of VEGF and exert antiangiogenic effects. EENL treatment could have a potential therapeutic role during cancer progression. PMID:22461839

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

PubMed

Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

2005-06-01

To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway.

PubMed

Tseng, Shih-Ya; Chao, Ting-Hsing; Li, Yi-Heng; Liu, Ping-Yen; Lee, Cheng-Han; Cho, Chung-Lung; Wu, Hua-Lin; Chen, Jyh-Hong

2016-04-01

Cilostazol is an antiplatelet agent with vasodilatory effects that works by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). This study investigated the effects of cilostazol in preventing high glucose (HG)-induced impaired angiogenesis and examined the potential mechanisms involving activation of AMP-activated protein kinase (AMPK). Assays for colony formation, adhesion, proliferation, migration, and vascular tube formation were used to determine the effect of cilostazol in HG-treated endothelial progenitor cells (EPCs) or human umbilical vein endothelial cells (HUVECs). Animal-based assays were performed in hyperglycemic ICR mice undergoing hind limb ischemia. An immnunoblotting assay was used to identify the expression and activation of signaling molecules in vitro and in vivo. Cilostazol treatment significantly restored endothelial function in EPCs and HUVECs through activation of AMPK/acetyl-coenzyme A carboxylase (ACC)-dependent pathways and cAMP/protein kinase A (PKA)-dependent pathways. Recovery of blood flow in the ischemic hind limb and the population of circulating CD34(+) cells were significantly improved in cilostazol-treated mice, and these effects were abolished by local AMPK knockdown. Cilostazol increased the phosphorylation of AMPK/ACC and Akt/endothelial nitric oxide synthase signaling molecules in parallel with or downstream of the cAMP/PKA-dependent signaling pathway in vitro and in vivo. Cilostazol prevents HG-induced endothelial dysfunction in EPCs and HUVECs and enhances angiogenesis in hyperglycemic mice by interactions with a broad signaling network, including activation of AMPK/ACC and probably cAMP/PKA pathways. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

Uncaria rhynchophylla induces angiogenesis in vitro and in vivo.

PubMed

Choi, Do-Young; Huh, Jeong-Eun; Lee, Jae-Dong; Cho, Eun-Mi; Baek, Yong-Hyeon; Yang, Ha-Ru; Cho, Yoon-Je; Kim, Kang-Il; Kim, Deog-Yoon; Park, Dong-Suk

2005-12-01

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, and angiogenic factors and matrix protein interactions modulate this process. The aim of this study was to determine the angiogenic properties of Uncaria rhynchophylla. Uncaria rhynchophylla significantly enhanced human umbilical vein endothelial cells (HUVECs) proliferation in a dose-dependent manner. Neutralization of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) by monoclonal antibody suppressed the Uncaria rhynchophylla stimulatory effect on proliferation. In addition, Uncaria rhynchophylla significantly increased chemotactic-migration on gelatin and tubular structures on Matrigel of HUVECs in a dose-dependent manner. Interestingly, Uncaria rhynchophylla dose-dependently increased VEGF, and bFGF gene expression and protein secretion of HUVEC. The angiogenic activity of Uncaria rhynchophylla was confirmed using an in vivo Matrigel angiogenesis model, showing promotion of blood vessel formation. These results suggest that Uncaria rhynchophylla could potentially used to accelerate vascular wound healing or to promote the growth of collateral blood vessel in ischemic tissues.

Selective Suppression of Endothelial Cell Apoptosis by the High Molecular Weight Form of Adiponectin

PubMed Central

Kobayashi, Hideki; Ouchi, Noriyuki; Kihara, Shinji; Walsh, Kenneth; Kumada, Masahiro; Abe, Yuki; Funahashi, Tohru; Matsuzawa, Yuji

2015-01-01

Adiponectin is an adipocyte-derived, antiatherogenic protein that is present in serum as three isoforms. Total adiponectin levels are decreased in obese or diabetic humans or animal models. This study was designed to elucidate the relative isoform distribution of adiponectin in human disease states and identify the active form of adiponectin toward vascular endothelial cells. The percentage of high molecular weight form (HMW) per total adiponectin was significantly lower in patients with coronary artery disease than control subjects, whereas the hexamer form was similar and the trimer form was significantly higher. During weight reduction in obese subjects, the HMW form increased and the trimer and hexamer forms decreased. Recombinant adiponectin dose-dependently suppressed apoptosis and caspase-3 activity in human umbilical vein endothelial cells (HUVECs). Transduction with dominant-negative AMP-activated protein kinase (AMPK) abolished the suppressive effect of adiponectin on HUVECs. Gel filtration chromatography was used to separate the adiponectin isoforms, and the antiapoptotic effect toward HUVECs was only observed with the HMW form. These data suggest that HMW adiponectin specifically confers the vascular-protective activities of this adipocytokine. PMID:14752031

Safflor yellow B suppresses angiotensin II-mediated human umbilical vein cell injury via regulation of Bcl-2/p22{sup phox} expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chaoyun; He, Yanhao; Department of Pharmacology, Xi'an Jiaotong University School of Medicine, Key Laboratory of Environment and Genes Related to Disease, Ministry of Education, Xi'an, Shaanxi 710061

Intracellular reactive oxygen species (ROS) are derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Angiotensin II (Ang II) can cause endothelial dysfunction by promoting intracellular ROS generation. Safflor yellow B (SYB) effectively inhibits ROS generation by upregulating Bcl-2 expression. In this study, we examined the effects of SYB on Ang II-induced injury to human umbilical vein endothelial cells (HUVECs), and elucidated the roles of NADPH oxidase and Bcl-2. We treated cultured HUVECs with Ang II, SYB, and Bcl-2 siRNA, and determined NADPH oxidase activity and ROS levels. Furthermore, cellular and mitochondrial physiological states were evaluated, and the expression levels ofmore » target proteins were analyzed. Ang II significantly enhanced intracellular ROS levels, caused mitochondrial membrane dysfunction, and decreased cell viability, leading to apoptosis. This was associated with increased expression of AT1R and p22{sup phox}, increased NADPH oxidase activity, and an increased ratio of Bax/Bcl-2, leading to decreases in antioxidant enzyme activities, which were further strengthened after blocking Bcl-2. Compared to Ang II treatment alone, co-treatment with SYB significantly reversed HUVEC injury. Taken together, these results demonstrate that SYB could significantly protect endothelial cells from Ang II-induced cell damage, and that it does so by upregulating Bcl-2 expression and inhibiting ROS generation. - Highlights: • Angiotensin II depresses mitochondria physiological function. • Angiotensin II activates NADPH oxidase via up-regulating expresion of p22{sup phox}. • Bcl-2 plays a pivotal role in improving mitochondria function and regulates ROS level. • Inhibitor of Bcl-2 promotes angiotensin II mediated HUVEC injury. • SYB attenuates angiotensin II mediated HUVEC injury via up regulating Bcl-2 expression.« less

Resveratrol inhibits proteinase-activated receptor-2-induced release of soluble vascular endothelial growth factor receptor-1 from human endothelial cells

PubMed Central

Al-Ani, Bahjat

2013-01-01

We recently reported that (i) activation of the proinflammatory receptor, proteinase-activated receptor-2 (PAR-2) caused the release of an important biomarker in preeclampsia, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, also known as sFlt-1) from human umbilical vein endothelial cells (HUVECs), and (ii) that the anti-oxidant and anti-inflammatory agent, resveratrol, is capable of inhibiting the proinflammatory cytokine-induced sVEGFR-1 release from human placenta. Based on these findings and because PAR-2 is upregulated by proinflammatory cytokines, we sought to determine whether resveratrol can inhibit PAR-2-induced sVEGFR-1 release. PAR-2 expressing cells, HUVECs and human embryonic kidney cells (HEK-293) transfected with a human VEGFR-1 promoter-luciferase reporter construct were incubated with PAR-2-activating peptide and/or resveratrol. Cell supernatants were assayed for sVEGFR-1 by enzyme-linked immunosorbent assay (ELISA), and VEGFR-1 promoter-luciferase assay was performed on the harvested cell lysates. Preincubation of HEK-293 cells with resveratrol significantly inhibited PAR-2-induced VEGFR-1 promoter activity without affecting cell viability as assessed by MTT assay. The addition of resveratrol also blocked PAR-2-mediated sVEGFR-1 release from HUVECs. The present study demonstrates that resveratrol suppressed both VEGFR-1 promoter activity and sVEGFR-1 protein release induced by PAR-2 activation, which further endorses our recent findings of a potential therapeutic role for resveratrol in preeclampsia. PMID:26933402

Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury.

PubMed

Xiao, Bing; Chai, Yi; Lv, Shigang; Ye, Minhua; Wu, Miaojing; Xie, Liyuan; Fan, Yanghua; Zhu, Xingen; Gao, Ziyun

2017-10-01

Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

Mechanisms of shock wave induced endothelial cell injury.

PubMed

Sondén, Anders; Svensson, Bengt; Roman, Nils; Brismar, Bo; Palmblad, Jan; Kjellström, B Thomas

2002-01-01

Medical procedures, for example, laser angioplasty and extracorporeal lithotripsy as well as high-energy trauma expose human tissues to shock waves (SWs) that may cause tissue injury. The mechanisms for this injury, often affecting blood vessel walls, are poorly understood. Here we sought to assess the role of two suggested factors, viz., cavitation or reactive oxygen species (ROS). A laser driven flyer-plate model was used to expose human umbilical cord vein endothelial cell (HUVEC) monolayers to SWs or to SWs plus cavitation (SWC). Cell injury was quantified with morphometry, trypan blue staining, and release of (51)Cr from labeled HUVECs. HUVECs, exposed to SWs only, could not be distinguished from controls in morphological appearance or ability to exclude trypan blue. Yet, release of (51)Cr, indicated a significant cell injury (P < 0.05). HUVEC cultures exposed to SWC, exhibited cell detachment and cell membrane damage detectable with trypan blue. Release of (51)Cr was fourfold compared to SW samples (P < 0.01). Signs of cell injury were evident at 15 minutes and did not change over the next 4 hours. No protective effects of ROS scavengers were demonstrated. Independent of ROS, SWC generated an immediate cell injury, which can explain, for example, vessel wall perturbation described in relation to SW treatments and trauma. Copyright 2002 Wiley-Liss, Inc.

Extract of Artemisia lavandulaefolia Inhibits In Vitro Angiogenesis in Human Umbilical Vein Endothelial Cells

PubMed Central

Yi, Eui-Yeun; Han, Kyung-Suk; Kim, Yung-Jin

2014-01-01

Angiogenesis is important processes for tumor growth and metastasis. Anti-angiogenesis target therapy has recently been known to be new anti-cancer therapeutic strategies. Natural products such as traditional medicine comprise a major source of angiogenesis inhibitors. Artemisia lavandulaefolia has been known to use in the traditional medical practices. However, its molecular mechanism on the tumor protection and therapy was not clearly elucidated. In this study, we investigated the possibility that extract of A. lavandulaefolia inhibits in vitro angiogenesis. Therefore, we examined the effect of extract of A. lavandulaefolia on the vascular network formation of human umbilical vein endothelial cells (HUVECs). We found that the treatment of A. lavandulaefolia extract suppressed the tube formation of HUVECs without any influence on the viability of HUVECs. In addition, extract of A. lavandulaefolia inhibited the migration and invasion of HUVECs. These results suggest that extract of A. lavandulaefolia could be act for an angiogenic inhibitor. PMID:25574458

Lipid-Polymer Nanoparticles Encapsulating Curcumin for Modulating the Vascular Deposition of Breast Cancer Cells

PubMed Central

Palange, Anna L.; Di Mascolo, Daniele; Carallo, Claudio; Gnasso, Agostino; Decuzzi, Paolo

2014-01-01

Vascular adhesion and endothelial transmigration are critical steps in the establishment of distant metastasis by circulating tumor cells (CTCs). Also, vascular inflammation plays a pivotal role in steering CTCs out of the blood stream. Here, long circulating lipid-polymer nanoparticles encapsulating curcumin (NANOCurc) are proposed for modulating the vascular deposition of CTCs. Upon treatment with NANOCurc, the adhesion propensity of highly metastatic breast cancer cells (MDA-MB-231) onto TNF-α stimulated endothelial cells (HUVECs) reduces by ~ 70%, in a capillary flow. Remarkably, the CTC vascular deposition already reduces up to ~ 50% by treating solely the inflamed HUVECs. The CTC arrest is mediated by the interaction between ICAM-1 on HUVECs and MUC-1 on cancer cells, and moderate doses of curcumin down-regulate the expression of both molecules. This suggests that NANOCurc could prevent metastasis and limit the progression of the disease by modulating vascular inflammation and impairing the CTC arrest. PMID:24566270

Bosutinib, dasatinib, imatinib, nilotinib, and ponatinib differentially affect the vascular molecular pathways and functionality of human endothelial cells.

PubMed

Gover-Proaktor, Ayala; Granot, Galit; Pasmanik-Chor, Metsada; Pasvolsky, Oren; Shapira, Saar; Raz, Oshrat; Raanani, Pia; Leader, Avi

2018-05-09

The tyrosine kinase inhibitors (TKIs), nilotinib, ponatinib, and dasatinib (but not bosutinib or imatinib), are associated with vascular adverse events (VAEs) in chronic myeloid leukemia (CML). Though the mechanism is inadequately understood, an effect on vascular cells has been suggested. We investigated the effect of imatinib, nilotinib, dasatinib, bosutinib, and ponatinib on tube formation, cell viability, and gene expression of human vascular endothelial cells (HUVECs). We found a distinct genetic profile in HUVECs treated with dasatinib, ponatinib, and nilotinib compared to bosutinib and imatinib, who resembled untreated samples. However, unique gene expression and molecular pathway alterations were detected between dasatinib, ponatinib, and nilotinib. Angiogenesis/blood vessel-related pathways and HUVEC function (tube formation/viability) were adversely affected by dasatinib, ponatinib, and nilotinib but not by imatinib or bosutinib. These results correspond to the differences in VAE profiles of these TKIs, support a direct effect on vascular cells, and provide direction for future research.

UL74 of Human Cytomegalovirus Contributes to Virus Release by Promoting Secondary Envelopment of Virions▿ †

PubMed Central

Jiang, Xiao Jing; Adler, Barbara; Sampaio, Kerstin Laib; Digel, Margarete; Jahn, Gerhard; Ettischer, Nicole; Stierhof, York-Dieter; Scrivano, Laura; Koszinowski, Ulrich; Mach, Michael; Sinzger, Christian

2008-01-01

The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC. PMID:18184717

Progesterone increases nitric oxide synthesis in human vascular endothelial cells through activation of membrane progesterone receptor-α.

PubMed

Pang, Yefei; Dong, Jing; Thomas, Peter

2015-05-15

Progesterone exerts beneficial effects on the human cardiovascular system by inducing rapid increases in nitric oxide (NO) production in vascular endothelial cells, but the receptors mediating these nongenomic progesterone actions remain unclear. Using human umbilical vein endothelial cells (HUVECs) as a model, we show that progesterone binds to plasma membranes of HUVECs with the characteristics of membrane progesterone receptors (mPRs). The selective mPR agonist Org OD 02-0 had high binding affinity for the progesterone receptor on HUVEC membranes, whereas nuclear PR (nPR) agonists R5020 and medroxyprogesterone acetate displayed low binding affinities. Immunocytochemical and Western blot analyses confirmed that mPRs are expressed in HUVECs and are localized on their plasma membranes. NO levels increased rapidly after treatment with 20 nM progesterone, Org OD 02-0, and a progesterone-BSA conjugate but not with R5020, suggesting that this progesterone action is at the cell surface and initiated through mPRs. Progesterone and Org OD 02-0 (20 nM) also significantly increased endothelial nitric oxide synthase (eNOS) activity and eNOS phosphorylation. Knockdown of mPRα expression by treatment with small-interfering RNA (siRNA) blocked the stimulatory effects of 20 nM progesterone on NO production and eNOS phosphorylation, whereas knockdown of nPR was ineffective. Treatment with PI3K/Akt and MAP kinase inhibitors blocked the stimulatory effects of progesterone, Org OD 02-0, and progesterone-BSA on NO production and eNOS phosphorylation and also prevented progesterone- and Org OD 02-0-induced increases in Akt and ERK phosphorylation. The results suggest that progesterone stimulation of NO production in HUVECs is mediated by mPRα and involves signaling through PI3K/Akt and MAP kinase pathways. Copyright © 2015 the American Physiological Society.

Organizational behavior of human umbilical vein endothelial cells

PubMed Central

1982-01-01

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization. PMID:6813338

Efficient generation of endothelial cells from human pluripotent stem cells and characterization of their functional properties.

PubMed

Song, Wei; Kaufman, Dan S; Shen, Wei

2016-03-01

Although endothelial cells (ECs) have been derived from human pluripotent stem cells (hPSCs), large-scale generation of hPSC-ECs remains challenging and their functions are not well characterized. Here we report a simple and efficient three-stage method that allows generation of approximately 98 and 9500 ECs on day 16 and day 34, respectively, from each human embryonic stem cell (hESC) input. The functional properties of hESC-ECs derived in the presence and absence of a TGFβ-inhibitory molecule SB431542 were characterized and compared with those of human umbilical vein endothelial cells (HUVECs). Confluent monolayers formed by SB431542 + hESC-ECs, SB431542 - hESC-ECs, and HUVECs showed similar permeability to 10,000 Da dextran, but these cells exhibited striking differences in forming tube-like structures in 3D fibrin gels. The SB431542 + hESC-ECs were most potent in forming tube-like structures regardless of whether VEGF and bFGF were present in the medium; less potent SB431542 - hESC-ECs and HUVECs responded differently to VEGF and bFGF, which significantly enhanced the ability of HUVECs to form tube-like structures but had little impact on SB431542 - hESC-ECs. This study offers an efficient approach to large-scale hPSC-EC production and suggests that the phenotypes and functions of hPSC-ECs derived under different conditions need to be thoroughly examined before their use in technology development. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 678-687, 2016. © 2015 Wiley Periodicals, Inc.

TSLP promotes angiogenesis of human umbilical vein endothelial cells by strengthening the crosstalk between cervical cancer cells and eosinophils.

PubMed

Zhang, Bing; Wei, Chun-Yan; Chang, Kai-Kai; Yu, Jia-Jun; Zhou, Wen-Jie; Yang, Hui-Li; Shao, Jun; Yu, Jin-Jin; Li, Ming-Qing; Xie, Feng

2017-12-01

Our previous study demonstrated that thymic stromal lymphopoietin (TSLP) secreted by cervical cancer cells promotes angiogenesis and recruitment, and regulates the function of eosinophils (EOS). However, the function of TSLP in the crosstalk between EOS and vascular endothelial cells in cancer lesions remains unknown. The aim of the present study was to investigate the effect of EOS caused by TSLP in in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). The results of the present study revealed that recombinant human TSLP protein (rhTSLP) increased the secretion of vascular endothelial growth factor (VEGF), but not fibroblast growth factors, in HL-60-eosinophils (HL-60E). Compared with cervical cancer cells (HeLa or CasKi cells) or HL-60E alone, there were increased levels of interleukin (IL)-8 and VEGF in the co-culture system between cervical cancer cells, and HL-60E cells. This effect was strengthened by rhTSLP, but inhibited by inhibiting the TSLP signal with anti-human TSLP or TSLP receptor neutralizing antibodies. The results of the tube formation assays revealed that treatment with the supernatant from cervical cancer cells and/or HL-60E resulted in an increase in angiogenesis in HUVECs, which could be decreased by TSLP or TSLPR inhibitors. The results of the present study suggested that TSLP derived of cervical cancer cells may indirectly stimulate angiogenesis of HUVECs, by upregulating IL-8 and VEGF production, in a co-culture model between cervical cancer cells and EOS, therefore promoting the development of cervical cancer.

Curcumin inhibits activation induced by urban particulate material or titanium dioxide nanoparticles in primary human endothelial cells

PubMed Central

Montiel-Dávalos, Angélica; Silva Sánchez, Guadalupe Jazmin; Huerta-García, Elizabeth; Rueda-Romero, Cristhiam; Soca Chafre, Giovanny; Mitre-Aguilar, Irma B.; Alfaro-Moreno, Ernesto; Pedraza-Chaverri, José

2017-01-01

Curcumin has protective effects against toxic agents and shows preventive properties for various diseases. Particulate material with an aerodynamic diameter of ≤10 μm (PM10) and titanium dioxide nanoparticles (TiO2-NPs) induce endothelial dysfunction and activation. We explored whether curcumin is able to attenuate different events related to endothelial activation. This includes adhesion, expression of adhesion molecules and oxidative stress induced by PM10 and TiO2-NPs. Human umbilical vein endothelial cells (HUVEC) were treated with 1, 10 and 100 μM curcumin for 1 h and then exposed to PM10 at 3 μg/cm2 or TiO2-NPs at 10 μg/cm2. Cell adhesion was evaluated by co-culture with U937 human myelomonocytic cells. Adhesion molecules expression was measured by flow cytometry after 3 or 24 h of exposure. Oxidative stress was determined by 2,7-dichlorodihydrofluorescein (H2DCF) oxidation. PM10 and TiO2-NPs induced the adhesion of U937 cells and the expression of E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1). The expression of E- and P-selectins matched the adhesion of monocytes to HUVEC after 3 h. In HUVEC treated with 1 or 10 μM curcumin, the expression of adhesion molecules and monocytes adhesion was significantly diminished. Curcumin also partially reduced the H2DCF oxidation induced by PM10 and TiO2-NPs. Our results suggest an anti-inflammatory and antioxidant role by curcumin attenuating the activation caused on endothelial cells by exposure to particles. Therefore, curcumin could be useful in the treatment of diseases where an inflammatory process and endothelial activation are involved. PMID:29244817

Guanylyl cyclase-dependent chemotaxis of endothelial cells in response to nitric oxide gradients.

PubMed

Isenberg, Jeff S; Ridnour, Lisa A; Thomas, Douglas D; Wink, David A; Roberts, David D; Espey, Michael Graham

2006-03-15

Nitric oxide (NO) is an important regulator of angiogenesis and neovascularization. The nature of endothelial cell motility responses to NO was examined using a Boyden chamber method. NO generated via decomposition of either DEA/NO or DETA/NO produced increases in human umbilical vein endothelial cell (HUVEC) chemotaxis, which were completely abrogated by ODQ, a soluble guanylyl cyclase inhibitor. Measurements of NO either directly by chemiluminescence or its chemistry with diaminofluorescein revealed that chemotaxis was driven by subtle NO gradients between the lower and the upper wells in this system. In addition to diffusion and volatilization from the upper chambers, the data showed that HUVEC consumption of NO contributed to these sustained gradients. Comparison of DEA/NO- and DETA/NO-mediated responses suggested that the persistence of spatial NO gradients is as significant as the absolute magnitude of NO exposure per unit time. The findings suggest that subnanomolar NO gradients are sufficient to mobilize endothelial cell migration into hypoxic tissue during neovascularization events, such as in wound healing and cancer.

HSP27 Inhibits Homocysteine-Induced Endothelial Apoptosis by Modulation of ROS Production and Mitochondrial Caspase-Dependent Apoptotic Pathway.

PubMed

Tian, Xin; Zhao, Lei; Song, Xianjing; Yan, Youyou; Liu, Ning; Li, Tianyi; Yan, Bingdi; Liu, Bin

2016-01-01

Objectives. Elevated plasma homocysteine (Hcy) could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. Heat shock protein 27 (HSP27), a small heat shock protein, is reported to exert protective effect against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell apoptosis in human umbilical vein endothelial cells (HUVECs) and to determine the underlying mechanisms. Methods. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) of normal or HSP27-overexpressing HUVECs in the presence of Hcy were analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results. We found that Hcy could induce cell apoptosis with corresponding decrease of nitric oxide (NO) level, increase of endothelin-1 (ET-1), intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels, elevation of ROS, and dissipation of MMP. In addition, HSP27 could protect the cell against Hcy-induced apoptosis and inhibit the effect of Hcy on HUVECs. Furthermore, HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 activity. Conclusions. Therefore, we concluded that HSP27 played a protective role against Hcy-induced endothelial apoptosis through modulation of ROS production and the mitochondrial caspase-dependent apoptotic pathway.

Alginate-gelatin encapsulation of human endothelial cells promoted angiogenesis in in vivo and in vitro milieu.

PubMed

Nemati, Sorour; Rezabakhsh, Aysa; Khoshfetrat, Ali Baradar; Nourazarian, Alireza; Biray Avci, Çığır; Goker Bagca, Bakiye; Alizadeh Sardroud, Hamed; Khaksar, Majid; Ahmadi, Mahdi; Delkhosh, Aref; Sokullu, Emel; Rahbarghazi, Reza

2017-12-01

Up to present, many advantages have been achieved in the field of cell-based therapies by applying sophisticated methodologies and delivery approaches. Microcapsules are capable to provide safe microenvironment for cells during transplantation in a simulated physiological 3D milieu. Here, we aimed to investigate the effect of alginate-gelatin encapsulation on angiogenic behavior of human endothelial cells over a period of 5 days. Human umbilical vein endothelial cells were encapsulated by alginate-gelatin substrate and incubated for 5 days. MTT and autophagy PCR array analysis were used to monitor cell survival rate. For in vitro angiogenesis analysis, cell distribution of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 were detected by ELISA. In addition to in vitro tubulogenesis assay, we monitored the expression of VE-cadherin by Western blotting. The migration capacity of encapsulated HUVECs was studied by measuring MMP-2 and MMP-9 via gelatin zymography. The in vivo angiogenic potential of encapsulated HUVECs was analyzed in immune-compromised mouse implant model during 7 days post-transplantation. We demonstrated that encapsulation promoted HUVECs cell survival and proliferation. Compared to control, no significant differences were observed in autophagic status of encapsulated cells (p > 0.05). The level of Tie-1, Tie-2, VEGFR-1, and VEGFR-2 were increased, but did not reach to significant levels. Encapsulation decreased MMP-2, -9 activity and increased the VE-cadherin level in enclosed cells (p < 0.05). Moreover, an enhanced in vivo angiogenic response of encapsulated HUVECs was evident as compared to non-capsulated cells (p < 0.05). These observations suggest that alginate-gelatin encapsulation can induce angiogenic response in in vivo and in vitro conditions. © 2017 Wiley Periodicals, Inc.

MiR-216a: a link between endothelial dysfunction and autophagy

PubMed Central

Menghini, R; Casagrande, V; Marino, A; Marchetti, V; Cardellini, M; Stoehr, R; Rizza, S; Martelli, E; Greco, S; Mauriello, A; Ippoliti, A; Martelli, F; Lauro, R; Federici, M

2014-01-01

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis. PMID:24481443

Angiogenic properties of endometrial mesenchymal stromal cells in endothelial co-culture: an in vitro model of endometriosis.

PubMed

Canosa, S; Moggio, A; Brossa, A; Pittatore, G; Marchino, G L; Leoncini, S; Benedetto, C; Revelli, A; Bussolati, B

2017-03-01

Can endometrial mesenchymal stromal cells (E-MSCs) differentiate into endothelial cells in an in vitro co-culture system with human umbilical vein endothelial cells (HUVECs)? E-MSCs can acquire endothelial markers and function in a direct co-culture system with HUVECs. E-MSCs have been identified in the human endometrium as well as in endometriotic lesions. E-MSCs appear to be involved in formation of the endometrial stromal vascular tissue and the support of tissue growth and vascularization. The use of anti-angiogenic drugs appears as a possible therapeutic strategy against endometriosis. This is an in vitro study comprising patients receiving surgical treatment of ovarian endometriosis (n = 9). E-MSCs were isolated from eutopic and ectopic endometrial tissue and were characterized for the expression of mesenchymal and endothelial markers by FACS analysis and Real-Time PCR. CD31 acquisition was evaluated by FACS analysis and immunofluorescence after a 48 h-direct co-culture with green fluorescent protein +-HUVECs. A tube-forming assay was set up in order to analyze the functional potential of their interaction. Finally, co-cultures were treated with the anti-angiogenic agent Cabergoline. A subpopulation of E-MSCs acquired CD31 expression and integrated into tube-like structures when directly in contact with HUVECs, as observed by both FACS analysis and immunofluorescence. The isolation of CD31+ E-MSCs revealed significant increases in CD31, vascular endothelial growth factor receptor 2, TEK receptor tyrosine kinase and vascular endothelial-Cadherin mRNA expression levels with respect to basal and to CD31neg cells (P < 0.05). On the other hand, the expression of mesenchymal genes such as c-Myc, Vimentin, neuronal-Cadherin and sushi domain containing 2 remained unchanged. Cabergoline treatment induced a significant reduction of the E-MSC angiogenic potential (P < 0.05 versus control). Not applicable. Further studies are necessary to investigate the cellular and molecular mechanisms underlying the endothelial cell differentiation. E-MSCs may undergo endothelial differentiation, and be potentially involved in the development of endometriotic implants. Cell culture systems that more closely mimic the cellular complexity typical of endometriotic tissues in vivo are required to develop novel strategies for treatment. This study was supported by the 'Research Fund ex-60%', University of Turin, Turin, Italy. All authors declare that their participation in the study did not involve actual or potential conflicts of interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

The Interplay of Dental Pulp Stem Cells and Endothelial Cells in an Injectable Peptide Hydrogel on Angiogenesis and Pulp Regeneration In Vivo

PubMed Central

Dissanayaka, Waruna Lakmal; Hargreaves, Kenneth M.; Jin, Lijian; Samaranayake, Lakshman P.

2015-01-01

Securing an adequate blood supply for the survival of cell transplants is critical for a successful outcome in tissue engineering. Interactions between endothelial and progenitor/stem cells are important for vascularization of regenerating tissue. Recently, self-assembling peptide nanofibers were described as a promising environment for pulp regeneration due to their synthetic nature and controlled physicochemical properties. In this study, the peptide hydrogel PuraMatrix™ was used as a scaffold system to investigate the role of dental pulp stem cells (DPSCs) in triggering angiogenesis and the potential for regenerating vascularized pulp in vivo. Human umbilical vein endothelial cells (HUVECs), DPSCs, or cocultures of both cell types were encapsulated in three-dimensional PuraMatrix. The peptide nanofiber microenvironment supported cell survival, cell migration, and capillary network formation in the absence of exogenous growth factors. DPSCs increased early vascular network formation by facilitating the migration of HUVECs and by increasing vascular endothelial growth factor (VEGF) expression. Both the DPSC-monoculture and coculture groups exhibited vascularized pulp-like tissue with patches of osteodentin after transplantation in mice. The cocultured groups exhibited more extracellular matrix, vascularization, and mineralization than the DPSC-monocultures in vivo. The DPSCs play a critical role in initial angiogenesis, whereas coordinated efforts by the HUVECs and DPSCs are required to achieve a balance between extracellular matrix deposition and mineralization. The findings of this study also highlighted the importance of a microenvironment that supports cell–cell interactions and cell migration, which contribute to successful dental pulp regeneration. PMID:25203774

Anti-Inflammatory Activity of Marine Ovothiol A in an In Vitro Model of Endothelial Dysfunction Induced by Hyperglycemia.

PubMed

Castellano, Immacolata; Di Tomo, Pamela; Di Pietro, Natalia; Mandatori, Domitilla; Pipino, Caterina; Formoso, Gloria; Napolitano, Alessandra; Palumbo, Anna; Pandolfi, Assunta

2018-01-01

Chronic hyperglycemia is associated with oxidative stress and vascular inflammation, both leading to endothelial dysfunction and cardiovascular disease that can be weakened by antioxidant/anti-inflammatory molecules in both healthy and diabetic subjects. Among natural molecules, ovothiol A, produced in sea urchin eggs to protect eggs/embryos from the oxidative burst at fertilization and during development, has been receiving increasing interest for its use as an antioxidant. Here, we evaluated the potential antioxidative/anti-inflammatory effect of purified ovothiol A in an in vitro cellular model of hyperglycemia-induced endothelial dysfunction employing human umbilical vein endothelial cells (HUVECs) from women affected by gestational diabetes (GD) and from healthy mothers. Ovothiol A was rapidly taken up by both cellular systems, resulting in increased glutathione values in GD-HUVECs, likely due to the formation of reduced ovothiol A. In tumor necrosis factor- α -stimulated cells, ovothiol A induced a downregulation of adhesion molecule expression and decrease in monocyte-HUVEC interaction. This was associated with a reduction in reactive oxygen and nitrogen species and an increase in nitric oxide bioavailability. These results point to the potential antiatherogenic properties of the natural antioxidant ovothiol A and support its therapeutic potential in pathologies related to cardiovascular diseases associated with oxidative/inflammatory stress and endothelial dysfunction.

Terrestrosin D, a steroidal saponin from Tribulus terrestris L., inhibits growth and angiogenesis of human prostate cancer in vitro and in vivo.

PubMed

Wei, Shihu; Fukuhara, Hideo; Chen, Guang; Kawada, Chiaki; Kurabayashi, Atsushi; Furihata, Mutsuo; Inoue, Keiji; Shuin, Taro

2014-01-01

The aim of this study was to investigate whether terrestrosin D (TED) inhibits the progression of castration-resistant prostate cancer and consider its mechanism. Cell cycle, mitochondrial membrane potential (ΔΨm) and apoptosis were determined by flow cytometry. Caspase-3 activity and vascular endothelial growth factor secretion were detected by a caspase-3 assay and human vascular endothelial growth factor kit, respectively. A PC-3 xenograft mouse model was used to evaluate the anticancer effect of TED in vivo. In vitro, TED strongly suppressed the growth of prostate cancer cells and endothelial cells in a dose-dependent manner. TED induced cell cycle arrest and apoptosis in PC-3 cells and human umbilical vascular endothelial cells (HUVECs). TED-induced apoptosis did not involve the caspase pathway. TED also decreased ΔΨm in PC-3 cells and HUVECs. In vivo, TED significantly suppressed tumor growth in nude mice bearing PC-3 cells, without any overt toxicity. Immunohistochemical analysis showed TED induced apoptotic cell death and inhibited angiogenesis in xenograft tumor cells. Cell cycle arrest and induction of apoptosis in cancer cells and endothelial cells might be plausible mechanisms of actions for the observed antitumor and antiangiogenic activities of TED. © 2014 S. Karger AG, Basel.

A butyrolactone derivative suppressed lipopolysaccharide-induced autophagic injury through inhibiting the autoregulatory loop of p8 and p53 in vascular endothelial cells.

PubMed

Meng, Ning; Zhao, Jing; Su, Le; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

2012-02-01

Lipopolysaccharide (LPS)-induced vascular endothelial cell (VEC) dysfunction is an important contributing factor in vascular diseases. Recently, we found that LPS impaired VEC by inducing autophagy. Our previous researches showed that a butyrolactone derivative, 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) selectively protected VEC function. The objective of the present study is to investigate whether and how 3BDO inhibits LPS-induced VEC autophagic injury. Our results showed that LPS induced autophagy and led to increase of reactive oxygen species (ROS) and decrease of mitochondrial membrane potential (MMP) in Human umbilical vein vascular endothelial cells (HUVECs). Furthermore, LPS significantly increased p8 and p53 protein levels and the nuclear translocation of p53. All of these effects of LPS on HUVECs were strongly inhibited by 3BDO. Importantly, the ROS scavenger N-acetylcysteine (NAC) could inhibited LPS-induced autophagy and knockdown of p8 by RNA interference inhibited the autophagy, p53 protein level increase, the translocation of p53 into nuclei and the ROS level increase induced by LPS in HUVECs. The data suggested that 3BDO inhibited LPS-induced autophagy in HUVECs through inhibiting the ROS overproduction, the increase of p8 and p53 expression and the nuclear translocation of p53. Our findings provide a potential tool for understanding the mechanism underlying LPS-induced autophagy in HUVECs and open the door to a novel therapeutic drug for LPS-induced vascular diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells.

PubMed

Izuta, Hiroshi; Shimazawa, Masamitsu; Tsuruma, Kazuhiro; Araki, Yoko; Mishima, Satoshi; Hara, Hideaki

2009-11-17

Vascular endothelial growth factor (VEGF) is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ), bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs). In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE)]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis > bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

Caveolae and caveolin-1 mediate endocytosis and transcytosis of oxidized low density lipoprotein in endothelial cells

PubMed Central

Sun, Shao-wei; Zu, Xu-yu; Tuo, Qin-hui; Chen, Lin-xi; Lei, Xiao-yong; Li, Kai; Tang, Chao-ke; Liao, Duan-fang

2010-01-01

Aim: To explore the mechanisms involved in ox-LDL transcytosis across endothelial cells and the role of caveolae in this process. Methods: An in vitro model was established to investigate the passage of oxidized low density lipoprotein (ox-LDL) through a tight monolayer of human umbilical vein endothelial cells (HUVEC) cultured on a collagen-coated filter. Passage of DiI-labeled ox-LDL through the monolayer was measured using a fluorescence spectrophotometer. The uptake and efflux of ox-LDL by HUVEC were determined using fluorescence microscopy and HPLC. Results: Caveolae inhibitors – carrageenan (250 μg/mL), filipin (5 μg/mL), and nocodazole (33 μmol/L)–decreased the transport of ox-LDL across the monolayer by 48.9%, 72.4%, and 79.8% as compared to the control group. In addition, they effectively decreased ox-LDL uptake and inhibited the efflux of ox-LDL. Caveolin-1 and LOX-1 were up-regulated by ox-LDL in a time-dependent manner and decreased gradually after depletion of ox-LDL (P<0.05). After treatment HUVEC with ox-LDL and silencing caveolin-1, NF-κB translocation to the nucleus was blocked and LOX-1 expression decreased (P<0.05). Conclusion: Caveolae can be a carrier for ox-LDL and may be involved in the uptake and transcytosis of ox-LDL by HUVEC. PMID:20835266

Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis

PubMed Central

Xiong, Si-qi; Jiang, Hai-bo; Li, Yan-xiu; Li, Hai-bo; Xu, Hui-zhuo; Wu, Zhen-kai; Zheng, Wei

2016-01-01

Objective To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. Methods siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor–reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. Results The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. Conclusions Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF. PMID:27994436

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

PubMed

Leckel, K; Beecken, W-D; Jonas, D; Oppermann, E; Coman, M C; Beck, K-F; Cinatl, J; Hailer, N P; Auth, M K H; Bechstein, W O; Shipkova, M; Blaheta, R A

2003-11-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

Endothelial cellular senescence is inhibited by nitric oxide: Implications in atherosclerosis associated with menopause and diabetes

PubMed Central

Hayashi, Toshio; Matsui-Hirai, Hisako; Miyazaki-Akita, Asaka; Fukatsu, Akiko; Funami, Jun; Ding, Qun-Fang; Kamalanathan, Sumitra; Hattori, Yuichi; Ignarro, Louis J.; Iguchi, Akihisa

2006-01-01

Senescence may contribute to the pathogenesis of atherosclerosis. Although the bioavailability of nitric oxide (NO) is limited in senescence, the effect of NO on senescence and its relationship to cardiovascular risk factors have not been investigated fully. We studied these factors by investigating senescence-associated β-galactosidase (SA-β-gal) and human telomerase activity in human umbilical venous endothelial cells (HUVECs). Treatment with NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) and transfection with endothelial NO synthase (eNOS) into HUVECs each decreased the number of SA-β-gal positive cells and increased telomerase activity. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) abolished the effect of eNOS transfection. The physiological concentration of 17β-estradiol activated hTERT, decreased SA-β-gal-positive cells, and caused cell proliferation. However, ICI 182780, an estrogen receptor-specific antagonist, and l-NAME each inhibited these effects. Finally, we investigated the effect of NO bioavailability on high glucose-promoted cellular senescence of HUVECs. Inhibition by eNOS transfection of this cellular senescence under high glucose conditions was less pronounced. Treatment with l-arginine or l-citrulline of eNOS-transfected cells partially inhibited, and combination of l-arginine and l-citrulline with antioxidants strongly prevented, high glucose-induced cellular senescence. These data demonstrate that NO can prevent endothelial senescence, thereby contributing to the anti-senile action of estrogen. The ingestion of NO-boosting substances, including l-arginine, l-citrulline, and antioxidants, can delay endothelial senescence under high glucose. We suggest that the delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the treatment of atherosclerosis in the elderly. PMID:17075048

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

PubMed Central

LECKEL, K; BEECKEN, W-D; JONAS, D; OPPERMANN, E; COMAN, M C; BECK, K-F; CINATL, J; HAILER, N P; AUTH, M K H; BECHSTEIN, W O; SHIPKOVA, M; BLAHETA, R A

2003-01-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell–endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 µm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 µm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy. PMID:14616783

Magnolol suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting Ras-dependent mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling pathways.

PubMed

Kim, Ki Mo; Kim, No Soo; Kim, Jinhee; Park, Jong-Shik; Yi, Jin Mu; Lee, Jun; Bang, Ok-Sun

2013-01-01

Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to possess anticancer activity. Recent studies have also demonstrated that magnolol inhibits cell growth and induces the apoptosis of cancer cells. However, the effects of magnolol on vascular endothelial growth factor (VEGF)-induced angiogenesis in endothelial cells have not been studied. In the present study, we have used human umbilical vein endothelial cells (HUVECs) to investigate the antiangiogenic effect and molecular mechanism of magnolol. Magnolol inhibited the VEGF-induced proliferation, chemotactic motility and tube formation of HUVECs in vitro as well as the vessel sprouting of the aorta ex vivo. Furthermore, magnolol inhibited VEGF-induced Ras activation and subsequently suppressed extracellular signal-regulated kinase (ERK), phosphatidylinositol-3-kinase (PI3K)/Akt and p38, but not Src and focal adhesion kinase (FAK). Interestingly, the knockdown of Ras by short interfering RNA produced inhibitory effects that were similar to the effects of magnolol on VEGF-induced angiogenic signaling events, such as ERK and Akt/eNOS activation, and resulted in the inhibition of proliferation, migration, and vessel sprouting in HUVECs. In combination, these results demonstrate that magnolol is an inhibitor of angiogenesis and suggest that this compound could be a potential candidate in the treatment of angiogenesis-related diseases.

Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chen-Si; School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan; He, Pei-Juin

2010-06-25

Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cellsmore » and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLC{beta}2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLC{beta}2/Ca2+ signal transduction in endothelial cells.« less

Endothelial Cell Bioenergetics and Mitochondrial DNA Damage Differ in Humans Having African or West Eurasian Maternal Ancestry

PubMed Central

Krzywanski, David M.; Moellering, Douglas R.; Westbrook, David G.; Dunham-Snary, Kimberly J.; Brown, Jamelle; Bray, Alexander W.; Feeley, Kyle P.; Sammy, Melissa J.; Smith, Matthew R.; Schurr, Theodore G.; Vita, Joseph A.; Ambalavanan, Namasivayam; Calhoun, David; Dell’Italia, Louis; Ballinger, Scott W.

2016-01-01

Background We hypothesized that endothelial cells having distinct mitochondrial genetic backgrounds would show variation in mitochondrial function and oxidative stress markers concordant with known differential cardiovascular disease susceptibilities. To test this hypothesis, mitochondrial bioenergetics were determined in endothelial cells from healthy individuals with African versus European maternal ancestries. Methods and Results Bioenergetics and mitochondrial DNA (mtDNA) damage were assessed in single donor human umbilical vein endothelial cells (HUVECs) belonging to mtDNA haplogroups H and L, representing West Eurasian and African maternal ancestry, respectively. HUVECs from haplogroup L utilized less oxygen for ATP production and had increased levels of mtDNA damage compared to those in haplogroup H. Differences in bioenergetic capacity were also observed in that HUVECs belonging to haplogroup L had decreased maximal bioenergetic capacities compared to haplogroup H. Analysis of peripheral blood mononuclear cells from age-matched healthy controls with West Eurasian or African maternal ancestries showed that haplogroups sharing an A to G mtDNA mutation at nucleotide pair (np) 10,398 had increased mtDNA damage compared to those lacking this mutation. Further study of angiographically proven coronary artery disease patients and age-matched healthy controls revealed that mtDNA damage was associated with vascular function and remodeling, and that age of disease onset was later in individuals from haplogroups lacking the A to G mutation at np 10,398. Conclusions Differences in mitochondrial bioenergetics and mtDNA damage associated with maternal ancestry may contribute to endothelial dysfunction and vascular disease. PMID:26787433

Vascular protective effects of aqueous extracts of Tribulus terrestris on hypertensive endothelial injury.

PubMed

Jiang, Yue-Hua; Guo, Jin-Hao; Wu, Sai; Yang, Chuan-Hua

2017-08-01

Angiotensin II (Ang II) is involved in endothelium injury during the development of hypertension. Tribulus terrestris (TT) is used to treat hypertension, arteriosclerosis, and post-stroke syndrome in China. The present study aimed to determine the effects of aqueous TT extracts on endothelial injury in spontaneously hypertensive rats (SHRs) and its protective effects against Ang II-induced injury in human umbilical vein endothelial cells (HUVECs). SHRs were administered intragastrically with TT (17.2 or 8.6 g·kg -1 ·d -1 ) for 6 weeks, using valsartan (13.5 mg·kg -1 ·d -1 ) as positive control. Blood pressure, heart rate, endothelial morphology of the thoracic aorta, serum levels of Ang II, endothelin-1 (ET-1), superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The endothelial injury of HUVECs was induced by 2 × 10 -6 mol·L -1 Ang II. Cell Apoptosisapoptosis, intracellular reactive oxygen species (ROS) was assessed. Endothelial nitric oxide synthase (eNOS), ET-1, SOD, and MDA in the cell culture supernatant and cell migration were assayed. The expression of hypertension-linked genes and proteins were analyzed. TT decreased systolic pressure, diastolic pressure, mean arterial pressure and heart rate, improved endothelial integrity of thoracic aorta, and decreased serum leptin, Ang II, ET-1, NPY, and Hcy, while increased NO in SHRs. TT suppressed Ang II-induced HUVEC proliferation and apoptosis and prolonged the survival, and increased cell migration. TT regulated the ROS, and decreased mRNA expression of Akt1, JAK2, PI3Kα, Erk2, FAK, and NF-κB p65 and protein expression of Erk2, FAK, and NF-κB p65. In conclusion, TT demonstrated anti-hypertensive and endothelial protective effects by regulating Erk2, FAK and NF-κB p65. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Impact of simulated microgravity on the secretory and adhesive activity of cultured human vascular endothelial cells.

NASA Astrophysics Data System (ADS)

Rudimov, Evgeny; Buravkova, Ludmila; Pogodina, Margarita; Andrianova, Irina

The layer of vascular endothelial cells (ECs) is a dynamic,disseminated organ that perform the function of an interface between the blood and vascular wall. The endothelial monolayer is able to quickly respond to changes in the microenvironment due to its synthesis of vasoactive substances, chemokines, adhesion molecules expression, etc. ECs are highly sensitive to gravitational changes and capable of short-term and long-term responses (Sangha et al., 2001; Buravkova et al., 2005; Infanger et al., 2006, 2007. However, the question remains how to reflect the impact of microgravity on endothelium under the inflammatory process. Therefore, the aim of this study was to investigate secretory and adhesive activity of human umbilical vein endothelial cells (HUVECs) during simulated microgravity and TNF-a activation. HUVECs were isolated according to Gimbrone et al. (1978) in modification A. Antonov (1981) and used for experiments at 2-4 passages. HUVECs were activated by low level of TNF-a (2 ng/ml). Microgravity was generated by Random Positioning Machine (RPM, Dutch Space, Leiden) placed into the thermostat at 37°C. After 24 hours of clinorotation we measured adhesion molecules expression on the cell surface (ICAM-1, VCAM-1, PECAM-1, E-selectin, CD144, endoglin (CD105)) and cell viability using a flow cytometry. To evaluate the level of target gene expression was used the real time RT-PCR. IL-6 and IL-8 concentration was measured in the conditioned medium of HUVECs by using the ELISA test. We found that simulated microgravity within 24 hours caused a decrease of ICAM-1, CD144, and E-selectin expression, at the same time not affect the cell viability, endoglin and PECAM-1 expression on the surface HUVEC. Furthermore, there were no changes of the level of IL-6 and IL-8 gene expression and their products in the culture medium. TNF-activated HUVECs showed an increase in gene expression of interleukins and molecules involved in the adhesion process, which also was confirmed by the higher level of cytokines in the medium and elevated share of CD144, ICAM-1 and VCAM-1-positive cells. Comparative analysis of the level TNF-induced secretion of IL-6 and IL-8, as well as the share of cells bearing ICAM-1 and VCAM-1, showed significant variability depending on the donors. Simultaneous exposure to simulated microgravity and proinflammatory activation did not potentiate and did not cancel the effect caused by TNF-a. In summary, our findings indicate that the simulated microgravity is not activating and additional pro-inflammatory stimulus to HUVEC in vitro model. This work was supported in part by Grant from RFBR No.12-04-31763 and Grant No.NSh-371.2014.4

Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells

PubMed Central

2012-01-01

Background Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro. Methods In human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation and on NO production. We also explored the impact of exogenous FABP4 on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt). Results We found that eNOS expression and activation and NO production are significantly inhibited by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4 induces endothelial dysfunction by inhibiting the activation of the insulin-signalling pathway resulting in decreased eNOS activation and NO production. Conclusion These findings provide a mechanistic linkage between FABP4 and impaired endothelial function in diabetes, which leads to an increased cardiovascular risk. PMID:22709426

A single-cell analysis platform for electrochemiluminescent detection of platelets adhesion to endothelial cells based on Au@DL-ZnCQDs nanoprobes.

PubMed

Long, Dongping; Shang, Yunfei; Qiu, Youyi; Zhou, Bin; Yang, Peihui

2018-04-15

A novel single-cell analysis platform (SCA) was developed for the investigation of platelets adhesion to single human umbilical vein endothelial cell (HUVEC) via using the adhesion molecule (E-selectin) on the damaged HUVEC as the marker site, and integrating electrochemiluminescence (ECL) with the ultrasensitive Au@DL-ZnCQDs nanoprobes. The Au@DL-ZnCQDs nanocomposite, a kind of double layer zinc-coadsorbed carbon quantum dot (ZnCQDs) core-shell nanoprobe, was firstly constructed by using gold nanoparticles (AuNPs) as the core to load with ZnCQDs and then the citrate-modified silver nanoparticles (AgNPs) as the bridge to link AuNPs-ZnCQDs with ZnCQDs to form the core-shell with double layer ZnCQDs (DL-ZnCQDs) nanoprobe, revealed a 10-fold signal amplification. The H 2 O 2 -induced oxidative damage HUVECs were utilized as the cellular model on which anti-E-selectin functionalized nanoprobes specially recognized E-selectin, the SCA showed that the ECL signals decreased with platelets adhesion to single HUVEC. The proposed SCA could effectively and dynamically monitor the adhesion between single HUVEC and platelets in the absence and presence of collagen activation, moreover, be able to quantitatively detect the number of platelets adhesion to single HUVEC, and show a good analytical performance with linear range from 1 to 15 platelets. In contrast, the HUVEC was down-regulated the expression of adhesion molecules by treating with quercetin inhibitor, and the SCA also exhibited the feasibility for analysis of platelets adhesion to single HUVEC. Therefore, the single-cell analysis platform provided a novel and promising protocol for analysis of the single intercellular adhesion, and it will be beneficial to elucidate the pathogenesis of cardiovascular diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Endothelial Activation by Platelets from Sickle Cell Anemia Patients

PubMed Central

Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T.; Costa, Fernando Ferreira; Conran, Nicola

2014-01-01

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD. PMID:24551209

Effects of Tacrolimus or Sirolimus on the adhesion of vascular wall cells: Controlled in-vitro comparison study.

PubMed

Krüger-Genge, A; Hiebl, B; Franke, R P; Lendlein, A; Jung, F

2017-01-01

In drug eluting stents the cytostatic drugs Sirolimus or Tacrolimus are used to inhibit blood vessel restenosis by limiting the proliferation of smooth muscle cells. However, the cytostatic activity of both drugs was shown to be not cell specific and could also affect the stent endothelialisation, respectively. Currently, only limited in vitro data are available about the impact of Sirolimus and Tacrolimus on endothelial cell proliferation over a broad concentration range. To answer this question the following study was performed.Commercially obtained HUVEC were expanded with DMEM cell culture medium (GIBCO, Germany) supplemented with 5 vol% fetal calf serum on non-coated regular polystyrene-based 24-multiwell plates. For drug testings 2×104 cells/cm2 were seeded and grown for 24 h until 30-40% of the multiwell surfaces were covered and then exposed to Sirolimus (1.0×10-11 - 1.0×10-5 mol/l) or Tacrolimus (2.0×10-8 - 6.2×10-5 mol/l), both dissolved in DMSO. 12, 24 and 48 h after adding the drugs cell numbers per area were quantified by counting the cells in six wells with four fields of view per well, representing 0.6 mm2, using a confocal laser microscope.After 48 h of cell growth in the drug-free cell culture medium, the HUVEC number increased from 2.0×104 to 3.55×104 cells/cm2 (mean cell doubling time: 53.6 h, n = 6). At lower concentrations (≤2.0×10-6 mol/l) Tacrolimus reduced the number of adherent HUVEC significantly less than Sirolimus (p < 0.05). However, at higher concentrations (≥2.07×10-5 mol/l) the effect of Tacrolimus on the number of adherent endothelial cells was significantly greater than that of Sirolimus (p < 0.05). At the highest concentration applied (6.22×10-5 mol/l), Tacrolimus induced detachment of all HUVECs within 12 h after drug application. The number of adherent HUVEC decreased only slightly (about 9%) after Sirolimus application at the highest concentration (1.09×10-5 mol/l).These data show that in a non-flow model the cytostatic drug Tacrolimus reduced the number of adherent endothelial cells less than Sirolimus, as long as the drug concentration did not surpass 10-6 mol/l. At the limits of solubility, Sirolimus (1×10-5 mol/l) reduced the number of adherent endothelial cells less than Tacrolimus (6×10-5 mol/l), which induced detachment of endothelial cells.

Suppression of tumor-induced angiogenesis by taspine isolated from Radix et Rhizoma Leonticis and its mechanism of action in vitro.

PubMed

Zhang, Yanmin; He, Langchong; Meng, Liang; Luo, Wenjuan; Xu, Xuemei

2008-04-08

The present study was to demonstrate the effect of taspine isolated from Radix et Rhizoma Leonticis on tumor angiogenesis and its mechanism of action. The anti-angiogenic effect in vivo was evaluated on chicken chorioallantoic membrane (CAM) neovascularisation model and CAM transplantation tumor model. Taspine exerted inhibitory influence on CAM angiogenesis and the growth and microvessel density (MVD) of CAM transplantation tumor at concentrations of 0.5-2μg/egg. The mechanism was demonstrated through detecting vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein secretion by enzyme-linked immunosorbent assay (ELISA), as well as mRNA expression of VEGF, Flt-1 and Flk-1/KDR by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that taspine down-regulated the VEGF and bFGF secretion in human non-small cell lung cancer cell (A549 cell) and human umbilical vein endothelial cell (HUVEC), and the VEGF and Flk-1/KDR mRNA expression in HUVEC. Additionally, the effect of taspine on HUVEC migration was detected with the method of cell scrape. The result indicated that taspine inhibited HUVEC migration in a dose-dependent manner. These findings suggest that taspine might be a promising candidate as angiogenesis inhibitors.

Selected Activities of Citrus Maxima Merr. Fruits on Human Endothelial Cells: Enhancing Cell Migration and Delaying Cellular Aging

PubMed Central

Buachan, Paiwan; Chularojmontri, Linda; Wattanapitayakul, Suvara K.

2014-01-01

Endothelial injury and damage as well as accumulated reactive oxygen species (ROS) in aging play a significant role in the development of cardiovascular disease (CVD). Recent studies show an association of high citrus fruit intake with a lower risk of CVD and stroke but the mechanisms involved are not fully understood. This study investigated the effects of pummelo (Citrus maxima Merr. var. Tubtim Siam, CM) fruit extract on human umbilical vein endothelial cell (HUVECs) migration and aging. The freeze-dried powder of fruit extract was characterized for antioxidant capacity (FRAP assay) and certain natural antioxidants, including ascorbic acid, gallic acid, hesperidin, and naringin (HPLC). Short-term (48 h) co-cultivation of HUVECs with CM enhanced cell migration as evaluated by a scratch wound assay and Boyden chamber assay. A long-term treatment with CM for 35 days significantly increased HUVEC proliferation capability as indicated by population doubling level (PDL). CM also delayed the onset of aging phenotype shown by senescence-associated β-galactosidase (SA-β-gal) staining. Furthermore, CM was able to attenuate increased ROS levels in aged cells when determined by 2′,7′-dichlorodihydrofluorescein diacetate (DCDHF) while eNOS mRNA expression was increased but the eNOS protein level was not changed. Thus, further in vivo and clinical studies are warranted to support the use of pummelo as a functional fruit for endothelial health and CVD risk reduction. PMID:24763109

Selected activities of Citrus maxima Merr. fruits on human endothelial cells: enhancing cell migration and delaying cellular aging.

PubMed

Buachan, Paiwan; Chularojmontri, Linda; Wattanapitayakul, Suvara K

2014-04-21

Endothelial injury and damage as well as accumulated reactive oxygen species (ROS) in aging play a significant role in the development of cardiovascular disease (CVD). Recent studies show an association of high citrus fruit intake with a lower risk of CVD and stroke but the mechanisms involved are not fully understood. This study investigated the effects of pummelo (Citrus maxima Merr. var. Tubtim Siam, CM) fruit extract on human umbilical vein endothelial cell (HUVECs) migration and aging. The freeze-dried powder of fruit extract was characterized for antioxidant capacity (FRAP assay) and certain natural antioxidants, including ascorbic acid, gallic acid, hesperidin, and naringin (HPLC). Short-term (48 h) co-cultivation of HUVECs with CM enhanced cell migration as evaluated by a scratch wound assay and Boyden chamber assay. A long-term treatment with CM for 35 days significantly increased HUVEC proliferation capability as indicated by population doubling level (PDL). CM also delayed the onset of aging phenotype shown by senescence-associated β-galactosidase (SA-β-gal) staining. Furthermore, CM was able to attenuate increased ROS levels in aged cells when determined by 2',7'-dichlorodihydrofluorescein diacetate (DCDHF) while eNOS mRNA expression was increased but the eNOS protein level was not changed. Thus, further in vivo and clinical studies are warranted to support the use of pummelo as a functional fruit for endothelial health and CVD risk reduction.

Korean Red Ginseng water extract inhibits COX-2 expression by suppressing p38 in acrolein-treated human endothelial cells

PubMed Central

Lee, Seung Eun; Park, Yong Seek

2013-01-01

Cigarette smoke is considered a major risk factor for vascular diseases. There are many toxic compounds in cigarette smoke, including acrolein and other α,β-unsaturated aldehydes, which are regarded as mediators of inflammation and vascular dysfunction. Furthermore, recent studies have revealed that acrolein, an α,β-unsaturated aldehyde in cigarette smoke, induces inflammatory mediator expression, which is known to be related to vascular diseases. In this study, we investigated whether Korean Red Ginseng (KRG) water extract suppressed acrolein-induced cyclooxygenase (COX)-2 expression in human umbilical vein endothelial cells (HUVECs). Acrolein-induced COX-2 expression was accompanied by increased levels of phosphorylated p38 in HUVECs and KRG inhibited COX-2 expression in HUVECs. These results suggest that KRG suppresses acrolein-induced COX-2 expression via inhibition of the p38 mitogen-activated protein kinase signaling pathway. In addition, KRG exhibited an inhibitory effect on acrolein-induced apoptosis, as demonstrated by annexin V–propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Consistent with these results, KRG may exert a vasculoprotective effect through inhibition of COX-2 expression in acrolein-stimulated human endothelial cells. PMID:24558308

Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.

PubMed

Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

2017-02-17

We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2'-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

PubMed Central

Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

2017-01-01

We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2′-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity. PMID:28218663

Effects of chlorogenic acid on intracellular calcium regulation in lysophosphatidylcholine-treated endothelial cells.

PubMed

Jung, Hye-Jin; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

2017-06-01

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ([Ca 2＋ ] i ) by releasing Ca 2＋ from intracellular stores and via Ca 2＋ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced Ca 2＋ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated Ca 2＋ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis. [BMB Reports 2017; 50(6): 323-328].

Effects of chlorogenic acid on intracellular calcium regulation in lysophosphatidylcholine-treated endothelial cells

PubMed Central

Jung, Hye-Jin; Im, Seung-Soon; Song, Dae-Kyu; Bae, Jae-Hoon

2017-01-01

Lysophosphatidylcholine (LPC) is a major phospholipid component of oxidized low-density lipoprotein (ox-LDL) and is implicated in its atherogenic activity. This study investigated the effects of LPC on cell viability, intracellular calcium homeostasis, and the protective mechanisms of chlorogenic acid (CGA) in human umbilical vein endothelial cells (HUVECs). LPC increased intracellular calcium ([Ca2+]i) by releasing Ca2+ from intracellular stores and via Ca2+ influx through store-operated channels (SOCs). LPC also increased the generation of reactive oxygen species (ROS) and decreased cell viability. The mRNA expression of Transient receptor potential canonical (TRPC) channel 1 was increased significantly by LPC treatment and suppressed by CGA. CGA inhibited LPC-induced Ca2+ influx and ROS generation, and restored cell viability. These results suggested that CGA inhibits SOC-mediated Ca2+ influx and ROS generation by attenuating TRPC1 expression in LPC-treated HUVECs. Therefore, CGA might protect endothelial cells against LPC injury, thereby inhibiting atherosclerosis. PMID:28088946

Vildagliptin Stimulates Endothelial Cell Network Formation and Ischemia-induced Revascularization via an Endothelial Nitric-oxide Synthase-dependent Mechanism*

PubMed Central

Ishii, Masakazu; Shibata, Rei; Kondo, Kazuhisa; Kambara, Takahiro; Shimizu, Yuuki; Tanigawa, Tohru; Bando, Yasuko K.; Nishimura, Masahiro; Ouchi, Noriyuki; Murohara, Toyoaki

2014-01-01

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production. PMID:25100725

Vildagliptin stimulates endothelial cell network formation and ischemia-induced revascularization via an endothelial nitric-oxide synthase-dependent mechanism.

PubMed

Ishii, Masakazu; Shibata, Rei; Kondo, Kazuhisa; Kambara, Takahiro; Shimizu, Yuuki; Tanigawa, Tohru; Bando, Yasuko K; Nishimura, Masahiro; Ouchi, Noriyuki; Murohara, Toyoaki

2014-09-26

Dipeptidyl peptidase-4 inhibitors are known to lower glucose levels and are also beneficial in the management of cardiovascular disease. Here, we investigated whether a dipeptidyl peptidase-4 inhibitor, vildagliptin, modulates endothelial cell network formation and revascularization processes in vitro and in vivo. Treatment with vildagliptin enhanced blood flow recovery and capillary density in the ischemic limbs of wild-type mice, with accompanying increases in phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS). In contrast to wild-type mice, treatment with vildagliptin did not improve blood flow in ischemic muscles of eNOS-deficient mice. Treatment with vildagliptin increased the levels of glucagon-like peptide-1 (GLP-1) and adiponectin, which have protective effects on the vasculature. Both vildagliptin and GLP-1 increased the differentiation of cultured human umbilical vein endothelial cells (HUVECs) into vascular-like structures, although vildagliptin was less effective than GLP-1. GLP-1 and vildagliptin also stimulated the phosphorylation of Akt and eNOS in HUVECs. Pretreatment with a PI3 kinase or NOS inhibitor blocked the stimulatory effects of both vildagliptin and GLP-1 on HUVEC differentiation. Furthermore, treatment with vildagliptin only partially increased the limb flow of ischemic muscle in adiponectin-deficient mice in vivo. GLP-1, but not vildagliptin, significantly increased adiponectin expression in differentiated 3T3-L1 adipocytes in vitro. These data indicate that vildagliptin promotes endothelial cell function via eNOS signaling, an effect that may be mediated by both GLP-1-dependent and GLP-1-independent mechanisms. The beneficial activity of GLP-1 for revascularization may also be partially mediated by its ability to increase adiponectin production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

6-Methylsulfinylhexyl isothiocyanate modulates endothelial cell function and suppresses leukocyte adhesion.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Nagai, Masashi; Hayashi, Tatsuya; Suzuki, Koji

2014-01-01

6-Methylsulfinylhexyl isothiocyanate (6-MSITC) is an active compound in wasabi (Wasabia japonica Matsum.), which is one of the most popular spices in Japan. 6-MSITC suppresses lipopolysaccharide-induced macrophage activation, arachidonic- or adenosine diphosphate-induced platelet activation, and tumor cell proliferation. These data indicate that 6-MSITC has several biological activities involving anti-inflammatory, anti-coagulant, and anti-apoptosis properties. Endothelial cells (ECs) maintain vascular homeostasis and play crucial roles in crosstalk between blood coagulation and vascular inflammation. In this study, we determined the anti-coagulant and anti-inflammatory effects of 6-MSITC on human umbilical vein endothelial cells (HUVECs). 6-MSITC slightly reduced tissue factor expression, but did not alter von Willebrand factor release in activated HUVECs. 6-MSITC modulated the generation of activated protein C, which is essential for negative regulation of blood coagulation, on normal ECs. In addition, 6-MSITC reduced tumor necrosis factor-α (TNF-α)-induced interleukin-6 and monocyte chemoattractant protein-1 expression. 6-MSITC markedly attenuated TNF-α-induced adhesion of human monoblast U937 cells to HUVECs and reduced vascular cell adhesion molecule-1 and E-selectin mRNA expression in activated ECs. These results showed that 6-MSITC modulates EC function and suppresses cell adhesion. This study provides new insight into the mechanism of the anti-inflammatory effect of 6-MSITC, suggesting that 6-MSITC has therapeutic potential as a treatment for vasculitis and vascular inflammation.

The synergistic effect on osteogenic differentiation of human mesenchymal stem cells by diode laser-treated stimulating human umbilical vein endothelial cells

NASA Astrophysics Data System (ADS)

Kao, Chia-Tze; Hsu, Tuan-Ti; Huang, Tsui-Hsien; Wu, Yu-Tin; Chen, Yi-Wen; Shie, Ming-You

2016-02-01

Angiogenesis plays an important role in determining the biostimulation of bone regeneration, in either new bone or blood vessel formation. Human umbilical cord cells (HUVECs) are important effector cells in angiogenesis and are indispensable for osteogenesis and for their heterogeneity and plasticity. However, there are very few studies about the effects of HUVECs on diode laser-stimulated/regulated osteogenesis. In this study, we used diode laser as a model biostimulation to examine the role of HUVECs on laser-stimulated osteogenesis. Several bone formation-related proteins were also significantly up-regulated by the diode laser stimulation, indicating that HUVECs may participate in diode laser-stimulated osteogenesis. Interestingly, when human mesenchymal stem cells (hMSCs) cultured with HUVECs were diode laser-treated, the osteogenesis differentiation of the hMSCs was significantly promoted, indicating the important role of HUVECs in diode laser-enhanced osteogenesis. Adequately activated HUVECs are vital for the success of diode laser-stimulated hard-tissue regeneration. These findings provided valuable insights into the mechanism of diode laser-stimulated osteogenic differentiation, and a strategy to optimize the evaluation system for the in vitro osteogenesis capacity of laser treatment in periodontal repair.

Functional Characterization of S100A8 and S100A9 in Altering Monolayer Permeability of Human Umbilical Endothelial Cells

PubMed Central

Wang, Liqun; Luo, Haihua; Chen, Xiaohuan; Jiang, Yong; Huang, Qiaobing

2014-01-01

S100A8, S100A9 and S100A8/A9 complexes have been known as important endogenous damage-associated molecular pattern (DAMP) proteins. But the pathophysiological roles of S100A8, S100A9 and S100A8/A9 in cardiovascular diseases are incompletely explained. In this present study, the effects of homo S100A8, S100A9 and their hetero-complex S100A8/A9 on endothelial barrier function were tested respectively in cultured human umbilical venous endothelial cells (HUVECs). The involvement of TLR4 and RAGE were observed by using inhibitor of TLR4 and blocking antibody of RAGE. The clarification of different MAPK subtypes in S100A8/A9-induced endothelial response was implemented by using specific inhibitors. The calcium-dependency was detected in the absence of Ca2+ or in the presence of gradient-dose Ca2+. The results showed that S100A8, S100A9 and S100A8/A9 could induce F-actin and ZO-1 disorganization in HUVECs and evoked the increases of HUVEC monolayer permeability in a dose- and time-dependent manner. The effects of S100A8, S100A9 and S100A8/A9 on endothelial barrier function depended on the activation of p38 and ERK1/2 signal pathways through receptors TLR4 and RAGE. Most importantly, we revealed the preference of S100A8 on TLR4 and S100A9 on RAGE in HUVECs. The results also showed the calcium dependency in S100A8- and S100A9-evoked endothelial response, indicating that calcium dependency on formation of S100A8 or A9 dimmers might be the prerequisite for this endothelial functional alteration. PMID:24595267

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

PubMed Central

Hunt, Michelle A.; Currie, Margaret J.; Robinson, Bridget A.; Dachs, Gabi U.

2010-01-01

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays. PMID:20592869

Mono-(2-Ethylhexyl) Phthalate Induces Injury in Human Umbilical Vein Endothelial Cells

PubMed Central

Huang, Qi; Li, Bin-Feng; Chen, Chen; Zhang, Hua-Chuan; Xu, Shun-Qing

2014-01-01

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a widespread environmental contaminant and has been proved to have potential adverse effects on the reproductive system, carcinogenicity, liver, kidney and developmental toxicities. However, the effect of MEHP on vascular system remains unclear. The main purpose of this study was to evaluate the cytotoxic effects of MEHP on human umbilical endothelial cells (HUVEC) and its possible molecular mechanism. HUVEC cells were treated with MEHP (0, 6.25, 12.5, 25,50 and 100 µM), and the cellular apoptosis and mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In present study, MEHP induced a dose-dependent cell injury in HUVEC cell via an apoptosis pathway as characterized by increased percentage of sub-G1, activation of caspase-3, -8and -9, and increased ratio of Bax/bcl-2 mRNA and protein expression as well as cytochrome C releasing. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N-Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, could effectively block MEHP-induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicated that MEHP induced apoptosis in HUVEC cells through a reactive oxygen species-mediated mitochondria-dependent pathway. PMID:24836450

Endosulfan inducing apoptosis and necroptosis through activation RIPK signaling pathway in human umbilical vascular endothelial cells.

PubMed

Zhang, Lianshuang; Wei, Jialiu; Ren, Lihua; Zhang, Jin; Yang, Man; Jing, Li; Wang, Ji; Sun, Zhiwei; Zhou, Xianqing

2017-01-01

Endosulfan, an organochlorine pesticide, was found in human blood, and its possible cardiovascular toxicity has been suggested. However, the mechanism about endothelial cell injuries induced by endosulfan has remained unknown. In the present study, human umbilical vein endothelial cells (HUVECs) were chosen to explore the toxicity mechanism and were treated with 0, 1, 6, and 12 μg/mL -1 endosulfan for 24 h, respectively. The results showed that exposure to endosulfan could inhibit the cell viability, increase the release of lactate dehydrogenase (LDH), damage the ultrastructure, and lead to apoptosis and necroptosis in HUVECs. Furthermore, endosulfan upregulated the expressions of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like (MLKL), caspase 8, and caspase 3, which means the activation of RIPK1 pathways. In addition, endosulfan promoted the increases of ROS, IL-1α, and IL-33 levels while antioxidant N-acetyl-L-cysteine (NAC) effectively attenuated the cytotoxicity from endosulfan. Taken together, these results have demonstrated that endosulfan induces the apoptosis and necroptosis of HUVECs, where the RIPK pathway plays a pro-necroptotic role and NAC plays an anti-necroptotic role. Our results may contribute to understanding cellular mechanisms for endosulfan-induced cardiovascular toxicity.

Controlled copper ion release from phosphate-based glasses improves human umbilical vein endothelial cell survival in a reduced nutrient environment.

PubMed

Stähli, Christoph; Muja, Naser; Nazhat, Showan N

2013-02-01

The success of tissue engineering is dependent on rapid scaffold vascularization after engraftment. Copper ions are well known to be angiogenic but exhibit cytotoxicity at elevated doses. The high sensitivity to copper concentration underlines the need of a controlled release mechanism. This study investigated the effect of copper ions released from phosphate-based glasses (PGs) on human umbilical vein endothelial cells (HUVECs) under standard growth conditions (SGC), as well as in a reduced nutrient environment (RNE) with decreased bovine serum and growth factor concentrations to approximate conditions in the core of large volume scaffolds where nutrient diffusion is limited. Initially, HUVECs were exposed to a range of CuCl(2) concentrations in order to identify an optimal response in terms of their metabolism, viability, and apoptotic activity. Under SGC, HUVEC metabolic activity and viability were reduced in a dose-dependent manner in the presence of 0.44-12 ppm Cu(2+). In contrast, HUVEC death induced by the RNE was delayed by an optimal dose of 4 ppm Cu(2+), which was associated with a down-regulation of apoptosis as evidenced by caspase-3/7 activity. Copper ion release from soluble PGs of the formulation 50P(2)O(5)-30CaO-(20-x)Na(2)O-xCuO [mol%] (x=0, 1, 5 and 10) demonstrated a controllable increase with CuO content. The presence of 4 ppm copper ions released from the 10% CuO PG composition reproduced the delay in HUVEC death in the RNE, suggesting the potential of these materials to extend survival of transplanted endothelial cells in large volume scaffolds.

Cytoprotective Effect of Hydroalcoholic Extract of Pinus eldarica Bark against H2O2-Induced Oxidative Stress in Human Endothelial Cells

PubMed Central

Babaee, Fatemeh; Safaeian, Leila; Zolfaghari, Behzad; Haghjoo Javanmard, Shaghayegh

2016-01-01

Background: Pinus eldarica is a widely growing pine in Iran consisting of biologically active constituents with antioxidant properties. This study investigates the effect of hydroalcoholic extract of P. eldarica bark against oxidative damage induced by hydrogen peroxide (H2O2) in human umbilical vein endothelial cells (HUVECs). Methods: The total phenolic content of P. eldarica extract was determined using Folin-Ciocalteu method. The cytotoxicity of P. eldarica extract (25-1000 µg/ml) on HUVECs was assessed using 3-(4,5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. Cytoprotective effect of P. eldarica extract (25-500 µg/ml) on H2O2-induced oxidative stress was also evaluated by MTT assay. The intra- and extra-cellular hydroperoxides concentration and ferric reducing antioxidant power (FRAP) were measured in pretreated cells. Results: The total phenolic content of P. eldarica extract was estimated as 37.04±1.8% gallic acid equivalent. P. eldarica extract (25-1000 µg/ml) had no cytotoxic effect on HUVECs viability. The pretreatment of HUVECs with P. eldarica extract at the concentrations of 50-500 µg/ml significantly reduced the cytotoxicity of H2O2. P. eldarica extract decreased hydroperoxides concentration and increased FRAP value in intra-cellular fluid at the concentration range of 100-500 µg/ml and in extra-cellular fluid at the concentration range of 25-500 µg/ml. Conclusions: This study revealed the antioxidant and cytoprotective effects of P. eldarica extract against H2O2-induced oxidative stress in HUVECs. Concerning the high content of phenolic compounds in P. eldarica, more research is needed to evaluate its clinical value in endothelial dysfunction and in other oxidative conditions. PMID:26931383

[Role of different scale structures of titanium implant in the biological behaviors of human umbilical vein endothelial cells].

PubMed

Liang, N W; Shi, L; Huang, Y; Deng, X L

2017-02-18

To study the role of different scale structure of Ti implants on the biological behaviors of human umbilical vein endothelial cell (HUVECs) and to reveal the role of material surface topographical features on peri-implant angiogenesis. Titanium (Ti) discs with different surface structures (Ti discs with smooth surface, Ti discs with nano scale structure, Ti discs with micro scale structure and Ti discs with micro/nano scale structure, named as SM-Ti, Nano-Ti, Micro-Ti and Micro/Nano-Ti, respectively) were prepared and their surface topographical features were confirmed via scanning electron microscopy (SEM) observation. HUVECs were cultured on these Ti discs. Biological outcomes of HUVECs on different surfaces were carried out, including cell adhesive capacity, proliferation, vascular endothelial growth factor (VEGF) production and intracellular expression of Ca(2+). The results of SEM images and immunofluorescence double staining of rhodamine-phalloidin and DAPI showed that compared with the SM-Ti and Nano-Ti group, the adhesive capacity and proliferation behavior of HUVECs on the surfaces of Micro-Ti and Micro/Nano-Ti was decreased. The results of culturing HUVECs on different groups of Ti discs after 24 hours showed that the cells number grew from (18±4) to (42±6)/ vision on SM-Ti, (28±6) to (52±10)/vision on Nano-Ti, (20±4) to (21±6)/vision on Micro-Ti and (16±4) to (18±6)/vision on Micro/Nano-Ti. Moreover, compared with the adhesion and proliferation of HUVECs on SM-Ti group and Nano-Ti, the adhesion and proliferation of HUVECs on Micro-Ti group and Micro/Nano-Ti group was significantly reduced (P<0.05).The results of enzyme-linked immunosorbent assay (ELISA) showed that the VEGF productions of SM-Ti, Nano-Ti, Micro-Ti and Micro/Nano-Ti were (690±35) ng/L, (560±20) ng/L, (474±43) ng/L and (517±29) ng/L, respectively. Moreover, compared with the VEGF production of HUVECs on SM-Ti group, the VEGF production of HUVECs on Micro-Ti group and Micro/Nano-Ti group was significantly reduced (P<0.05). The results of Ca(2+) ion detection showed that the Ca(2+) expression of HUVECs on Micro-Ti and Micro/Nano-Ti was significantly higher than that on the surface of SM-Ti and Nano-Ti. These results implied that the over expressed Ca(2+) might contributed to the impaired biological function of HUVECs on Micro-Ti and Micro/Nano-Ti. Different topographical features on titanium influenced the biological behaviors of the HUVECs, which may illustrate how topographical features of Ti implant affect peri-implant angiogenesis. These results also suggest that the biological behaviors of HUVECs might be relative to the changed expression of intracellular Ca(2+).

Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montgomery, K.F.; Tarr, P.I.; Bomsztyk, K.

1991-08-01

Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor {alpha} (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), the authors demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5{prime} flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-{kappa}B and AP-1. Gel mobility shiftmore » assays demonstrate that TNF, IL-1, or LPS induces activation of NF-{kappa}B-like DNA binding activity in HUVEC. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-{kappa}B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-{kappa}B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kB-like proteins but does inhibit ELAM-1 gene transcription. They conclude that PKC-independent activation of NF-{kappa}B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.« less

Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

PubMed

Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

2017-11-01

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

Cellular redox homeostasis in endothelial cells treated with nonmodified and Fenton-modified nanodiamond powders.

PubMed

Solarska-Ściuk, K; Gajewska, A; Skolimowski, J; Gajek, A; Bartosz, G

2014-01-01

Diamond nanoparticles find numerous applications in pharmacy, medicine, cosmetics, and biotechnology. However, possible adverse cellular effects of diamond nanoparticle cells have been reported, which may limit their use. The aim of this study was to compare the effect of nonmodified diamond nanoparticles (D) and diamond nanoparticles modified by the Fenton reaction (D+OH) on human umbilical cord endothelial cells (HUVEC-ST). We found that both D and D+OH show time- and concentration-dependent cytotoxicity, inducing apoptosis and necrosis of HUVEC-ST. Interaction with D and D+OH also induced changes in the production of reactive oxygen and nitrogen species and changes in the level of glutathione and activities of antioxidant enzymes in the cells. These data demonstrate that diamond nanoparticles may induce oxidative stress in human endothelial cells, which contributes to their cytotoxic effects seen at higher concentrations of D and D+OH. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Impact of immobilizing of low molecular weight hyaluronic acid within gelatin-based hydrogel through enzymatic reaction on behavior of enclosed endothelial cells.

PubMed

Khanmohammadi, Mehdi; Sakai, Shinji; Taya, Masahito

2017-04-01

The hydrogels having the ability to promote migration and morphogenesis of endothelial cells (ECs) are useful for fabricating vascularized dense tissues in vitro. The present study explores the immobilization of low molecular weight hyaluronic acid (LMWHA) derivative within gelatin-based hydrogel to stimulate migration of ECs. The LMWHA derivative possessing phenolic hydroxyl moieties (LMWHA-Ph) was bound to gelatin-based derivative hydrogel through the horseradish peroxidase-catalyzed reaction. The motility of ECs was analyzed by scratch migration assay and microparticle-based cell migration assay. The incorporated LMWHA-Ph molecules within hydrogel was found to be preserved stably through covalent bonds during incubation. The free and immobilized LMWHA-Ph did not lose an inherent stimulatory effect on human umbilical vein endothelial cells (HUVECs). The immobilized LMWHA-Ph within gelatin-based hydrogel induced the high motility of HUVECs, accompanied by robust cytoskeleton extension, and cell subpopulation expressing CD44 cell receptor. In the presence of immobilized LMWHA-Ph, the migration distance and the number of existing HUVECs were demonstrated to be encouraged in dose-dependent and time-dependent manners. Based on the results obtained in this work, it was concluded that the enzymatic immobilization of LMWHA-Ph within gelatin-based hydrogel represents a promising approach to promote ECs' motility and further exploitation for vascular tissue engineering applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells.

PubMed

Hofbauer, Pablo; Riedl, Sabrina; Witzeneder, Karin; Hildner, Florian; Wolbank, Susanne; Groeger, Marion; Gabriel, Christian; Redl, Heinz; Holnthoner, Wolfgang

2014-09-01

As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells. The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs). Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities. The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton

NASA Technical Reports Server (NTRS)

Sawyer, S. J.; Norvell, S. M.; Ponik, S. M.; Pavalko, F. M.

2001-01-01

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

Newly synthesized quinazolinone HMJ-38 suppresses angiogenetic responses and triggers human umbilical vein endothelial cell apoptosis through p53-modulated Fas/death receptor signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiang, Jo-Hua; Yang, Jai-Sing; Lu, Chi-Cheng

The current study aims to investigate the antiangiogenic responses and apoptotic death of human umbilical vein endothelial cells (HUVECs) by a newly synthesized compound named 2-(3′-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone (HMJ-38). This work attempted to not only explore the effects of angiogenesis on in vivo and ex vivo studies but also hypothesize the implications for HUVECs (an ideal cell model for angiogenesis in vitro) and further undermined apoptotic experiments to verify the underlying molecular signaling by HMJ-38. Our results demonstrated that HMJ-38 significantly inhibited blood vessel growth and microvessel formation by the mouse Matrigel plug assay of angiogenesis, and the suppression of microsprouting frommore » the rat aortic ring assay was observed after HMJ-38 exposure. In addition, HMJ-38 disrupted the tube formation and blocked the ability of HUVECs to migrate in response to VEGF. We also found that HMJ-38 triggered cell apoptosis of HUVECs in vitro. HMJ-38 concentration-dependently suppressed viability and induced apoptotic damage in HUVECs. HMJ-38-influenced HUVECs were performed by determining the oxidative stress (ROS production) and ATM/p53-modulated Fas and DR4/DR5 signals that were examined by flow cytometry, Western blotting, siRNA and real-time RT-PCR analyses, respectively. Our findings demonstrate that p53-regulated extrinsic pathway might fully contribute to HMJ-38-provoked apoptotic death in HUVECs. In view of these observations, we conclude that HMJ-38 reduces angiogenesis in vivo and ex vivo as well as induces apoptosis of HUVECs in vitro. Overall, HMJ-38 has a potent anti-neovascularization effect and could warrant being a vascular targeting agent in the future. - Highlights: • HMJ-38 suppresses angiogenic actions in vivo and ex vivo. • Inhibitions of blood vessel and microvessel formation by HMJ-38 are acted. • Cytotoxic effects of HUVECs occur by HMJ-38 challenge. • p53-modulated extrinsic pathway contributes to HMJ-38-provoked apoptosis in HUVECs. • Novel HMJ-38 possesses anti-angiogenic properties and pro-apoptotic processes.« less

Effect of benzo[a]pyrene on the production of vascular endothelial growth factor by human eosinophilic leukemia EoL-1 cells.

PubMed

Gu, Jie; Chan, Lai-Sheung; Wong, Chris Kong-Chu; Wong, Ngok-Shun; Wong, Chun-Kwok; Leung, Kok-Nam; Mak, Naiki K

2011-01-01

Benzo[a]pyrene (BaP) has been shown to affect both the development and response of T and B cells in the immune system. However, the effect of BaP on other immune cells, such as eosionophils, is unknown. In this study, we investigated the effect of BaP on the production of vascular endothelial growth factor (VEGF) using an in vitro eosinophilic EoL-1 cell and human umbilical vein endothelial cell (HUVEC) co-culture system. EoL-1-conditioned medium was found to promote the growth of HUVEC in a time-dependent manner. The growth stimulating activity was due to the production of VEGF by the EoL-1 cells. The production of VEGF was correlated with the enhanced expression of the phosphorylated form of extracellular signal-regulated kinases (p-ERKs) and the upregulated expression of VEGF mRNA. Furthermore, BaP-induced expression of VEGF mRNA was reduced by the ERK inhibitor PD98059. Results from this study suggested that BaP might affect the growth of endothelial cells through the modulation of VEGF production by eosinophils.

Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

PubMed Central

2009-01-01

Background Vascular endothelial growth factor (VEGF) is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ), bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs). Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE)]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases. PMID:19917137

Evaluation of the in vitro and in vivo angiogenic effects of exendin-4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Hye-Min; Kang, Yujung; Chun, Hyung J.

2013-04-26

Highlights: •We investigated the effects of exendin-4 on the angiogenic process. •Exendin-4 increased migration, sprouting, and tube formation by HUVECs in in vitro. •Exendin-4 increased sprouts in aortic rings and induced new vessels in Matrigel in in vivo. •Exendin-4 may be of potential use for the treatment of vascular complications of diabetes. -- Abstract: Exendin-4, an analog of glucagon-like peptide (GLP)-1, has beneficial effects on cardiovascular disease induced by diabetes mellitus (DM). Recently, exendin-4 was reported to induce the proliferation of endothelial cells. However, its angiogenic effect on endothelial cells has not been clearly evaluated. Therefore, we investigated the effectsmore » of exendin-4 on the angiogenic process with respect to migration, sprouting, and neovascularization using in vitro and in vivo assays. Treatment with exendin-4 increased the migration of human umbilical vein endothelial cells (HUVECs) in in vitro scratch wound assays, as well as the number of lumenized vessels sprouting from HUVECs in in vitro 3D bead assays. These responses were abolished by co-treatment with exendin (9–39), a GLP-1 receptor antagonist, which suggests that exendin-4 regulates endothelial cell migration and tube formation in a GLP-1 receptor-dependent manner. In an ex vivo assay, treatment of aortic rings with exendin-4 increased the sprouting of endothelial cells. Exendin-4 also significantly increased the number of new vessels and induced blood flow in Matrigel plugs in in vivo assays. Our results provide clear evidence for the angiogenic effect of exendin-4 in in vitro and in vivo assays and provide a mechanism underlying the cardioprotective effects of exendin-4.« less

Role of adenosine transport in gestational diabetes-induced l-arginine transport and nitric oxide synthesis in human umbilical vein endothelium

PubMed Central

Vásquez, Gustavo; Sanhueza, Felipe; Vásquez, Rodrigo; González, Marcelo; Martín, Rody San; Casanello, Paola; Sobrevia, Luis

2004-01-01

Gestational diabetes is associated with increased l-arginine transport and nitric oxide (NO) synthesis, and reduced adenosine transport in human umbilical vein endothelial cells (HUVEC). Adenosine increases endothelial l-arginine/NO pathway via A2 purinoceptors in HUVEC from normal pregnancies. It is unknown whether the effect of gestational diabetes is associated with activation of these purinoceptors or altered expression of human cationic amino acid transporter 1 (hCAT-1) or human equilibrative nucleoside transporter 1 (hENT1), or endothelial NO synthase (eNOS) in HUVEC. Cells were isolated from normal or gestational diabetic pregnancies and cultured up to passage 2. Gestational diabetes increased hCAT-1 mRNA expression (2.4-fold) and activity, eNOS mRNA (2.3-fold), protein level (2.1-fold), and phosphorylation (3.8-fold), but reduced hENT1 mRNA expression (32%) and activity. Gestational diabetes increased extracellular adenosine (2.7 μm), and intracellular l-arginine (1.9 mm) and l-citrulline (0.7 mm) levels compared with normal cells (0.05 μm, 0.89 mm, 0.35 mm, respectively). Incubation of HUVEC from normal pregnancies with 1 μm nitrobenzylthioinosine (NBMPR) mimicked the effect of gestational diabetes, but NBMPR was ineffective in diabetic cells. Gestational diabetes and NBMPR effects involved eNOS, PKC and p42/44mapk activation, and were blocked by the A2a purinoceptor antagonist ZM-241385. Thus, gestational diabetes increases the l-arginine/NO pathway involving activation of mitogen-activated protein (MAP) kinases, protein kinase C (PKC) and NO cell signalling cascades following activation of A2a purinoceptors by extracellular adenosine. A functional relationship is proposed between adenosine transport and modulation of l-arginine transport and NO synthesis in HUVEC, which could be determinant in regulating vascular reactivity in diabetes mellitus. PMID:15272035

Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)-Streptavidin and Biotinylated Fibronectin

PubMed Central

Anamelechi, Charles C.; Clermont, Edward C.; Novak, Matthew T.; Reichert, William M.

2014-01-01

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately. PMID:19348476

Heparin use in a rat hemorrhagic shock model induces biologic activity in mesenteric lymph separate from shock.

PubMed

Qin, Yong; Prescott, Lauriston M; Deitch, Edwin A; Kaiser, Vicki L

2011-04-01

Experimental data have shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock induces neutrophil activation, cytotoxicity, decreased red blood cell (RBC) deformability, and bone marrow colony growth suppression. These data have led to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of multiple organ failure after THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cell toxicity was not induced by lymph when alternate anticoagulants (citrate and EDTA) were used in THS. Human umbilical vein endothelial cell toxicity was induced by lymph after heparin but not saline or citrate injection into trauma-sham shock and naive animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein and hepatic) were detected in the plasma and lymph from THS and naive animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified lipoprotein lipase added to naive lymph-induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models, thus necessitating a review of previous work in this field.

Shape Memory Polymers Containing Higher Acrylate Content Display Increased Endothelial Cell Attachment

PubMed Central

Govindarajan, Tina; Shandas, Robin

2018-01-01

Shape Memory Polymers (SMPs) are smart materials that can recall their shape upon the application of a stimulus, which makes them appealing materials for a variety of applications, especially in biomedical devices. Most prior SMP research has focused on tuning bulk properties; studying surface effects of SMPs may extend the use of these materials to blood-contacting applications, such as cardiovascular stents, where surfaces that support rapid endothelialization have been correlated to stent success. Here, we evaluate endothelial attachment onto the surfaces of a family of SMPs previously developed in our group that have shown promise for biomedical devices. Nine SMP formulations containing varying amounts of tert-Butyl acrylate (tBA) and Poly(ethylene glycol) dimethacrylate (PEGDMA) were analyzed for endothelial cell attachment. Dynamic mechanical analysis (DMA), contact angle studies, and atomic force microscopy (AFM) were used to verify bulk and surface properties of the SMPs. Human umbilical vein endothelial cell (HUVEC) attachment and viability was verified using fluorescent methods. Endothelial cells preferentially attached to SMPs with higher tBA content, which have rougher, more hydrophobic surfaces. HUVECs also displayed an increased metabolic activity on these high tBA SMPs over the course of the study. This class of SMPs may be promising candidates for next generation blood-contacting devices. PMID:29707382

Microfluidic perfusion culture chip providing different strengths of shear stress for analysis of vascular endothelial function.

PubMed

Hattori, Koji; Munehira, Yoichi; Kobayashi, Hideki; Satoh, Taku; Sugiura, Shinji; Kanamori, Toshiyuki

2014-09-01

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm(2) in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 10(5)) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm(2). Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm(2). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Placental growth factor expression is reversed by antivascular endothelial growth factor therapy under hypoxic conditions.

PubMed

Zhou, Ai-Yi; Bai, Yu-Jing; Zhao, Min; Yu, Wen-Zhen; Huang, Lv-Zhen; Li, Xiao-Xin

2014-08-01

Clinical trials have revealed that the antivascular endothelial growth factor (VEGF) therapies are effective in retinopathy of prematurity (ROP). But the low level of VEGF was necessary as a survival signal in healthy conditions, and endogenous placental growth factor (PIGF) is redundant for development. The purpose of this study was to elucidate the PIGF expression under hypoxia as well as the influence of anti-VEGF therapy on PIGF. CoCl2-induced hypoxic human umbilical vein endothelial cells (HUVECs) were used for an in vitro study, and oxygen-induced retinopathy (OIR) mice models were used for an in vivo study. The expression patterns of PIGF under hypoxic conditions and the influence of anti-VEGF therapy on PIGF were evaluated by quantitative reverse transcription-polymerase chain reaction (RTPCR). The retinal avascular areas and neovascularization (NV) areas of anti-VEGF, anti-PIGF and combination treatments were calculated. Retina PIGF concentration was evaluated by ELISA after treatment. The vasoactive effects of exogenous PIGF on HUVECs were investigated by proliferation and migration studies. PIGF mRNA expression was reduced by hypoxia in OIR mice, in HUVECs under hypoxia and anti-VEGF treatment. However, PIGF expression was reversed by anti-VEGF therapy in the OIR model and in HUVECs under hypoxia. Exogenous PIGF significantly inhibited HUVECs proliferation and migration under normal conditions, but it stimulated cell proliferation and migration under hypoxia. Anti-PIGF treatment was effective for neovascular tufts in OIR mice (P<0.05). The finding that PIGF expression is iatrogenically up-regulated by anti-VEGF therapy provides a consideration to combine it with anti-PIGF therapy.

Quantum dot interactions and flow effects in angiogenic zebrafish (Danio rerio) vessels and human endothelial cells.

PubMed

Jiang, Xiao-Yu; Sarsons, Christopher D; Gomez-Garcia, M Juliana; Cramb, David T; Rinker, Kristina D; Childs, Sarah J

2017-04-01

Nanoparticle (NP) interactions with biological tissues are affected by the size, shape and surface chemistry of the NPs. Here we use in vivo (zebrafish) and in vitro (HUVEC) models to investigate association of quantum dots (QDs) with endothelial cells and the effect of fluid flow. After injection into the developing zebrafish, circulating QDs associate with endothelium and penetrate surrounding tissue parenchyma over time. Amino-functionalized QDs cluster, interact with cells, and clear more rapidly than carboxy-functionalized QDs in vivo, highlighting charge influences. QDs show stronger accumulation in slow-flowing, small caliber venous vessels than in fast-flowing high caliber arterial vessels. Parallel-plate flow experiments with HUVEC support these findings, showing reduced QD-EC association with increasing flow. In vivo, flow arrest after nanoparticle injection still results in venous accumulation at 18 h. Overall our results suggest that both QD charge and blood flow modulate particle-endothelial cell interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

Autophagic effects of Hibiscus sabdariffa leaf polyphenols and epicatechin gallate (ECG) against oxidized LDL-induced injury of human endothelial cells.

PubMed

Chen, Jing-Hsien; Lee, Ming-Shih; Wang, Chi-Ping; Hsu, Cheng-Chin; Lin, Hui-Hsuan

2017-08-01

Oxidized low-density lipoprotein (ox-LDL) contributes to the pathogenesis of atherosclerosis by promoting vascular endothelial cell injury. Hibiscus sabdariffa leaf polyphenols (HLP), rich in flavonoids, have been shown to possess antioxidant and antiatherosclerotic activities. In this study, we examined the protective role of HLP and its main compound (-)-epicatechin gallate (ECG) in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL in vitro. In a model of ox-LDL-impaired HUVECs, assessments of cell viability, cytotoxicity, cell proliferation, apoptosis, and autophagy were detected. To highlight the mechanisms of the antiapoptotic effects of HLP and ECG, the expressions of molecular proteins were measured by Western blotting, real-time PCR, and so on. HLP or ECG improved the survival of HUVECs from ox-LDL-induced viability loss. In addition, HLP or ECG showed potential in reducing ox-LDL-dependent apoptosis. Next, the ox-LDL-induced formation of acidic vesicular organelles and upregulation of the autophagy-related genes were increased by HLP or ECG. The HLP-triggered autophagic flux was further confirmed by increasing the LC3-II level under the pretreatment of an autophagy inhibitor chloroquine. Molecular data indicated the autophagic effect of HLP or ECG might be mediated via class III PI3K/Beclin-1 and PTEN/class I PI3K/Akt cascade signaling, as demonstrated by the usage of a class III PI3K inhibitor 3-methyladenine (3-MA) and a PTEN inhibitor SF1670. Our data imply that ECG-enriched HLP upregulates the autophagic pathway, which in turn led to reduce ox-LDL-induced HUVECs injury and apoptosis and provide a new mechanism for its antiatherosclerotic activity.

Aspirin prevents resistin-induced endothelial dysfunction by modulating AMPK, ROS, and Akt/eNOS signaling.

PubMed

Ou, Hsiu-Chung; Lee, Wen-Jane; Wu, Ching-Mei; Chen, Judy Fuh-Meei; Sheu, Wayne Huey-Herng

2012-04-01

Resistin, an adipocytokine, plays a potential role in cardiovascular disease and may contribute to increased atherosclerotic risk by modulating the activity of endothelial cells. A growing body of evidence suggests that aspirin is a potent antioxidant. We investigated whether aspirin mitigates resistin-induced endothelial dysfunction via modulation of reactive oxygen species (ROS) generation and explored the role that AMP-activated protein kinase (AMPK), a negative regulator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, plays in the suppressive effects of aspirin on resistin-induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) were pretreated with various doses of aspirin (10-500 μg/mL) for 2 hours and then incubated with resistin (100 ng/mL) for an additional 48 hours. Fluorescence produced by the oxidation of dihydroethidium (DHE) was used to quantify the production of superoxide in situ; superoxide dismutase (SOD) and catalase activities were determined by an enzymatic assay; and protein levels of AMPK-mediated downstream signaling were investigated by Western blot. Treatment of HUVECs with resistin for 48 hours resulted in a 2.9-fold increase in superoxide production; however, pretreatment with aspirin resulted in a dose-dependent decrease in production of superoxide (10-500 μg/mL; n = 3 experiments; all P < .05). Resistin also suppressed the activity of superoxide dismutase and catalase by nearly 50%; that result, however, was not observed in HUVECs that had been pretreated with aspirin at a concentration of 500 μg/mL. The membrane translocation assay showed that the levels of NADPH oxidase subunits p47(phox)and Rac-1 in membrane fractions of HUVECs were threefold to fourfold higher in cells that had been treated with resistin for 1 hour than in untreated cells; however, pretreatment with aspirin markedly inhibited resistin-induced membrane assembly of NADPH oxidase via modulating AMPK-suppressed PKC-α activation. Application of AMPKα1-specific siRNA resulted in increased activation of PKC-α and p47(phox). In addition, resistin significantly decreased AMPK-mediated downstream Akt/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling and induced the phosphorylation of p38 mitogen-activated protein kinases, which in turn activated NF-κB-mediated inflammatory responses such as the release of interleukin (IL)-6 and IL-8, the overexpression of adhesion molecules, and stimulation of monocytic THP-1 cell attachment to HUVECs (2.5-fold vs control; n = 3 experiments). Furthermore, resistin downregulated eNOS and upregulated inducible NO synthase (iNOS) expression, thereby augmenting the formation of NO and protein nitrosylation. Pretreatment with aspirin, however, exerted significant cytoprotective effects in a dose-dependent manner (P < .05). Our findings suggest a direct connection between adipocytokines and endothelial dysfunction and provide further insight into the protective effects of aspirin in obese individuals with endothelial dysfunction. Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF.

PubMed

Schoenfeld, Jonathan; Lessan, Khashayar; Johnson, Nicola A; Charnock-Jones, D Stephen; Evans, Amanda; Vourvouhaki, Ekaterini; Scott, Laurie; Stephens, Richard; Freeman, Tom C; Saidi, Samir A; Tom, Brian; Weston, Gareth C; Rogers, Peter; Smith, Stephen K; Print, Cristin G

2004-01-01

We recently published a review in this journal describing the design, hybridisation and basic data processing required to use gene arrays to investigate vascular biology (Evans et al. Angiogenesis 2003; 6: 93-104). Here, we build on this review by describing a set of powerful and robust methods for the analysis and interpretation of gene array data derived from primary vascular cell cultures. First, we describe the evaluation of transcriptome heterogeneity between primary cultures derived from different individuals, and estimation of the false discovery rate introduced by this heterogeneity and by experimental noise. Then, we discuss the appropriate use of Bayesian t-tests, clustering and independent component analysis to mine the data. We illustrate these principles by analysis of a previously unpublished set of gene array data in which human umbilical vein endothelial cells (HUVEC) cultured in either rich or low-serum media were exposed to vascular endothelial growth factor (VEGF)-A165 or placental growth factor (PlGF)-1(131). We have used Affymetrix U95A gene arrays to map the effects of these factors on the HUVEC transcriptome. These experiments followed a paired design and were biologically replicated three times. In addition, one experiment was repeated using serial analysis of gene expression (SAGE). In contrast to some previous studies, we found that VEGF-A and PlGF consistently regulated only small, non-overlapping and culture media-dependant sets of HUVEC transcripts, despite causing significant cell biological changes.

K20E, an oxidative-coupling compound of methyl caffeate, exhibits anti-angiogenic activities through down-regulations of VEGF and VEGF receptor-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Chun-Hsu; Lin, Wen-Hsin; Chien, Yi-Chung

Anti-angiogenesis is one of the most popular clinical interventions for cancer chemotherapy. A series of synthesized derivative of methyl caffeate were used to evaluate the anti-angiogenic activity and to investigate possible pharmacological mechanisms in the present study. The most potent anti-angiogenic compound was evaluated in the experiments of murine allograft tumor model and Matrigel plug assay as well as cell models in the human umbilical vascular endothelial cells (HUVECs) and the LLC1 lung cancer cells. Our results suggested that K20E suppressed the tumor growth in the allograft tumor model and exhibited anti-angiogenic activity in Matrigel plug assay. Besides, HUVEC viabilitymore » was found to be significantly reduced by arresting cell cycle at G{sub 2}/M phase and apoptosis. Cell migration, invasion, and tube formation of the HUVECs were also markedly suppressed by K20E treatment. K20E largely down-regulated the intracellular and secreted vascular endothelial growth factor (VEGF) in the LLC1 cancer cells. Besides, VEGF receptor-2 (VEGFR-2) and its downstream signaling cascades (AKT-mTOR and MEK1/2-ERK1/2) as well as gelatinases were all evidently reduced in the HUVECs treated with K20E. Inversely, K20E can up-regulate the expression levels of p53 and p21 proteins in the HUVECs. Based on these results, our study suggested that K20E possessed inhibiting angiogenesis through regulation of VEGF/VEGFR-2 and its downstream signaling cascades in the vascular endothelial cells (VECs). - Highlights: • K20E is an oxidative-coupling compound of methyl caffeate. • K20E exhibits anti-tumor and anti-angiogenesis effects. • K20E suppresses the expressions of VEGF and VEGF receptor-2 (VEGFR-2) proteins. • K20E deactivates VEGFR-2-mediated downstream signaling pathways to inhibit angiogenesis. • K20E up-regulates p53-p21 pathway to induce apoptosis and cell arrest at G2/M phase.« less

Alteration of human umbilical vein endothelial cell gene expression in different biomechanical environments.

PubMed

Shoajei, Shahrokh; Tafazzoli-Shahdpour, Mohammad; Shokrgozar, Mohammad Ali; Haghighipour, Nooshin

2014-05-01

Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm(2) , 1 Hz) and uniform (5 dyn/cm(2) ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm(2) ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes. © 2013 International Federation for Cell Biology.

Flavonoids from the leaves of Carya cathayensis Sarg. inhibit vascular endothelial growth factor-induced angiogenesis.

PubMed

Tian, Sha-Sha; Jiang, Fu-Sheng; Zhang, Kun; Zhu, Xue-Xin; Jin, Bo; Lu, Jin-Jian; Ding, Zhi-Shan

2014-01-01

The total flavonoids (TFs) were isolated from the leaves of Carya cathayensis Sarg. (LCC), a well-known Chinese medicinal herb commercially cultivated in Tianmu Mountain district, a cross area of Zhejiang and Anhui provinces in China. Five flavonoids, i.e. cardamonin, pinostrobin chalcone (PC), wogonin, chrysin, and pinocembrin were the main components of the TFs. The TFs and these pure compounds suppressed vascular endothelial growth factor (VEGF)-induced angiogenesis as detected in the mouse aortic ring assay, and cardamonin showed the best effect among them. To further elucidate the mechanisms for suppressing angiogenesis of these flavonoids, assays of VEGF-induced proliferation and migration in human umbilical vein endothelial cells (HUVECs) were performed. The TFs, cardamonin, pinocembrin, and chrysin obviously suppressed both VEGF-induced HUVEC proliferation and migration. However, PC and wogonin not only slightly inhibited VEGF-induced proliferation but also remarkably suppressed those of migration in HUVECs. Our further study showed that cardamonin decreased the phosphorylation of ERK and AKT induced by VEGF with a dose-dependent manner in HUVECs. Our findings indicate that the TFs and these pure flavonoids may become potential preventive and/or therapeutic agents against angiogenesis-related diseases. © 2013.

High glucose increases the formation and pro-oxidative activity of endothelial microparticles.

PubMed

Burger, Dylan; Turner, Maddison; Xiao, Fengxia; Munkonda, Mercedes N; Akbari, Shareef; Burns, Kevin D

2017-09-01

Individuals with diabetes exhibit increases in circulating endothelial microparticles (eMPs, also referred to as endothelial microvesicles), which are associated with endothelial dysfunction and a heightened risk of cardiovascular complications. We have shown that eMPs are markers and mediators of vascular injury although their role in diabetes is unclear. We hypothesised that the composition and biological activity of eMPs are altered in response to high glucose exposure. We assessed the effects of high glucose on eMP formation, composition and signalling in cultured HUVECs. eMPs were isolated from the media of HUVECs cultured under conditions of normal glucose (eMP NG ), high glucose (eMP HG ) or osmotic control of L-glucose (eMP LG ). eMP size, concentration and surface charge were assessed by nanoparticle tracking analysis and flow cytometry. eMP protein composition was assessed by liquid chromatography-tandem mass spectrometry, and eMP-mediated effects on coagulation, reactive oxygen species (ROS) production and vessel function were assessed. Exposure of HUVECs to high glucose for 24 h caused a threefold increase in eMP formation, increased mean particle size (269 ± 18 nm vs 226 ± 11 nm) and decreased surface charge. Compared with eMP NG or eMP LG , eMP HG possessed approximately threefold greater pro-coagulant activity, stimulated HUVEC ROS production to a greater extent (~250% of eMP NG ) and were more potent inhibitors of endothelial-dependent relaxation. Proteomic analysis of eMPs identified 1212 independent proteins of which 68 were exclusively found in eMP HG . Gene ontology analysis revealed that eMP HG -exclusive proteins were associated with signalling pathways related to blood coagulation, cell signalling and immune cell activation. Our results indicate that elevated glucose is a potent stimulus for eMP formation that also alters their molecular composition leading to increased bioactivity. Such effects may contribute to progressive endothelial injury and subsequent cardiovascular complications in diabetes.

Sirt6 mRNA-incorporated endothelial microparticles (EMPs) attenuates DM patient-derived EMP-induced endothelial dysfunction

PubMed Central

Jing, Tong; Ya-Shu, Kuang; Xue-Jun, Wang; Han-Jing, Hou; Yan, Lai; Yi-An, Yao; Fei, Chen; Xue-Bo, Liu

2017-01-01

Background Endothelial microparticles (EMPs) are small vesicles released by endothelial cells (ECs); they are considered biomarkers for endothelial dysfunction and therapeutic targets in diabetes-related vascular disease. Sirtuins have also been shown to play important roles in diabetes by regulating endothelial dysfunction. However, the effect of sirtuin-incorporated EMPs on their parental ECs remains unknown. Aim The present study aims to investigate the diagnostic value of EMPs in diabetes and detect the protective effects of sirtuin 6 (Sirt6) mRNA -incorporated EMPs on endothelial dysfunction. Methods EMPs were prepared from cultured HUVECs and venous blood from patients with diabetes (n=10) and from healthy volunteers (n=6) after sequential centrifugation. Adv-Sirt6 or Sirt6 siRNA was used to alter Sirt6 expression. EC angiogenesis, inflammatory phenotypes, nitric oxide (NO) formation and eNOS phosphorylation were used to evaluate endothelial dysfunction. Results The levels of EMPs in diabetic patients and high glucose-cultured HUVECs are high, whereas Sirt6 expression in plasma and EMPs is low. EMPs generated from diabetic patients or high glucose-cultured HUVECs increase inflammatory chemokine release and blunt EC angiogenesis. Furthermore, EMPs enriched with Sirt6 mRNA induces EC angiogenesis, increases eNOS phosphorylation and impedes inflammatory chemokine release. Inhibition of Sirt6 mRNA expression in EMPs by siRNA hinders angiogenesis and eNOS phosphorylation but increases cellular inflammation. Conclusion The Sirt6 mRNA-carrying EMPs may ameliorate endothelial dysfunction in diabetic patients. PMID:29371988

Sirt6 mRNA-incorporated endothelial microparticles (EMPs) attenuates DM patient-derived EMP-induced endothelial dysfunction.

PubMed

Jing, Tong; Ya-Shu, Kuang; Xue-Jun, Wang; Han-Jing, Hou; Yan, Lai; Yi-An, Yao; Fei, Chen; Xue-Bo, Liu

2017-12-26

Endothelial microparticles (EMPs) are small vesicles released by endothelial cells (ECs); they are considered biomarkers for endothelial dysfunction and therapeutic targets in diabetes-related vascular disease. Sirtuins have also been shown to play important roles in diabetes by regulating endothelial dysfunction. However, the effect of sirtuin-incorporated EMPs on their parental ECs remains unknown. The present study aims to investigate the diagnostic value of EMPs in diabetes and detect the protective effects of sirtuin 6 ( Sirt6 ) mRNA -incorporated EMPs on endothelial dysfunction. EMPs were prepared from cultured HUVECs and venous blood from patients with diabetes (n=10) and from healthy volunteers (n=6) after sequential centrifugation. Adv- Sirt6 or Sirt6 siRNA was used to alter Sirt6 expression. EC angiogenesis, inflammatory phenotypes, nitric oxide (NO) formation and eNOS phosphorylation were used to evaluate endothelial dysfunction. The levels of EMPs in diabetic patients and high glucose-cultured HUVECs are high, whereas Sirt6 expression in plasma and EMPs is low. EMPs generated from diabetic patients or high glucose-cultured HUVECs increase inflammatory chemokine release and blunt EC angiogenesis. Furthermore, EMPs enriched with Sirt6 mRNA induces EC angiogenesis, increases eNOS phosphorylation and impedes inflammatory chemokine release. Inhibition of Sirt6 mRNA expression in EMPs by siRNA hinders angiogenesis and eNOS phosphorylation but increases cellular inflammation. The Sirt6 mRNA -carrying EMPs may ameliorate endothelial dysfunction in diabetic patients.

β-Escin Effectively Modulates HUVECS Proliferation and Tube Formation.

PubMed

Varinská, Lenka; Fáber, Lenka; Kello, Martin; Petrovová, Eva; Balážová, Ľudmila; Solár, Peter; Čoma, Matúš; Urdzík, Peter; Mojžiš, Ján; Švajdlenka, Emil; Mučaji, Pavel; Gál, Peter

2018-01-17

In the present study we evaluated the anti-angiogenic activities of β-escin (the major active compound of Aesculus hippocastanum L. seeds). Human umbilical-vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying the anti-angiogenic effect of β-escin. We investigated the in vitro effects on proliferation, migration, and tube formation of HUVECs and in vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) angiogenesis assay. Moreover, the effect on gene expressions was determined by the RT2 ProfilerTM human angiogenesis PCR Array. It was found that β-escin exerts inhibitory effect on the basic fibroblast growth factor (bFGF)-induced proliferation, migration and tube formation, as well as CAM angiogenesis in vivo. The inhibition of critical steps of angiogenic process observed with β-escin could be partially explained by suppression of Akt activation in response to bFGF. Moreover, the anti-angiogenic effects of β-escin could also be mediated via inhibition of EFNB2 and FGF-1 gene expressions in endothelial cells. In conclusion, β-escin affects endothelial cells as a negative mediator of angiogenesis in vitro and in vivo and may therefore be considered as a promising candidate for further research elucidating its underlying mechanism of action.

Inhibition of Hydrogen Sulfide-induced Angiogenesis and Inflammation in Vascular Endothelial Cells: Potential Mechanisms of Gastric Cancer Prevention by Korean Red Ginseng.

PubMed

Choi, Ki-Seok; Song, Heup; Kim, Eun-Hee; Choi, Jae Hyung; Hong, Hua; Han, Young-Min; Hahm, Ki Baik

2012-04-01

Previously, we reported that Helicobacter pylori-associated gastritis and gastric cancer are closely associated with increased levels of hydrogen sulfide (H2S) and that Korean red ginseng significantly reduced the severity of H. pylori-associated gastric diseases by attenuating H2S generation. Because the incubation of endothelial cells with H2S has been known to enhance their angiogenic activities, we hypothesized that the amelioration of H2S-induced gastric inflammation or angiogenesis in human umbilical vascular endothelial cells (HUVECs) might explain the preventive effect of Korean red ginseng on H. pylori-associated carcinogenesis. The expression of inflammatory mediators, angiogenic growth factors, and angiogenic activities in the absence or presence of Korean red ginseng extracts (KRGE) were evaluated in HUVECs stimulated with the H2S generator sodium hydrogen sulfide (NaHS). KRGE efficiently decreased the expression of cystathionine β-synthase and cystathionine γ-lyase, enzymes that are essential for H2S synthesis. Concomitantly, a significant decrease in the expression of inflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase, and several angiogenic factors, including interleukin (IL)-8, hypoxia inducible factor-1a, vascular endothelial growth factor, IL-6, and matrix metalloproteinases, was observed; all of these factors are normally induced after NaHS. An in vitro angiogenesis assay demonstrated that NaHS significantly increased tube formation in endothelial cells, whereas KRGE pretreatment significantly attenuated tube formation. NaHS activated p38 and Akt, increasing the expression of angiogenic factors and the proliferation of HUVECs, whereas KRGE effectively abrogated this H2S-activated angiogenesis and the increase in inflammatory mediators in vascular endothelial cells. In conclusion, KRGE was able to mitigate H2S-induced angiogenesis, implying that antagonistic action against H2S-induced angiogenesis may be the mechanism underlying the gastric cancer preventive effects of KRGE in H. pylori infection.

The effect of artichoke (Cynara scolymus L.) extract on ROS generation in HUVEC cells.

PubMed

Juzyszyn, Z; Czerny, B; Pawlik, A; Droździk, M

2008-09-01

The effect of an artichoke extract on induced reactive oxygen species (ROS) generation in cultured human umbilical endothelial cells (HUVECs) and its reductive properties were evaluated. Preincubation of HUVEC cells with the artichoke extract at concentrations of 25-100 microg/mL for 24 h abolished ROS generation induced by LPS and oxyLDL as evaluated by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF). Potent, concentration-dependent reductive properties of the artichoke extract were demonstrated by the reduction kinetics of cytochrome c in reference to ascorbate were also revealed. The results of the present study the warrant application of artichoke extracts as endothelium protecting agents.

Similar NF-κB Gene Signatures in TNF-α Treated Human Endothelial Cells and Breast Tumor Biopsies

PubMed Central

Perrot-Applanat, Martine; Vacher, Sophie; Toullec, Aurore; Pelaez, Irma; Velasco, Guillaume; Cormier, Françoise; Saad, Hanan El Sheikh; Lidereau, Rosette; Baud, Véronique; Bièche, Ivan

2011-01-01

Background Endothelial dysfunction has been implicated in the pathogenesis of diverse pathologies ranging from vascular and immune diseases to cancer. TNF-α is one of the mediators of endothelial dysfunction through the activation of transcription factors, including NF-κB. While HUVEC (macrovascular cells) have been largely used in the past, here, we documented an NF-κB gene signature in TNFα-stimulated microvascular endothelial cells HMEC often used in tumor angiogenesis studies. Methodology/Principal Findings We measured mRNA expression of 55 NF-κB related genes using quantitative RT-PCR in HUVEC and HMEC. Our study identified twenty genes markedly up-regulated in response to TNFα, including adhesion molecules, cytokines, chemokines, and apoptosis regulators, some of them being identified as TNF-α-inducible genes for the first time in endothelial cells (two apoptosis regulators, TNFAIP3 and TNFRSF10B/Trail R2 (DR5), the chemokines GM-CSF/CSF2 and MCF/CSF1, and CD40 and TNF-α itself, as well as NF-κB components (RELB, NFKB1or 50/p105 and NFKB2 or p52/p100). For eight genes, the fold induction was much higher in HMEC, as compared to HUVEC. Most importantly, our study described for the first time a connection between NF-κB activation and the induction of most, if not all, of these genes in HMEC as evaluated by pharmacological inhibition and RelA expression knock-down by RNA interference. Moreover, since TNF-α is highly expressed in tumors, we further applied the NF-κB gene signature documented in TNFα-stimulated endothelial cells to human breast tumors. We found a significant positive correlation between TNF and the majority (85 %) of the identified endothelial TNF-induced genes in a well-defined series of 96 (48 ERα positive and 48 ERα negative) breast tumors. Conclusion/Significance Taken together these data suggest the potential use of this NF-κB gene signature in analyzing the role of TNF-α in the endothelial dysfunction, as well as in breast tumors independently of the presence of ERα. PMID:21754991

Dynamics of VEGF matrix-retention in vascular network patterning

NASA Astrophysics Data System (ADS)

Köhn-Luque, A.; de Back, W.; Yamaguchi, Y.; Yoshimura, K.; Herrero, M. A.; Miura, T.

2013-12-01

Vascular endothelial growth factor (VEGF) is a central regulator of blood vessel morphogenesis, although its role in patterning of endothelial cells into vascular networks is not fully understood. It has been suggested that binding of soluble VEGF to extracellular matrix components causes spatially restricted cues that guide endothelial cells into network patterns. Yet, current evidence for such a mechanism remains indirect. In this study, we quantitatively analyse the dynamics of VEGF retention in a controlled in vitro situation of human umbilical vascular endothelial cells (HUVECs) in Matrigel. We show that fluorescent VEGF accumulates in pericellular areas and colocalizes with VEGF binding molecules. Analysis of fluorescence recovery after photobleaching reveals that binding/unbinding to matrix molecules dominates VEGF dynamics in the pericellular region. Computational simulations using our experimental measurements of kinetic parameters show that matrix retention of chemotactic signals can lead to the formation of reticular cellular networks on a realistic timescale. Taken together, these results show that VEGF binds to matrix molecules in proximity of HUVECs in Matrigel, and suggest that bound VEGF drives vascular network patterning.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhengfu, He; Hu, Zhang; Huiwen, Miao

The search for safe, effective and affordable therapeutics against non-small cell lung cancer (NSCLC) and other lung cancers is important. Here we explored the potential effect of 1-o-acetylbritannilactone (ABL), a novel extract from Inula britannica-F, on angiogenesis and lung cancer cell growth. We demonstrated that ABL dose-dependently inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, and capillary structure formation of cultured human umbilical vascular endothelial cells (HUVECs). In vivo, ABL administration suppressed VEGF-induced new vasculature formation in Matrigel plugs. For the mechanism investigations, we found that ABL largely inhibited VEGF-mediated activation of Src kinase and focal adhesion kinase (FAK) in HUVECs.more » Furthermore, treatment of A549 NSCLC cells with ABL resulted in cell growth inhibition and Src-FAK in-activation. Significantly, administration of a single dose of ABL (12 mg/kg/day) remarkably suppressed growth of A549 xenografts in nude mice. In vivo microvessels formation and Src activation were also significantly inhibited in ABL-treated xenograft tumors. Taken together, our findings suggest that ABL suppresses angiogenesis and lung cancer cell growth possibly via regulating the VEGFR-Src-FAK signaling. - Highlights: • 1-o-acetylbritannilactone (ABL) inhibits VEGF-induced angiogenesis in vivo. • ABL inhibits VEGF-induced HUVEC migration, proliferation, capillary tube formation. • ABL inhibits VEGF-mediated activation of Src and FAK in HUVECs. • ABL inhibits growth and Src-FAK activation in A549 cells. • ABL administration inhibits A549 tumor angiogenesis and growth in nude mice.« less

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

PubMed

Behjati, Mohaddeseh; Hashemi, Mohammad; Kazemi, Mohammad; Salehi, Mansoor; Javanmard, Shaghayegh Haghjooy

2017-01-01

Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis. Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured. ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels ( P = 0.09) and vWF ( P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue. The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor/c-kit pathway

PubMed Central

Elmasri, Harun; Ghelfi, Elisa; Yu, Chen-wei; Traphagen, Samantha; Cernadas, Manuela; Cao, Haiming; Shi, Guo-Ping; Plutzky, Jorge; Sahin, Mustafa; Hotamisligil, Gokhan; Cataltepe, Sule

2013-01-01

Fatty acid binding protein 4 (FABP4) plays an important role in regulation of glucose and lipid homeostasis as well as inflammation through its actions in adipocytes and macrophages. FABP4 is also expressed in a subset of endothelial cells, but its role in this cell type is not known. We found that FABP4-deficient human umbilical vein endothelial cells (HUVECs) demonstrate a markedly increased susceptibility to apoptosis as well as decreased migration and capillary network formation. Aortic rings from FABP4−/− mice demonstrated decreased angiogenic sprouting, which was recovered by reconstitution of FABP4. FABP4 was strongly regulated by mTORC1 and inhibited by Rapamycin. FABP4 modulated activation of several important signaling pathways in HUVECs, including downregulation of P38, eNOS, and stem cell factor (SCF)/c-kit signaling. Of these, the SCF/c-kit pathway was found to have a major role in attenuated angiogenic activity of FABP4-deficient ECs as provision of exogenous SCF resulted in a significant recovery in cell proliferation, survival, morphogenesis, and aortic ring sprouting. These data unravel a novel pro-angiogenic role for endothelial cell-FABP4 and suggest that it could be exploited as a potential target for diseases associated with pathological angiogenesis. PMID:22562362

Characteristics of tumor and host cells in 3-D simulated microgravity environment

NASA Astrophysics Data System (ADS)

Chopra, V.; Dinh, T.; Wood, T.; Pellis, N.; Hannigan, E.

Co-cultures of three-dimensional (3-D) constructs of one cell type with dispersed cells of a second cell type in low-shear rotating suspension cultures in simulated microgravity environment have been used to investigate invasive properties of normal and malignant cell types. We have shown that the epithelial and endothelial cells undergo a switch in characteristics when grown in an in vitro 3-D environment, that mimics the in vivo host environment as compared with conventional two-dimensional (2-D) monolayer cultures. Histological preparations and immunohistochemical staining procedures of cocultured harvests demonstrated various markers of interest: like collagen vimentin, mucin, elastin, fibrin, fibrinogen, cytokeratin, adhesion molecules and various angiogenic factors by tumor cells from gynecological cancer patients along with fibroblasts, endothelial cells and patient-derived mononuclear cells (n=8). The growth rate was enhanced 10-15 folds by 3-D cocultures of patient-derived cells as compared with 2-D monolayer cultures and 3-D monocultures. The production of interleukin-2, interleukin-6, interleukin -8, vascular endothelial cell growth factor, basic fibroblast growth factor, and angiogenin was studied by using ELISA and RT- PCR. Human umbilical vein-derived endothelial cell (HUVEC) were used to study the mitogenic response of the conditioned medium collected from 3-D monocultures and cocultures during proliferation and migration assays. The conditioned medium collected from 3-D cocultures of cancer cells also 1) increased the expression of message levels of vascular endothelial growth factor and its receptor flt-1 and KDR was observed by HUVEC, and 2) increased the expression of intracellular and vascular cell adhesion molecules on the surface of HUVEC, when measured by using Live cell ELISA assays and immunofluorescent staining as compared with 3-D monocultures of normal epithelial cells. There was an increase in production of 1) enzymatic activity that could generate bioactive angiostatin from purified human plasminogen, and 2) fibrin (red), mucin (blue), and elastic fiber (black) by cell aggregates of 3-D monocultures of patient-derived cells as compared with 3-D monocultures of normal epithelial cells. This coculture system can be used to study the effectiveness of various antiangiogenic agents on endothelial cell proliferation and migration and also the interaction of multiple cell types in a cost effective fashion, since it provides new insights into the invasive process and its effects on both invading and invaded cells.

An affordable method to obtain cultured endothelial cells from peripheral blood

PubMed Central

Bueno-Betí, Carlos; Novella, Susana; Lázaro-Franco, Macarena; Pérez-Cremades, Daniel; Heras, Magda; Sanchís, Juan; Hermenegildo, Carlos

2013-01-01

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols. PMID:24118735

Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms.

PubMed

Meschiari, Cesar A; Izidoro-Toledo, Tatiane; Gerlach, Raquel F; Tanus-Santos, Jose E

2013-06-01

Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10-400 μM) or SNAP (50-400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs' (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases.

Muramyl dipeptide (MDP) induces reactive oxygen species (ROS) generation via the NOD2/COX-2/NOX4 signaling pathway in human umbilical vein endothelial cells (HUVECs).

PubMed

Kong, Ling-Jun; Liu, Xiao-Qian; Xue, Ying; Gao, Wei; Lv, Qian-Zhou

2018-03-20

Vascular endothelium dysfunction caused by oxidative stress accelerates the pathologic process of cardiovascular diseases. NOD2, an essential receptor of innate immune system, has been demonstrated to play a critical role in atherosclerosis. Here, the aim of our study was to investigate the effect and underlying molecular mechanism of muramyl dipeptide (MDP) on NOX4-mediated ROS generation in human umbilical vein endothelial cells (HUVECs). 2,7-dichlorofluorescein diacetate staining was to measure the intracellular ROS level and showed MDP promoted ROS production in a time- and dose-dependent manner. The mRNA and protein levels of NOX4 and COX-2 were detected by real-time PCR and western blot. Small interfering RNA (siRNA) was used to silence NOD2 or COX-2 gene expression and investigate the mechanism of NOD2-mediated signaling pathway in HUVECs. Data showed that MDP induced NOX4 and COX-2 expression in a time- and dose-dependent manner. NOD2 knock-down suppressed up-regulation of COX-2 and NOX4 in HUVECs treated with MDP. Furthermore, silence of COX-2 in HUVECs down-regulated the NOX4 expression after MDP stimulation. Collectively, we indicated that NOD2 played a leading role in MDP-induced COX-2/NOX4/ROS signaling pathway in HUVECs, which was a novel regulatory mechanism in the progress of ROS generation.

Synthesis and preliminary biological evaluation of novel taspine derivatives as anticancer agents.

PubMed

Zhang, Jie; Zhang, Yanmin; Shan, Yuanyuan; Li, Na; Ma, Wei; He, Langchong

2010-07-01

Antiangiogenic therapy might represent a new promising anticancer therapeutic strategy. Taspine can significantly inhibit cell proliferation of human umbilical vein endothelial cells (HUVECs) induced by vascular endothelial growth factor-165, which is crucial for angiogenesis. In this study, a series of novel taspine derivatives were synthesized and screened for in vitro anticancer and antiangiogenesis activities. The majority of the derivatives demonstrated a moderate degree of cytotoxicity against human cancer cell lines. One of them (14) exhibited much better antiproliferative activity against CACO-2 (IC(50)=52.5microM) and ECV304 (IC(50)=2.67microM) cells than taspine did. Some of them were also effective in antiproliferative assays against HUVECs. The in silico estimate of solubility of title compounds were higher than that of taspine. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

N-butanol extracts of Morinda citrifolia suppress advanced glycation end products (AGE)-induced inflammatory reactions in endothelial cells through its anti-oxidative properties.

PubMed

Ishibashi, Yuji; Matsui, Takanori; Isami, Fumiyuki; Abe, Yumi; Sakaguchi, Tatsuya; Higashimoto, Yuichiro; Yamagishi, Sho-Ichi

2017-03-04

Advanced glycation end products (AGEs), senescent macroprotein derivatives formed during a normal aging process and acceleratedly under diabetic conditions, play a role in atherosclerotic cardiovascular disease. AGEs cause endothelial cell (EC) damage, an initial trigger for atherosclerosis through the interaction with a receptor for AGEs (RAGE). We have previously shown that n-butanol extracts of Morinda citrifolia (noni), a plant belonging to the family Rubiaceae, block the binding of AGEs to RAGE in vitro. In this study, we examined the effects of n-butanol extracts of noni on reactive oxygen species (ROS) generation and inflammatory reactions on AGE-exposed human umbilical vein ECs (HUVECs). HUVECs were treated with 100 μg/ml AGE-bovine serum albumin (AGE-BSA) or non-glycated BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni for 4 h. Then ROS generation and inflammatory and gene expression in HUVECs were evaluated by dihydroethidium staining and real-time reverse transcription-polymerase chain reaction analyses, respectively. THP-1 cell adhesion to HUVECs was measured after 2-day incubation of AGE-BSA or BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni. N-butanol extracts of noni at 670 ng/ml significantly inhibited the AGE-induced ROS generation and RAGE, intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 gene expressions in HUVECs. AGEs significantly increased monocytic THP-1 cell adhesion to HUVECs, which was also prevented by 670 ng/ml n-butanol extracts of noni. The present study demonstrated for the first time that N-butanol extracts of noni could suppress the AGE-induced inflammatory reactions in HUVECs through its anti-oxidative properties via blocking of the interaction of AGEs with RAGE. Inhibition of the AGE-RAGE axis by n-butanol extracts of noni may be a novel nutraceutical strategy for the treatment of cardiovascular disease.

Nicotinic acid inhibits NLRP3 inflammasome activation via SIRT1 in vascular endothelial cells.

PubMed

Li, Yanxiang; Yang, Guangde; Yang, Xiaofeng; Wang, Weirong; Zhang, Jiye; He, Yanhao; Zhang, Wei; Jing, Ting; Lin, Rong

2016-11-01

Emerging evidences indicated that NLRP3 inflammasome initiates inflammatory response involved in cardiovascular disease. Nicotinic acid (NA) has been known to possess potential anti-inflammatory property. The aim of this study was to investigate the effect of NA on the activation of NLRP3 inflammasome and the underlying mechanisms. It was found that lipopolysaccharide (LPS) and adenosine triphosphate (ATP) triggered the activation of NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs). NA inhibited NLRP3 inflammasome activation and subsequent caspase-1 cleavage as well as interleukin (IL)-1β secretion. Moreover, NA administration up-regulated SIRT1 expression in HUVECs stimulated with LPS plus ATP. Importantly, knockdown of SIRT1 reversed the inhibitory effect of NA on the activation of NLRP3 inflammasome. Further study revealed that NA also decreased the generation of reactive oxygen species (ROS) in HUVECs. In addition, NA inhibited NLRP3 inflammasome activation partly through suppression of ROS. Taken together, these findings indicate that NA is able to regulate the activation of NLRP3 inflammasome in HUVECs, which may be partly mediated by SIRT1 and ROS. Copyright © 2016 Elsevier B.V. All rights reserved.

Protective effects of 6-Gingerol on vascular endothelial cell injury induced by high glucose via activation of PI3K-AKT-eNOS pathway in human umbilical vein endothelial cells.

PubMed

Liu, Dan; Wu, Mengqing; Lu, Yi; Xian, Tao; Wang, Yupeng; Huang, Bowei; Zeng, Guohua; Huang, Qiren

2017-09-01

6-Gingerol (6-Gin), an active constituent of Zingiber officinale, has been reported to have anti-inflammatory, anti-oxidative, anti-cancerous etc. bioactivities. However, little is known about its endothelial protective effects and the underlying mechanisms. In this study, our purpose was to investigate the protective effects of 6-Gin and its underlying mechanisms. HUVECs were exposed to high glucose (HG, 33mM glucose) for 48h, followed by 50μM 6-Gin with or without LY294002 (10μM), AKT inhibitor IV (0.5μM) or L-NAME (5mM) for another 24h. Cell viability, levels of NO, LDH and ROS were detected. In addition, the expression levels of IKK, IRS-1, PI3K, AKT, eNOS and their phosphorylated proteins were measured by western blots. Compared with the control, HUVECs were significantly impaired by HG, characterized by decreased levels of the cell viability, NO, pY458-PI3K, pS473-AKT and pS1177-eNOS while increased levels of LDH, pS176-IKK, and p-S312-IRS-1. Conversely, 6-Gin remarkably protected HUVECs against HG-induced injury in a concentration- and time-dependent manner. However, the protective effects of 6-Gin were abolished by co-treatment with LY294002, AKT inhibitor IV or L-NAME at the HG state. Collectively, 6-Gin attenuated the injury of HUVECs induced by HG through the activation of PI3K-AKT-eNOS signal pathway. The findings provide a novel potential for 6-Gin to prevent and treat the angiopathy resulting from diabetes mellitus. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Circular RNA hsa_circ_0010729 regulates vascular endothelial cell proliferation and apoptosis by targeting the miR-186/HIF-1α axis.

PubMed

Dang, Rui-Ying; Liu, Feng-Li; Li, Yan

2017-08-19

Circular RNAs (circRNAs) are a group of non-protein-coding RNAs generated from back splicing. Emerging evidence has demonstrated its vital regulation on angiogenesis. However, the underlying mechanism responsible for circRNAs effects on vascular endothelial cells is still unclear. In the present study, we screened the expression profiles and investigated the physiological role of circRNAs in hypoxia-induced human umbilical vein endothelial cells (HUVECs). Using circRNA microarray analysis, we identified 36 circRNAs that were significantly dysregulated including 14 down-regulated circRNAs and 22 up-regulated with 2-fold change (P < 0.05). From the over-expressed circRNAs, hsa_circ_0010729 was selected as candidate circRNA and which was validated to be significantly up-regulated using RT-PCR. In loss-of-function experiments of HUVECs, hsa_circ_0010729 knockdown suppressed the proliferation and migration ability and enhanced apoptosis. Bioinformatic prediction and luciferase assay revealed that hsa_circ_0010729 and hypoxia inducible factor 1 alpha (HIF-1α) were targeted by miR-186. Validation experiments verified that hsa_circ_0010729 was co-expressed with HIF-1α, being negatively correlated with miR-186. Moreover, rescue experiments demonstrated that miR-186 inhibitor could reverse the role of hsa_circ_0010729 knockdown on HUVECs progression. Overall, the present study identifies the crucial regulation of hsa_circ_0010729 on vascular endothelial cell proliferation and apoptosis via targeting miR-186/HIF-1α axis. Copyright © 2017 Elsevier Inc. All rights reserved.

Progranulin protects vascular endothelium against atherosclerotic inflammatory reaction via Akt/eNOS and nuclear factor-κB pathways.

PubMed

Hwang, Hwan-Jin; Jung, Tae Woo; Hong, Ho Cheol; Choi, Hae Yoon; Seo, Ji-A; Kim, Sin Gon; Kim, Nan Hee; Choi, Kyung Mook; Choi, Dong Seop; Baik, Sei Hyun; Yoo, Hye Jin

2013-01-01

Atherosclerosis is considered a chronic inflammatory disease, initiated by activation and dysfunction of the endothelium. Recently, progranulin has been regarded as an important modulator of inflammatory processes; however, the role for prgranulin in regulating inflammation in vascular endothelial cells has not been described. Signaling pathways mediated by progranulin were analyzed in human umbilical vein endothelial cells (HUVECs) treated with progranulin. Progranulin significantly induced Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs, an effect that was blocked with Akt inhibitor. Furthermore, nitric oxide (NO) level, the end product of Akt/eNOS pathway, was significantly upregulated after progranulin treatment. Next, we showed that progranulin efficiently inhibited lipopolysaccharide (LPS)-mediated pro-inflammatory signaling. LPS-induced phosphorylation of IκB and nuclear factor-κB (NF-κB) levels decreased after progranulin treatment. Also, progranulin blocked translocation of NF-κB from the cytosol to the nucleus. In addition, progranulin significantly reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by inhibiting binding of NF- κB to their promoter regions and blocked attachment of monocytes to HUVECs. Progranulin also significantly reduced the expression of tumor necrosis factor receptor-α (TNF-α) and monocyte chemo-attractant protein-1 (MCP-1), the crucial inflammatory molecules known to aggravate atherosclerosis. Progranulin efficiently inhibited LPS-mediated pro-inflammatory signaling in endothelial cells through activation of the Akt/eNOS pathway and attenuation of the NF-κB pathway, suggesting its protective roles in vascular endothelium against inflammatory reaction underlying atherosclerosis.

[Effect of microRNA-34a/SIRT1/p53 signal pathway on notoginsenoside R₁ delaying vascular endothelial cell senescence].

PubMed

Lai, Xiao-Hua; Lei, Yan; Yang, Jing; Xiu, Cheng-Kui

2018-02-01

This study aimed to investigate the effect of notoginsenoside R₁ in delaying H₂O₂-induced vascular endothelial cell senescence through microRNA-34a/SIRT1/p53 signal pathway. In this study， human umbilical vein endothelial cells(HUVECs) were selected as the study object; the aging model induced by hydrogen peroxide(H₂O₂) was established， with resveratrol as the positive drug. HUVECs were randomly divided into four groups， youth group， senescence model group， notoginsenoside R₁ group and resveratrol group. Notoginsenoside R₁ group and resveratrol group were modeled with 100 μmoL·L⁻¹ H₂O₂ for 4 h after 24 h treatment with notoginsenoside R₁(30 μmoL·L⁻¹) and resveratrol(10 μmoL·L⁻¹) respectively. At the end， each group was cultured with complete medium for 24 h. The degree of cellular senescence was detected by senescence-associated β-galactosidase(SA-β-Gal) staining kit， the cell viability was detected by cell counting kit-8， the cell cycle distribution was analyzed by flow cytometry， and the cellular SOD activity was detected by WST-1 method in each group. The expressions of SIRT1， p53， p21 and p16 proteins in HUVECs were detected by Western blot. In addition， the mRNA expressions of miRNA-34a， SIRT1 and p53 in HUVECs were assayed by Real-time PCR. These results indicated that notoginsenoside R₁ significantly reduced the positive staining rate of senescent cells， enhanced the cell proliferation capacity and intracellular SOD activity， decreased the proportion of cells in G₀/G₁ phase， and increased the percentage of cells in S phase simultaneously compared with the senescence model group. Moreover， notoginsenoside R₁ decreased the mRNA expressions of miRNA-34a and p53 and the protein expression of p53， p21 and p16.At the same time， notoginsenoside R₁ increased the protein and mRNA expressions of SIRT1. The differences in these results between the senescence model group and the notoginsenoside R₁ group were statistically significant( P <0.05). However， there was not statistically significant difference in these results between the notoginsenoside R₁ group and the resveratrol group. In conclusion， the senescence of endothelial cells induced by H₂O₂ can be used as a model for studying aging. Notoginsenoside R₁ has an obvious anti-aging effect on vascular endothelial cells in this study. The possible mechanism is that notoginsenoside R₁ can delay the senescence process of vascular endothelial cells induced by H₂O₂ by regulating microRNA-34a/SIRT1/p53 signal pathway. Copyright© by the Chinese Pharmaceutical Association.

Knockdown of long noncoding RNA XIST alleviates oxidative low-density lipoprotein-mediated endothelial cells injury through modulation of miR-320/NOD2 axis.

PubMed

Xu, Xiaohui; Ma, Congmin; Liu, Chao; Duan, Zhihui; Zhang, Li

2018-06-14

Atherosclerosis remains to be one of the most common vascular disorders resulting in morbidity and mortality in the world. Recent studies suggested that endothelial cells (ECs) injury caused by oxidative low-density lipoprotein (ox-LDL) is an early marker for atherosclerosis. Nevertheless, the mechanisms of ox-LDL-induced ECs injury are complicated and largely unknown. Here, we found lncRNA XIST (X-inactive specific transcript) was upregulated in human umbilical vein endothelial cells (HUVECs) stimulated by ox-LDL. Knockdown of XIST boosted the cell viability and suppressed cell apoptosis under ox-LDL stimuli. Further experiments identified XIST regulated the expression of Nucleotide-Binding Oligomerization Domain 2 (NOD2) by sponging miR-320. XIST silencing exerted a protective effect on ox-LDL-induced HUVECs injury via miR-320/NOD2 regulatory network. Our data provide insight into the role of the lncRNA XIST in ox-LDL mediated ECs injury, which can aid in developing new therapeutic strategies for the treatment of atherosclerosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Presence of MUC4 in human milk and at the luminal surfaces of blood vessels.

PubMed

Zhang, Jin; Perez, Aymee; Yasin, Mohammad; Soto, Pedro; Rong, Min; Theodoropoulos, George; Carothers Carraway, Coralie A; Carraway, Kermit L

2005-07-01

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. Surprisingly, development and characterization of a new monoclonal antibody (mAb), called 1G8, against ASGP-2 demonstrated by immunohistochemistry the presence of MUC4 at the luminal surfaces of blood vessels of both normal tissues and tumors. Muc4 was detected with 1G8 and other Muc4 antibodies in blood vessels from humans, rats and mice. This expression of MUC4 in endothelial cells was confirmed by immunoblotting with 1G8 in human umbilical vein endothelial cells (HUVECs), human iliac artery endothelial cells (HIAECs), and human microvascular endothelial cells (HMVECs). MUC4 could be observed on HUVECs grown on either plastic or Matrigel. Finally, MUC4 expression in the three types of endothelial cell lines was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These results provide, to our knowledge, the first demonstration of a member of the MUC gene family and membrane mucin in blood vessels. As a luminal surface component, the MUC4 is situated to contribute to the non-adhesive luminal surface and to act as an intrinsic protection and survival factor. (c) 2004 Wiley-Liss, Inc.

Longxuetongluo capsule inhibits atherosclerosis progression in high-fat diet-induced ApoE-/- mice by improving endothelial dysfunction.

PubMed

Zheng, Jiao; Liu, Binglin; Lun, Qixing; Gu, Xiaopan; Pan, Bo; Zhao, Yunfang; Xiao, Wei; Li, Jun; Tu, Pengfei

2016-12-01

Chinese dragon's blood has been used to treat blood stasis for thousands of years. Its total phenolic extract (Longxuetongluo capsule, LTC) is used for the treatment of ischemic stroke; however, its protective effect against atherosclerosis remains poorly understood. This paper aims to investigate the antiatherosclerotic effect of LTC and the underlying mechanisms in high-fat diet (HFD)-induced ApoE -/- mice. The levels of plasma lipid and areas of atherosclerotic lesions in the aortic sinus in ApoE -/- mice were evaluated. The effect of LTC on the nitric oxide (NO) production in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs) was determined. The adhesion of monocytes to ox-LDL-stimulated HUVECs was further studied. LTC at low, medium, and high doses markedly decreased the atherosclerotic lesion areas of the aortic sinus in HFD-induced ApoE -/- mice by 26.4% (p < 0.05), 30.1% (p < 0.05), and 46.5% (p < 0.01), respectively, although it did not improve the dyslipidemia. Furthermore, LTC restored the diminished NO production of ox-LDL-stimulated HUVECs (p < 0.001) and inhibited the adhesion between monocytes and endothelial cells (p < 0.01). LTC appeared to alleviate ox-LDL-stimulated dysfunction of HUVECs, and inhibit the adhesion of monocytes to HUVECs via the MAPK/IKK/IκB/NF-κB signaling pathway, thus decrease atherosclerotic lesions in the aortic sinus in HFD-induced ApoE -/- mice. These findings suggest the potential of LTC for use as an effective agent against atherosclerosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia.

PubMed

Ferrelli, Francesca; Pastore, Donatella; Capuani, Barbara; Lombardo, Marco F; Blot-Chabaud, Marcel; Coppola, Andrea; Basello, Katia; Galli, Angelica; Donadel, Giulia; Romano, Maria; Caratelli, Sara; Pacifici, Francesca; Arriga, Roberto; Di Daniele, Nicola; Sbraccia, Paolo; Sconocchia, Giuseppe; Bellia, Alfonso; Tesauro, Manfredi; Federici, Massimo; Della-Morte, David; Lauro, Davide

2015-02-01

Diabetic hyperglycaemia causes endothelial dysfunction mainly by impairing endothelial nitric oxide (NO) production. Moreover, hyperglycaemia activates several noxious cellular pathways including apoptosis, increase in reactive oxygen species (ROS) levels and diminishing Na(+)-K(+) ATPase activity which exacerbate vascular damage. Serum glucocorticoid kinase (SGK)-1, a member of the serine/threonine kinases, plays a pivotal role in regulating NO production through inducible NO synthase activation and other cellular mechanisms. Therefore, in this study, we aimed to investigate the protective role of SGK-1 against hyperglycaemia in human umbilical endothelial cells (HUVECs). We used retrovirus to infect HUVECs with either SGK-1, SGK-1Δ60 (lacking of the N-60 amino acids-increase SGK-1 activity) or SGK-1Δ60KD (kinase-dead constructs). We tested our hypothesis in vitro after high glucose and glucosamine incubation. Increase in SGK-1 expression and activity (SGK-1Δ60) resulted in higher production of NO, inhibition of ROS synthesis and lower apoptosis in endothelial cell after either hyperglycaemia or glucosamine treatments. Moreover, in this study, we showed increased GLUT-1 membrane translocation and Na(+)-K(+) ATPase activity in cell infected with SGK-1Δ60 construct. These results suggest that as in endothelial cells, an increased SGK-1 activity and expression reduces oxidative stress, improves cell survival and restores insulin-mediated NO production after different noxae stimuli. Therefore, SGK-1 may represent a specific target to further develop novel therapeutic options against diabetic vascular disease.

Distributed vasculogenesis from modular agarose-hydroxyapatite-fibrinogen microbeads.

PubMed

Rioja, Ana Y; Daley, Ethan L H; Habif, Julia C; Putnam, Andrew J; Stegemann, Jan P

2017-06-01

Critical limb ischemia impairs circulation to the extremities, causing pain, disrupted wound healing, and potential tissue necrosis. Therapeutic angiogenesis seeks to repair the damaged microvasculature directly to restore blood flow. In this study, we developed modular, micro-scale constructs designed to possess robust handling qualities, allow in vitro pre-culture, and promote microvasculature formation. The microbead matrix consisted of an agarose (AG) base to prevent aggregation, combined with cell-adhesive components of fibrinogen (FGN) and/or hydroxyapatite (HA). Microbeads encapsulating a co-culture of human umbilical vein endothelial cells (HUVEC) and fibroblasts were prepared and characterized. Microbeads were generally 80-100µm in diameter, and the size increased with the addition of FGN and HA. Addition of HA increased the yield of microbeads, as well as the homogeneity of distribution of FGN within the matrix. Cell viability was high in all microbead types. When cell-seeded microbeads were embedded in fibrin hydrogels, HUVEC sprouting and inosculation between neighboring microbeads were observed over seven days. Pre-culture of microbeads for an additional seven days prior to embedding in fibrin resulted in significantly greater HUVEC network length in AG+HA+FGN microbeads, as compared to AG, AG+HA or AG+FGN microbeads. Importantly, composite microbeads resulted in more even and widespread endothelial network formation, relative to control microbeads consisting of pure fibrin. These results demonstrate that AG+HA+FGN microbeads support HUVEC sprouting both within and between adjacent microbeads, and can promote distributed vascularization of an external matrix. Such modular microtissues may have utility in treating ischemic tissue by rapidly re-establishing a microvascular network. Critical limb ischemia (CLI) is a chronic disease that can lead to tissue necrosis, amputation, and death. Cell-based therapies are being explored to restore blood flow and prevent the complications of CLI. In this study, we developed small, non-aggregating agarose-hydroxyapatite-fibrinogen microbeads that contained endothelial cells and fibroblasts. Microbeads were easy to handle and culture, and endothelial sprouts formed within and between microbeads. Our data demonstrates that the composition of the microbead matrix altered the degree of endothelial sprouting, and that the addition of hydroxyapatite and fibrinogen resulted in more distributed sprouting compared to pure fibrin microbeads. The microbead format and control of the matrix formulation may therefore be useful in developing revascularization strategies for the treatment of ischemic disease. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

DDAH1 deficiency attenuates endothelial cell cycle progression and angiogenesis.

PubMed

Zhang, Ping; Xu, Xin; Hu, Xinli; Wang, Huan; Fassett, John; Huo, Yuqing; Chen, Yingjie; Bache, Robert J

2013-01-01

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS). ADMA is eliminated largely by the action of dimethylarginine dimethylaminohydrolase1 (DDAH1). Decreased DDAH activity is found in several pathological conditions and is associated with increased risk of vascular disease. Overexpression of DDAH1 has been shown to augment endothelial proliferation and angiogenesis. To better understand the mechanism by which DDAH1 influences endothelial proliferation, this study examined the effect of DDAH1 deficiency on cell cycle progression and the expression of some cell cycle master regulatory proteins. DDAH1 KO decreased in vivo Matrigel angiogenesis and depressed endothelial repair in a mouse model of carotid artery wire injury. DDAH1 deficiency decreased VEGF expression in HUVEC and increased NF1 expression in both HUVEC and DDAH1 KO mice. The expression of active Ras could overcome the decreased VEGF expression caused by the DDAH1 depletion. The addition of VEGF and knockdown NF1 could both restore proliferation in cells with DDAH1 depletion. Flow cytometry analysis revealed that DDAH1 sRNAi knockdown in HUVEC caused G1 and G2/M arrest that was associated with decreased expression of CDC2, CDC25C, cyclin D1 and cyclin E. MEF cells from DDAH1 KO mice also demonstrated G2/M arrest that was associated with decreased cyclin D1 expression and Akt activity. Our findings indicate that DDAH1 exerts effects on cyclin D1 and cyclin E expression through multiple mechanisms, including VEGF, the NO/cGMP/PKG pathway, the Ras/PI3K/Akt pathway, and NF1 expression. Loss of DDAH1 effects on these pathways results in impaired endothelial cell proliferation and decreased angiogenesis. The findings provide background information that may be useful in the development of therapeutic strategies to manipulate DDAH1 expression in cardiovascular diseases or tumor angiogenesis.

Soluble CD40 Ligand Stimulates the Pro-Angiogenic Function of Peripheral Blood Angiogenic Outgrowth Cells via Increased Release of Matrix Metalloproteinase-9

PubMed Central

Bou Khzam, Lara; Boulahya, Rahma; Abou-Saleh, Haissam; Hachem, Ahmed; Zaid, Younes; Merhi, Yahye

2013-01-01

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway. PMID:24358353

Soluble CD40 ligand stimulates the pro-angiogenic function of peripheral blood angiogenic outgrowth cells via increased release of matrix metalloproteinase-9.

PubMed

Bou Khzam, Lara; Boulahya, Rahma; Abou-Saleh, Haissam; Hachem, Ahmed; Zaid, Younes; Merhi, Yahye

2013-01-01

The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway.

DDAH1 Deficiency Attenuates Endothelial Cell Cycle Progression and Angiogenesis

PubMed Central

Zhang, Ping; Xu, Xin; Hu, Xinli; Wang, Huan; Fassett, John; Huo, Yuqing; Chen, Yingjie; Bache, Robert J.

2013-01-01

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase (NOS). ADMA is eliminated largely by the action of dimethylarginine dimethylaminohydrolase1 (DDAH1). Decreased DDAH activity is found in several pathological conditions and is associated with increased risk of vascular disease. Overexpression of DDAH1 has been shown to augment endothelial proliferation and angiogenesis. To better understand the mechanism by which DDAH1 influences endothelial proliferation, this study examined the effect of DDAH1 deficiency on cell cycle progression and the expression of some cell cycle master regulatory proteins. DDAH1 KO decreased in vivo Matrigel angiogenesis and depressed endothelial repair in a mouse model of carotid artery wire injury. DDAH1 deficiency decreased VEGF expression in HUVEC and increased NF1 expression in both HUVEC and DDAH1 KO mice. The expression of active Ras could overcome the decreased VEGF expression caused by the DDAH1 depletion. The addition of VEGF and knockdown NF1 could both restore proliferation in cells with DDAH1 depletion. Flow cytometry analysis revealed that DDAH1 sRNAi knockdown in HUVEC caused G1 and G2/M arrest that was associated with decreased expression of CDC2, CDC25C, cyclin D1 and cyclin E. MEF cells from DDAH1 KO mice also demonstrated G2/M arrest that was associated with decreased cyclin D1 expression and Akt activity. Our findings indicate that DDAH1 exerts effects on cyclin D1 and cyclin E expression through multiple mechanisms, including VEGF, the NO/cGMP/PKG pathway, the Ras/PI3K/Akt pathway, and NF1 expression. Loss of DDAH1 effects on these pathways results in impaired endothelial cell proliferation and decreased angiogenesis. The findings provide background information that may be useful in the development of therapeutic strategies to manipulate DDAH1 expression in cardiovascular diseases or tumor angiogenesis. PMID:24260221

Naringin Protects Against High Glucose-Induced Human Endothelial Cell Injury Via Antioxidation and CX3CL1 Downregulation.

PubMed

Li, Guilin; Xu, Yurong; Sheng, Xuan; Liu, Hua; Guo, Jingjing; Wang, Jiayue; Zhong, Qi; Jiang, Huaide; Zheng, Chaoran; Tan, Mengxia; Rao, Shenqiang; Yu, Yanling; Gao, Yun; Li, Guodong; Liang, Shangdong; Zhu, Gaochun

2017-01-01

The induction of endothelial injury by hyperglycemia in diabetes has been widely accepted. Naringin is a bio-flavonoid. Some studies showed that naringin alleviates diabetic complications, but the exact mechanisms by which naringin improves diabetic anomalies are not yet fully understood. The aim of this research was to study the protective effect of naringin on high glucose-induced injury of human umbilical vein endothelial cells (HUVECs). HUVECs were cultured with or without high glucose in the absence or presence of naringin for 5 days. The expression of CX3CL1 was determined by quantitative real-time RT-PCR (qPCR) and western blot. The cellular bioenergetic analysis oxygen consumption rate (OCR) was measured with a Seahorse Bioscience XF analyzer. The production of reactive oxygen species (ROS), the expression of CX3CL1 and the level of AKT phosphorylation were increased in HUVECs cultured with high glucose compared with controls. However, naringin rescued these increases in ROS production, CX3CL1 expression and AKT phosphorylation. Nitric oxide (NO) production and OCR were lower in the high glucose group, and naringin restored the changes induced by high glucose. Molecular docking results suggested that Naringin might interact with the CX3CL1 protein. Naringin protects HUVECs from high-glucose-induced damage through its antioxidant properties by downregulating CX3CL1 and by improving mitochondrial function. © 2017 The Author(s). Published by S. Karger AG, Basel.

Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling

PubMed Central

Baljinnyam, Erdene; Umemura, Masanari; Chuang, Christine; De Lorenzo, Mariana S; Iwatsubo, Mizuka; Chen, Suzie; Goydos, James S; Ishikawa, Yoshihiro; Whitelock, John M; Iwatsubo, Kousaku

2014-01-01

Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2-mediated cell–cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM-induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N-sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS–FGF2-mediated cell–cell communication. PMID:24725364

The role of cPLA2 in Methylglyoxal-induced cell apoptosis of HUVECs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Jie; Zhu, Chao; Hong, Yali

2017-05-15

Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is mainly formed as a byproduct of glycolysis. Elevated MGO level is known to induce apoptosis of vascular endothelial cells, which is implicated with progression of atherosclerosis and diabetic complications. However, the underlying mechanisms have not been exhaustively investigated yet. Here, we further characterized the mechanisms how MGO induced apoptosis in human umbilical vein endothelial cells (HUVECs). Our data revealed that cytosolic phospholipase A2 (cPLA2) played an important role in MGO-induced cell apoptosis. It was found that MGO could increase both the activity and expression of cPLA2. Inhibition of cPLA2 by Pyrrophenone (PYR)more » or siRNA significantly attenuated the MGO-induced apoptosis. Additionally, MGO time-dependently decreased the phosphorylation of nuclear factor κB (NF-κB). Pretreatment of the cells with NF-κB inhibitor, BAY11-7082, further increased MGO-induced apoptosis of HUVECs, indicating that NF-κB played a survival role in this MGO-induced apoptosis. Furthermore, in the presence of si-cPLA2 or PYR, MGO no longer decreased NF-κB phosphorylation. Beyond that, the antioxidant N-acetyl cysteine (NAC) could reverse the changes of both cPLA2 and NF-κB caused by MGO. p38, the upstream of cPLA2, was also significantly phosphorylated by MGO. However, p38 inhibitor failed to reverse the apoptosis induced by MGO. This study gives an important insight into the downstream signaling mechanisms of MGO, cPLA2-NF-κB, in endothelial apoptosis. - Highlights: • cPLA2 participated in MGO-induced HUVECs apoptosis. • Inhibition of NF-κB was involved in MGO-cPLA2-mediated cell apoptosis. • Antioxidant NAC attenuated MGO-induced cPLA2 activation and cell apoptosis.« less

Irisin protects against endothelial injury and ameliorates atherosclerosis in apolipoprotein E-Null diabetic mice.

PubMed

Lu, Junyan; Xiang, Guangda; Liu, Min; Mei, Wen; Xiang, Lin; Dong, Jing

2015-12-01

The circulating irisin increases energy expenditure and improves insulin resistance in mice and humans. The improvement of insulin resistance ameliorates atherosclerosis. Therefore, we hypothesized that irisin alleviates atherosclerosis in diabetes. Endothelial function was measured by acetylcholine-induced endothelium-dependent vasodilation using aortic rings in apolipoprotein E-Null (apoE(-/-)) streptozotocin-induced diabetic mice. Atherosclerotic lesion was evaluated by plaque area and inflammatory response in aortas. In addition, the endothelium-protective effects of irisin were also further investigated in primary human umbilical vein endothelial cells (HUVECs) in vitro. The in vivo experiments showed that irisin treatment significantly improved endothelial dysfunction, decreased endothelial apoptosis, and predominantly decreased atherosclerotic plaque area of both en face and cross sections when compared with normal saline-treated diabetic mice. Moreover, the infiltrating macrophages and T lymphocytes within plaque and the mRNA expression levels of inflammatory cytokines in aortas were also significantly reduced by irisin treatment in mice. The in vitro experiments revealed that irisin inhibited high glucose-induced apoptosis, oxidative stress and increased antioxidant enzymes expression in HUVECs, and pretreatment with LY294002, l-NAME, AMPK-siRNA or eNOS-siRNA, attenuated the protection of irisin on HUVECs apoptosis induced by high glucose. In addition, the in vivo and in vitro experiments showed that irisin increased the phosphorylation of AMPK, Akt and eNOS in aortas and cultured HUVECs. The present study indicates that systemic administration of irisin may be protected against endothelial injury and ameliorated atherosclerosis in apoE(-/-) diabetic mice. The endothelium-protective action of irisin was through activation of AMPK-PI3K-Akt-eNOS signaling pathway. Irisin could be therapeutic for atherosclerotic vascular diseases in diabetes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

PubMed

Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

2009-09-01

Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

Cannabidiol inhibits angiogenesis by multiple mechanisms

PubMed Central

Solinas, M; Massi, P; Cantelmo, AR; Cattaneo, MG; Cammarota, R; Bartolini, D; Cinquina, V; Valenti, M; Vicentini, LM; Noonan, DM; Albini, A; Parolaro, D

2012-01-01

BACKGROUND AND PURPOSE Several studies have demonstrated anti-proliferative and pro-apoptotic actions of cannabinoids on various tumours, together with their anti-angiogenic properties. The non-psychoactive cannabinoid cannabidiol (CBD) effectively inhibits the growth of different types of tumours in vitro and in vivo and down-regulates some pro-angiogenic signals produced by glioma cells. As its anti-angiogenic properties have not been thoroughly investigated to date, and given its very favourable pharmacological and toxicological profile, here, we evaluated the ability of CBD to modulate tumour angiogenesis. EXPERIMENTAL APPROACH Firstly, we evaluated the effect of CBD on human umbilical vein endothelial cell (HUVEC) proliferation and viability – through [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and FACS analysis – and in vitro motility – both in a classical Boyden chamber test and in a wound-healing assay. We next investigated CBD effects on different angiogenesis-related proteins released by HUVECs, using an angiogenesis array kit and an ELISA directed at MMP2. Then we evaluated its effects on in vitro angiogenesis in treated HUVECs invading a Matrigel layer and in HUVEC spheroids embedded into collagen gels, and further characterized its effects in vivo using a Matrigel sponge model of angiogenesis in C57/BL6 mice. KEY RESULTS CBD induced HUVEC cytostasis without inducing apoptosis, inhibited HUVEC migration, invasion and sprouting in vitro, and angiogenesis in vivo in Matrigel sponges. These effects were associated with the down-modulation of several angiogenesis-related molecules. CONCLUSIONS AND IMPLICATIONS This study reveals that CBD inhibits angiogenesis by multiple mechanisms. Its dual effect on both tumour and endothelial cells supports the hypothesis that CBD has potential as an effective agent in cancer therapy. PMID:22624859

Cannabidiol inhibits angiogenesis by multiple mechanisms.

PubMed

Solinas, M; Massi, P; Cantelmo, A R; Cattaneo, M G; Cammarota, R; Bartolini, D; Cinquina, V; Valenti, M; Vicentini, L M; Noonan, D M; Albini, A; Parolaro, D

2012-11-01

Several studies have demonstrated anti-proliferative and pro-apoptotic actions of cannabinoids on various tumours, together with their anti-angiogenic properties. The non-psychoactive cannabinoid cannabidiol (CBD) effectively inhibits the growth of different types of tumours in vitro and in vivo and down-regulates some pro-angiogenic signals produced by glioma cells. As its anti-angiogenic properties have not been thoroughly investigated to date, and given its very favourable pharmacological and toxicological profile, here, we evaluated the ability of CBD to modulate tumour angiogenesis. Firstly, we evaluated the effect of CBD on human umbilical vein endothelial cell (HUVEC) proliferation and viability - through [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and FACS analysis - and in vitro motility - both in a classical Boyden chamber test and in a wound-healing assay. We next investigated CBD effects on different angiogenesis-related proteins released by HUVECs, using an angiogenesis array kit and an ELISA directed at MMP2. Then we evaluated its effects on in vitro angiogenesis in treated HUVECs invading a Matrigel layer and in HUVEC spheroids embedded into collagen gels, and further characterized its effects in vivo using a Matrigel sponge model of angiogenesis in C57/BL6 mice. CBD induced HUVEC cytostasis without inducing apoptosis, inhibited HUVEC migration, invasion and sprouting in vitro, and angiogenesis in vivo in Matrigel sponges. These effects were associated with the down-modulation of several angiogenesis-related molecules. This study reveals that CBD inhibits angiogenesis by multiple mechanisms. Its dual effect on both tumour and endothelial cells supports the hypothesis that CBD has potential as an effective agent in cancer therapy. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Surgical option for the correction of Peyronie's disease: an autologous tissue-engineered endothelialized graft.

PubMed

Imbeault, Annie; Bernard, Geneviève; Ouellet, Gabrielle; Bouhout, Sara; Carrier, Serge; Bolduc, Stéphane

2011-11-01

Surgical treatment is indicated in severe cases of Peyronie's disease. Incision of the plaque with subsequent graft material implantation is the option of choice. Ideal graft tissue is not yet available. To evaluate the use of an autologous tissue-engineered endothelialized graft by the self-assembly method, for tunica albuginea (TA) reconstruction in Peyronie's disease. Two TA models were created. Human fibroblasts were isolated from a skin biopsy and cultured in vitro until formation of fibroblast sheets. After 4 weeks of maturation, human umbilical vein endothelial cells (HUVEC) were seeded on fibroblasts sheets and wrapped around a tubular support to form a cylinder of about 10 layers. After 21 days of tube maturation, HUVEC were seeded into the lumen of the fibroblast tubes for the endothelialized tunica albuginea (ETA). No HUVEC were seeded into the lumen for the TA model. Both constructs were placed under perfusion in a bioreactor for 1 week. Histology, immunohistochemistry, and burst pressure were performed to characterize mature tubular graft. Animal manipulations were also performed to demonstrate the impact of endothelial cells in vivo. Histology showed uniform multilayered fibroblasts. Extracellular matrix, produced entirely by fibroblasts, presented a good staining for collagen 1. Some elastin fibers were also present. For the TA model, anti-human von Willebrand antibody revealed the endothelial cells forming capillary-like structures. TA model reached a burst pressure of 584 mm Hg and ETA model obtained a burst pressure of 719 mm Hg. This tissue-engineered endothelialized tubular graft is structurally similar to normal TA and presents an adequate mechanical resistance. The self-assembly method used and the autologous property of this model could represent an advantage comparatively to other available grafts. Further evaluation including functional testing will be necessary to characterize in vivo implantation and behavior of the graft. © 2011 International Society for Sexual Medicine.

Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.

2008-05-15

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstratedmore » that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.« less

Sulforaphane reduces advanced glycation end products (AGEs)-induced inflammation in endothelial cells and rat aorta.

PubMed

Matsui, T; Nakamura, N; Ojima, A; Nishino, Y; Yamagishi, S-I

2016-09-01

Advanced glycation end products (AGEs)-receptor RAGE interaction evokes oxidative stress and inflammatory reactions, thereby being involved in endothelial cell (EC) damage in diabetes. Sulforaphane is generated from glucoraphanin, a naturally occurring isothiocyanate found in widely consumed cruciferous vegetables, by myrosinase. Sulforaphane has been reported to protect against oxidative stress-mediated cell and tissue injury. However, effects of sulforaphane on AGEs-induced vascular damage remain unclear. In this study, we investigated whether and how sulforaphane could inhibit inflammation in AGEs-exposed human umbilical vein ECs (HUVECs) and AGEs-injected rat aorta. Sulforaphane treatment for 4 or 24 h dose-dependently inhibited the AGEs-induced increase in RAGE, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecular-1 (VCAM-1) gene expression in HUVECs. AGEs significantly stimulated MCP-1 production by, and THP-1 cell adhesion to, HUVECs, both of which were prevented by 1.6 μM sulforaphane. Sulforaphane significantly suppressed oxidative stress generation and NADPH oxidase activation evoked by AGEs in HUVECs. Furthermore, aortic RAGE, ICAM-1 and VCAM-1 expression in AGEs-injected rats were increased, which were suppressed by simultaneous infusion of sulforaphane. The present study demonstrated for the first time that sulforaphane could inhibit inflammation in AGEs-exposed HUVECs and AGEs-infused rat aorta partly by suppressing RAGE expression through its anti-oxidative properties. Inhibition of the AGEs-RAGE axis by sulforaphane might be a novel therapeutic target for vascular injury in diabetes. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

Analysis of biological effects in human endothelial cells after stimulated microgravity

NASA Astrophysics Data System (ADS)

Min, Zhang; Sun, Yeqing; Xu, Dan

Space environment is characterized by strong radiation, ultra-high vacuum, weak magnetic field and microgravity. Among them, microgravity (10-4-10-6g) in space is different from gravity (1g) on earth, possibly causing visual disorders, muscle alterations, bone loss and dysfunction of cardiovascular systems. To study about microgravity environment, the most advanced rotary cell culture system (RCCS-1) was used to do stimulated microgravity (SMG) experiments in the ground. Up to now, most of studies focus on the biological effects under stimulated microgravity, but it is less known about the cellular response after stimulated microgravity. In the present study, we explored the subsequent effects of stimulated microgravity on human endothelial cells (HUVEC-C) after these cells were cultured on RCCS-1 for 48 hours. We co-cultured HUVEC-C cells with Hillex-microcarriers in 60-mm culture dishes for 24h, followed by transferring them to RCCS-1 so that cells remain to be the state of SMG. In parallel, HUVEC-C cells were co-cultured with microcarriers in the ground condition. We found that stimulated microgravity induced cytoskeleton remodeling, cell cycle G2/M arrest and cellular senescence, consistent with previous reports. To study the subsequent effects of stimulated microgravity, we make cells detach from microcarriers and observed various effects including cell growth, cell adhesion, cytoskeleton, cell cycle, apoptosis and senescence. The results showed that those cells undergoing stimulated microgravity appeared obvious growth inhibition, a transition from the decrease in cell adhesion ability and cytoskeleton remodeling within 24h to induction of apoptosis and senescence-like phenotype in the later time with slight changes in cell cycle. Analysis of protein expression in western blot demonstrated that apoptosis-related protein PTEN was up-regulated on the time-dependent pattern after stimulated microgravity, indicating that PTEN-PI3K-Akt pathway might play an important role in apoptosis. Our study suggests that stimulated microgravity has the subsequent biological effects of HUVEC-C, providing new insight of understanding the global effect of microgravity on cellular response in human endothelial cells.

New Natural Pigment Fraction Isolated from Saw Palmetto: Potential for Adjuvant Therapy of Hepatocellular Carcinoma.

PubMed

Tan, Hor-Yue; Wang, Ning; Takahashi, Masao; Feng, Yigang; Li, Hongyun; Feng, Yibin

2016-08-05

For the first time, we discovered a small proportion of aqueous fraction from Saw Palmetto apart from the fatty acid-rich fraction exhibited pharmacological activity. Therefore, this study aims to explore the anti-tumor potential of red pigmented aqueous fraction of Saw Palmetto, NYG on human hepatocellular carcinoma and its possible targets. Subcutaneous xenograft and orthotopic implantation models of HCC were used to evaluate the tumor inhibitory effect of NYG. Human hepatocellular carcinoma (HCC) cell lines and human umbilical vein endothelial cells (HUVEC) were used as in vitro model. The mRNA expression was conducted by qPCR. Protein expression was monitored by immunoblotting and immunohistochemistry. Cell migration and blood vessel formation were determined by chamber assay and tube formation assay, respectively. Significant tumor inhibition of NYG in dose-dependent manner was observed on subcutaneous xenograft and orthotopic HCC model. NYG has no direct action on cell viability or VEGF secretion of HCC cells. However, NYG reduced in vitro migration and vessel formation activities of HUVEC cells, as well as in vivo intratumoral neovascularization. NYG attenuated extracellular signal-regulated kinases (ERK) activation in endothelial cells, which may be associated with the suppression of migration and tube formation of HUVEC. NYG suppressed tumor expansion of HCC via inhibiting neovascularization, and may be potential adjuvant treatment for HCC.

The Role of Membrane-Derived Second Messengers and Bmx/Etk in Response to Radiation Treatment of Prostate Cancer

DTIC Science & Technology

2008-01-01

enhanced HUVEC radiosensitization. Furthermore, pretreatment of HUVEC with a pharmacological inhibitor of Bmx, LFM-A13, produced significant...Prostate cancer, Bmx, tyrosine kinase, kinase inhibitors , angiogenesis, tumor vasculature, radiation 16. SECURITY CLASSIFICATION OF: 17...activation and that a small molecule inhibitor of Bmx modulates the cellular viability of endothelial and prostate cancer cells, particularly with radiation

[Interaction between glycogen synthase kinase-3β and endoplasmic reticulum stress is involved in high glucose-induced injury in human umbilical vein endothelial cells].

PubMed

Xu, Wen-Ming; Lin, Jian-Cong; Chen, Mei-Ji; Zhang, Chang-Ran; Li, Yan-Bing

2018-05-20

To explore the role of the interaction between glycogen synthase kinase-3β (GSK-3β) and endoplasmic reticulum stress (ERS) in the high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVECs). HUVECs treated with 40 mmol/L glucose for 24 h were examined for expression levels of GSK-3β, GRP78, CHOP and cleaved caspase-3 protein using Western blotting. The cell viability was examined using CCK-8 assay and cell apoptosis was detected with Hoechst 33258 nuclear staining and photofluorography. The intracellular level of reactive oxygen species (ROS) was measured with dichlorfluoresein staining and photofluorography. Mitochondrial membrane potential (MMP) was tested by rhodamine 123 (Rh123) staining and photofluorography. Treatment of HUVECs with 40 µmol/L glucose for 3-24 h activated GSK-3β in a time-dependent manner, leading to significantly down-regulated expression of phosphorylated (p)-GSK-3β (P<0.05). HG exposure of the cells for 1-24 h induced ERS, evidenced by time-dependently up-regulated expression of GRP78 and CHOP (P<0.05). LiCl, an inhibitor of GSK-3β, attenuated HG-induced ERS and significantly lowered the expression levels of GRP78 and CHOP (P<0.01). 4-PBA, an inhibitor of ERS, obviously ameliorated the activation of GSK-3β by HG as shown by the increase in p-GSK-3β expression level (P<0.01). HG exposure for 24 h induced obvious injuries in HUVECs, which exhibited decreased cell viability, increased cell apoptosis, increased expression of cleaved caspase-3 and ROS generation, and loss of MMP. Pretreatment of the cells with LiCl or 4-PBA for 60 min before HG exposure significantly lessened the cell injuries (P<0.01). Interactions between GSK-3β and ERS occur in HUVECs exposed to HG and participate in HG-induced cell injuries.

Characterization of Adsorbents for Cytokine Removal from Blood in an In Vitro Model.

PubMed

Harm, Stephan; Gabor, Franz; Hartmann, Jens

2015-01-01

Cytokines are basic targets that have to be removed effectively in order to improve the patient's health status in treating severe inflammation, sepsis, and septic shock. Although there are different adsorbents commercially available, the success of their clinical use is limited. Here, we tested different adsorbents for their effective removal of cytokines from plasma and the resulting effect on endothelial cell activation. The three polystyrene divinylbenzene (PS-DVB) based adsorbents Amberchrom CG161c and CG300m and a clinically approved haemoperfusion adsorbent (HAC) were studied with regard to cytokine removal in human blood. To induce cytokine release from leucocytes, human blood cells were stimulated with 1 ng/ml LPS for 4 hours. Plasma was separated and adsorption experiments in a dynamic model were performed. The effect of cytokine removal on endothelial cell activation was evaluated using a HUVEC-based cell culture model. The beneficial outcome was assessed by measuring ICAM-1, E-selectin, and secreted cytokines IL-8 and IL-6. Additionally the threshold concentration for HUVEC activation by TNF-α and IL-1β was determined using this cell culture model. CG161c showed promising results in removing the investigated cytokines. Due to its pore size the adsorbent efficiently removed the key factor TNF-α, outperforming the commercially available adsorbents. The CG161c treatment reduced cytokine secretion and expression of cell adhesion molecules by HUVEC which underlines the importance of effective removal of TNF-α in inflammatory diseases. These results confirm the hypothesis that cytokine removal from the blood should approach physiological levels in order to reduce endothelial cell activation.

Synthesis and cytotoxicity of 2,5-dihydroxychalcones and related compounds.

PubMed

Nam, Nguyen-Hai; Hong, Dong-Ho; You, Young-Jae; Kim, Yong; Bang, Seong-Cheol; Kim, Hwan-Mook; Ahn, Byung-Zun

2004-06-01

A series of 2, 5-dihydroxychalcones and related compounds were synthesized, and their cytotoxicities against tumor cell lines and human umbilical venous endothelial cells (HUVEC) evaluated. It was found that chalcones, with electron-withdrawing substituents on an A ring, exhibited significant cytotoxicities. Among the synthesized compounds, 2'-chloro-2, 5-dihydroxychalcone (9) was most potent, with an IC50 value as low as 0.31 microg/mL. This compound also exhibited a significant cytotoxic selectivity toward HUVEC.

Anti-Cancer Activity of an Osthole Derivative, NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

PubMed Central

Chiu, Pei-Ting; Ho, Shiau-Jing; Wang, Chi-Han; Chi, Chih-Chin; Huang, Yu-Han; Lee, Cheng-Feng; Li, Ying-Shiuan; Ou, George; Hsu, Ming-Jen

2013-01-01

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer. PMID:24312323

Regulator of Calcineurin 1 in Periodontal Disease

PubMed Central

Peters, Ulrike; Solominidou, Eleni; Korkmaz, Yüksel; Rüttermann, Stefan; Klocke, Astrid; Flemmig, Thomas Frank; Beikler, Thomas

2016-01-01

Nuclear factor of activated T-cells (NFAT) and NF-kB pathway associated processes are involved in the pathogenesis of various inflammatory disorders, for example, periodontal disease. The activation of these pathways is controlled by the regulator of calcineurin 1 (RCAN1). The aim of this study was to elucidate the role of RCAN1 in periodontal disease. Healthy and inflamed periodontal tissues were analyzed by immunohistochemistry and immunofluorescence using specific rabbit polyclonal anti-RCAN1 antibodies. For expression analysis human umbilical vein endothelial cells (HUVEC) were used. HUVEC were incubated for 2 h with Vascular Endothelial Growth Factor (VEGF) or with wild type and laboratory strains of Porphyromonas gingivalis (P. gingivalis). Expression analysis of rcan1 and cox2 was done by real time PCR using specific primers for rcan1.4 and cox2. The expression of rcan1 was found to be significantly suppressed in endothelial cells of chronically inflamed periodontal tissues compared to healthy controls. Rcan1 and cox2 were significantly induced by VEGF and wild type and laboratory P. gingivalis strains. Interestingly, the magnitude of the rcan1 and cox2 induction was strain dependent. The results of this study indicate that RCAN1 is suppressed in endothelial cells of chronically inflamed periodontal tissues. During an acute infection, however, rcan1 seems to be upregulated in endothelial cells, indicating a modulating role in immune homeostasis of periodontal tissues. PMID:27403036

Inhibition of angiogenic attributes by decursin in endothelial cells and ex vivo rat aortic ring angiogenesis model.

PubMed

Bhat, Tariq A; Moon, Jung S; Lee, Sookyeon; Yim, Dongsool; Singh, Rana P

2011-11-01

The present study was undertaken to observe the inhibition of angiogenesis by decursin. It was the first time to show that decursin offered strong anti-angiogenic activities under the biologically relevant growth (with serum) conditions. Decursin significantly inhibited human umbilical vein endothelial cell (HUVEC) proliferation concomitant with G1 phase cell cycle arrest. Decursin also inhibited HUVEC-capillary tube formation and invasion/migration in a dose-dependant manner which was associated with the suppression of matrix metalloproteinase (MMP) -2 and -9 activities. Decursin suppressed angiogenesis in ex vivo rat aortic ring angiogenesis model where it significantly inhibited blood capillary-network sprouting from rat aortic sections. Taken together, these findings suggested anti-angiogenic activity of decursin in biologically relevant condition, and warrants further pre-clinical studies for its potential clinical usefulness.

Indoxyl sulfate enhances IL-1β-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

PubMed

Shen, Wen-Ching; Liang, Chan-Jung; Huang, Tao-Ming; Liu, Chen-Wei; Wang, Shu-Huei; Young, Guang-Huar; Tsai, Jaw-Shiun; Tseng, Ying-Chin; Peng, Yu-Sen; Wu, Vin-Cent; Chen, Yuh-Lien

2016-11-01

Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1β were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1β expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1β-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1β-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1β-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1β-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.

Angiogenic and wound healing potency of fermented virgin coconut oil: in vitro and in vivo studies.

PubMed

Ibrahim, Ahmad H; Li, Haibo; Al-Rawi, Sawsan S; Majid, Aman Shah Abdul; Al-Habib, Omar Am; Xia, Xiaobo; Majid, Amin Ms Abdul; Ji, Dan

2017-01-01

The process of wound healing involves activation of keratinocytes, fibroblasts, endothelial cells, etc. Angiogenesis is crucial during the process of wound healing. Virgin coconut oil is widely utilized in South Asia for various purposes including food, medicinal and industrial applications. This study aimed to evaluate the potency of fermented virgin coconut oil (FVCO) in angiogenesis and wound healing via both in vitro and in vivo assays. Human umbilical vein endothelial (HUVEC), fibroblast (CCD-18) and retinal ganglion (RGC-5) cells were cultured in medium containing different concentrations of FVCO. The proliferation, migration and morphological changes of cells were determined. The angiogenic effect of FVCO was evaluated by rat aortic assay. The therapeutic effect of FVCO on wound healing was further assessed in a wound excision model in Sprague Dawley rats. The expression of phospho-VEGFR2 (vascular endothelial growth factor receptor 2) in HUVECs was detected by Western blot. FVCO (6 and 12 µg/mL) significantly improved the proliferation of HUVEC, CCD-18 and RGC-5 cells ( P < 0.05 or 0.01). FVCO (25 µg/mL) markedly increased the migration ability of CCD-18 and RGC-5 cells ( P < 0.05). FVCO did not affect cell morphology as indicated by fluorescein diacetate (FDA), rhodamine 123 and Hoechst staining. FVCO (25, 50 and 100 µg/mL) significantly stimulated the ex vivo blood vessel formation as compared with negative control ( P < 0.05). Rats in FVCO group had significantly smaller wound size, higher wound healing percentage, and shorter wound closure time when compared with control group since day 8 ( P < 0.05), suggesting that oral FVCO administration notably promoted the wound healing process. FVCO treatment (6 and 12 µg/mL) significantly enhanced the phospho-VEGFR2 expression in HUVECs ( P = 0.006 and 0.000, respectively). Our study confirms a high angiogenic and wound healing potency of FVCO that might be mediated by the regulation of VEGF signing pathway.

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture

PubMed Central

Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2017-01-01

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation. PMID:28783133

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture.

PubMed

Freyer, Nora; Greuel, Selina; Knöspel, Fanny; Strahl, Nadja; Amini, Leila; Jacobs, Frank; Monshouwer, Mario; Zeilinger, Katrin

2017-08-07

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase ( p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.

The Biological Properties of OGI Surfaces Positively Act on Osteogenic and Angiogenic Commitment of Mesenchymal Stem Cells

PubMed Central

Bressan, Eriberto; Gardin, Chiara; Ferroni, Letizia; Soldini, Maria Costanza; Mandelli, Federico; Soldini, Claudio

2017-01-01

Osteogenesis process displays a fundamental role during dental implant osteointegration. In the present work, we studied the influence of Osteon Growth Induction (OGI) surface properties on the angiogenic and osteogenic behaviors of Mesenchymal Stem cells (MSC). MSC derived from dental pulp and HUVEC (Human Umbilical Vein Endothelial Cells) were grown in on OGI titanium surfaces, and cell proliferation and DNA synthesis were evaluated by MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] test and DNA quantification. Gene expression has been performed in order to evaluate the presence of mRNA related to endothelial and osteogenesis markers. Moreover, morphological and biochemical analyses of osteogenesis commitments has been performed. On OGI surfaces, MSC and HUVEC are able to proliferate. Gene expression profiler confirms that MSC on OGI surfaces are able to express endothelial and osteogenic markers, and that these expression are higher compared the expression on control surfaces. In conclusion On OGI surfaces proliferation, expression and morphological analyses of angiogenesis-associated markers in MSC are promoted. This process induces an increasing on their osteogenesis commitment. PMID:29149082

Syndecan-4 shedding impairs macrovascular angiogenesis in diabetes mellitus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ran; Xie, Jun; Wu, Han

Purpose: Syndecan-4 (synd4) is a ubiquitous heparan sulfate proteoglycan cell surface receptor that modulates cell proliferation, migration, mechanotransduction, and endocytosis. The extracellular domain of synd4 sheds heavily in acute inflammation, but the shedding of synd4 in chronic inflammation, such as diabetes mellitus (DM), is still undefined. We investigated the alterations of synd4 endothelial expression in DM and the influence of impaired synd4 signaling on angiogenesis in human umbilical vein endothelial cells (HUVECs), diabetic rats, synd4 null mice, and db/db mice. Material and methods: HUVECs were incubated with advanced glycation end products (AGEs). Western blot analysis was used to determine synd4more » protein expression and ELISA was used to detect soluble synd4 fragments. The concentration of synd4 in the aortic endothelia of diabetic rats was detected by immunohistochemical staining. Aortic ring assays were performed to study the process of angiogenesis in the diabetic rats and in synd4 null and db/db mice. Recombinant adenoviruses containing the synd4 gene or null were constructed to enhance synd4 aortic expression in db/db mice. Results: Western blot analysis showed decreased expression of the synd4 extracellular domain in HUVECs, and ELISA detected increased soluble fragments of synd4 in the media. Synd4 endothelial expression in the aortas of diabetic rats was decreased. Aortic ring assay indicated impaired angiogenesis in synd4 null and db/db mice, which was partially reversed by synd4 overexpression in db/db mice. Conclusion: Synd4 shedding from vascular endothelial cells played an important role in the diabetes-related impairment of angiogenesis. -- Highlights: •Synd4 shedding from endothelial cells is accelerated under the stimulation of AGEs. •Extracellular domain of synd4 is diminished in the endothelium of DM rats. •Aortic rings of synd4 null mice showed impaired angiogenesis. •Overexpression of synd4 partly rescues macrovascular angiogenesis in db/db mice.« less

Influence of shear stress and size on viability of endothelial cells exposed to gold nanoparticles

NASA Astrophysics Data System (ADS)

Fede, C.; Albertin, Giovanna; Petrelli, L.; De Caro, R.; Fortunati, I.; Weber, V.; Ferrante, Camilla

2017-09-01

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size.

Inhibition of angiogenesis by leflunomide via targeting the soluble ephrin-A1/EphA2 system in bladder cancer.

PubMed

Chu, Maolin; Zhang, Chunying

2018-01-24

Angiogenesis plays an important role in bladder cancer (BCa). The immunosuppressive drug leflunomide has attracted worldwide attention. However, the effects of leflunomide on angiogenesis in cancer remain unclear. Here, we report the increased expression of soluble ephrin-A1 (sEphrin-A1) in supernatants of BCa cell lines (RT4, T24, and TCCSUP) co-cultured with human umbilical vein endothelial cells (HUVECs) compared with that in immortalized uroepithelial cells (SV-HUC-1) co-cultured with HUVECs. sEphrin-A1 is released from BCa cells as a monomeric protein that is a functional form of the ligand. The co-culture supernatants containing sEphrin-A1 caused the internalization and down-regulation of EphA2 on endothelial cells and dramatic functional activation of HUVECs. This sEphrin-A1/EphA2 system is mainly functional in regulating angiogenesis in BCa tissue. We showed that leflunomide (LEF) inhibited angiogenesis in a N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced bladder carcinogenesis model and a tumor xenograft model, as well as in BCa cell and HUVEC co-culture systems, via significant inhibition of the sEphrin-A1/EphA2 system. Ephrin-A1 overexpression could partially reverse LEF-induced suppression of angiogenesis and subsequent tumor growth inhibition. Thus, LEF has a significant anti-angiogenesis effect on BCa cells and BCa tissue via its inhibition of the functional angiogenic sEphrin-A1/EphA2 system and may have potential for treating BCa beyond immunosuppressive therapy.

Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells.

PubMed

Brouillet, Sophie; Hoffmann, Pascale; Benharouga, Mohamed; Salomon, Aude; Schaal, Jean-Patrick; Feige, Jean-Jacques; Alfaidy, Nadia

2010-08-15

Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.

Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway

PubMed Central

Busceti, Carla L.; Marchitti, Simona; Bianchi, Franca; Di Pietro, Paola; Riozzi, Barbara; Stanzione, Rosita; Cannella, Milena; Battaglia, Giuseppe; Bruno, Valeria; Volpe, Massimo; Fornai, Francesco; Nicoletti, Ferdinando; Rubattu, Speranza

2017-01-01

Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs. PMID:28352232

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth.

PubMed

Lyu, Junfang; Yang, Eun Ju; Head, Sarah A; Ai, Nana; Zhang, Baoyuan; Wu, Changjie; Li, Ruo-Jing; Liu, Yifan; Yang, Chen; Dang, Yongjun; Kwon, Ho Jeong; Ge, Wei; Liu, Jun O; Shim, Joong Sup

2017-11-28

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

PubMed

Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

2016-01-01

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Solena amplexicaulis induces cell cycle arrest, apoptosis and inhibits angiogenesis in hepatocarcinoma cells and HUVECs.

PubMed

Ren, Jie; Xu, Yuan Yuan; Jiang, He Fei; Yang, Meng; Huang, Qian Hui; Yang, Jie; Hu, Kun; Wei, Kun

2014-01-01

Solena amplexicaulis (Lam.) Gandhi (SA) has been used as a traditional medicine for the treatment of dysentery, multiple abscess, gastralgia, urethritis, and eczema in the minority area of China. This study was aimed to examine the cell proliferation inhibitory activity of the SA extract (SACE) and its mechanism of action in human hepatoma cell line (HepG2) and evaluate its anti-angiogenesis activity in human umbilical vein endothelial cell line (HUVEC). SACE could inhibit the growth of HepG2 cells in a dose- and time-dependent manner. FCM analysis showed that SACE could induce G2/M phase arrest, cell apoptosis, the mitochondrial membrane potential loss (ΔΨm) and increase the production of intracellular ROS of HepG2 cells. After treatment with SACE, topical morphological changes of apoptotic body formation, obvious increase of apoptosis-related protein expressions, such as Bax, cytochrome c, caspase-3, PARP-1, and decrease of Bcl-2, procaspase-9 protein expressions were observed at the same time. Moreover, SACE caused the significant inhibition of endothelial cell migration and tube formation in HUVEC cells. The results suggested that SACE could act as an angiogenesis inhibitor and induce cell apoptosis via a caspase-dependent mitochondrial pathway. Therefore, SACE could be a potent candidate for the prevention and treatment of liver cancer.

The glutathione mimic ebselen inhibits oxidative stress but not endoplasmic reticulum stress in endothelial cells.

PubMed

Ahwach, Salma Makhoul; Thomas, Melanie; Onstead-Haas, Luisa; Mooradian, Arshag D; Haas, Michael J

2015-08-01

Reactive oxygen species are associated with cardiovascular disease, diabetes, and atherosclerosis, yet the use of antioxidants in clinical trials has been ineffective at improving outcomes. In endothelial cells, high-dextrose-induced oxidative stress and endoplasmic reticulum stress promote endothelial dysfunction leading to the recruitment and activation of peripheral blood lymphocytes and the breakdown of barrier function. Ebselen, a glutathione peroxidase 1 (GPX1) mimic, has been shown to improve β-cell function in diabetes and prevent atherosclerosis. To determine if ebselen inhibits both oxidative stress and endoplasmic reticulum (ER) stress in endothelial cells, we examined its effects in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) with and without high-dextrose. Oxidative stress and ER stress were measured by 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence and ER stress alkaline phosphatase assays, respectively. GPX1 over-expression and knockdown were performed by transfecting cells with a GPX1 expression construct or a GPX1-specific siRNA, respectively. Ebselen inhibited dextrose-induced oxidative stress but not ER stress in both HUVEC and HCAEC. Ebselen also had no effect on tunicamycin-induced ER stress in HCAEC. Furthermore, augmentation of GPX1 activity directly by sodium selenite supplementation or transfection of a GPX1 expression plasmid decreased dextrose-induced oxidative stress but not ER stress, while GPX1 knockout enhanced oxidative stress but had no effect on ER stress. These results suggest that ebselen targets only oxidative stress but not ER stress. Copyright © 2015. Published by Elsevier Inc.

The recovery time course of the endothelial-cell glycocalyx in vivo and its implications in vitro

PubMed Central

Potter, Daniel R.; Jiang, John; Damiano, Edward R.

2009-01-01

Compelling evidence continues to emerge suggesting that the glycocalyx surface layer on vascular endothelial cells plays a determining role in numerous physiological processes including inflammation, microvascular permeability, and endothelial mechanotransduction. Previous research has shown that enzymes degrade the glycocalyx, while inflammation causes shedding of the layer. To track the endogenous recovery of the glycocalyx in vivo, we used fluorescent micro-particle image velocimetry (µ-PIV) in mouse cremaster-muscle venules to estimate the hydrodynamically relevant glycocalyx thickness 1, 3, 5, and 7 days after enzymatic or cytokine-mediated degradation of the layer. Results indicate that after acute degradation of the glycocalyx, 5–7 days are required for the layer to endogenously restore itself to its native hydrodynamically relevant thickness in vivo. In light of these findings, and since demonstrable evidence has emerged that standard cell-culture conditions are not conducive to providing the environment and/or cellular conditions necessary to produce and maintain a physiologically relevant cell-surface glycocalyx in vitro, we sought to determine if merely the passage of time would be sufficient to promote the production of a hydrodynamically relevant glycocalyx on a confluent monolayer of human umbilical vein endothelial cells (HUVECs). Using µ-PIV, we found that the hydrodynamically relevant glycocalyx was substantially absent 7 days post-confluence on HUVEC-lined cylindrical collagen microchannels maintained under standard culture conditions. Thus it remains to be determined how a hydrodynamically relevant glycocalyx surface layer can be synthesized and maintained in culture before the endothelial-cell culture model can be used to elucidate glycocalyx-mediated mechanisms of endothelial-cell function. PMID:19443840

The innate defense antimicrobial peptides hBD3 and RNase7 are induced in human umbilical vein endothelial cells by classical inflammatory cytokines but not Th17 cytokines.

PubMed

Burgey, Christine; Kern, Winfried V; Römer, Winfried; Sakinc, Türkan; Rieg, Siegbert

2015-05-01

Antimicrobial peptides are multifunctional effector molecules of innate immunity. In this study we investigated whether endothelial cells actively contribute to innate defense mechanisms by expression of antimicrobial peptides. We therefore stimulated human umbilical vein endothelial cells (HUVEC) with inflammatory cytokines, Th17 cytokines, heat-inactivated bacteria, bacterial conditioned medium (BCM) of Staphylococcus aureus and Streptococcus sanguinis, and lipoteichoic acid (LTA). Stimulation with single cytokines induced discrete expression of human β-defensin 3 (hBD3) by IFN-γ or IL-1β and of ribonuclease 7 (RNase7) by TNF-α without any effects on LL-37 gene expression. Stronger hBD3 and RNase7 induction was observed after combined stimulation with IL-1β, TNF-α and IFN-γ and was confirmed by high hBD3 and RNase7 peptide levels in cell culture supernatants. In contrast, Th17 cytokines or stimulation with LTA did not result in AMP production. Moreover, only BCM of an invasive S. aureus bacteremia isolate induced hBD3 in HUVEC. We conclude that endothelial cells actively contribute to prevent dissemination of pathogens at the blood-tissue-barrier by production of AMPs that exhibit microbicidal and immunomodulatory functions. Further investigations should focus on tissue-specific AMP induction in different endothelial cell types, on pathogen-specific induction patterns and potentially involved pattern-recognition receptors of endothelial cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Nicotine promotes vascular endothelial growth factor secretion by human trophoblast cells under hypoxic conditions and improves the proliferation and tube formation capacity of human umbilical endothelial cells.

PubMed

Zhao, Hongbo; Wu, Lanxiang; Wang, Yahui; Zhou, Jiayi; Li, Ruixia; Zhou, Jiabing; Wang, Zehua; Xu, Congjian

2017-04-01

Pre-eclampsia, characterized as defective uteroplacental vascularization, remains the major cause of maternal and fetal mortality and morbidity. Previous epidemiological studies demonstrated that cigarette smoking reduced the risk of pre-eclampsia. However, the molecular mechanism remains elusive. In the present study, it is demonstrated that a low dose of nicotine decreased soluble vascular endothelial growth factor receptor 1 (sFlt1) secretion in human trophoblast cells under hypoxic conditions. Nicotine was then observed to promote vascular endothelial growth factor (VEGF) secretion by reducing sFlt1 secretion and increasing VEGF mRNA transcription. Further data showed that nicotine enhanced hypoxia-mediated hypoxia-inducible factor-1α (HIF-1α) expression and HIF-1α small interfering RNA abrogated nicotine-induced VEGF secretion, indicating that HIF-1α may be responsible for nicotine-mediated VEGF transcription under hypoxic conditions. Moreover, conditioned medium from human trophoblast cells treated with nicotine under hypoxic conditions promoted the proliferation and tube formation capacity of human umbilical endothelial cells (HUVEC) by promoting VEGF secretion. These findings indicate that nicotine may promote VEGF secretion in human trophoblast cells under hypoxic conditions by reducing sFlt1 secretion and up-regulating VEGF transcription and improve the proliferation and tube formation of HUVEC cells, which may contribute to elucidate the protective effect of cigarette smoking against pre-eclampsia. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells

PubMed Central

Yang, Hana; Lee, Seung Eun; Kim, Gun-Dong; Park, Hye Rim; Park, Yong Seek

2013-01-01

Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death. PMID:23983896

Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

PubMed

Ramírez, Marco A; Morales, Jorge; Cornejo, Marcelo; Blanco, Elias H; Mancilla-Sierpe, Edgardo; Toledo, Fernando; Beltrán, Ana R; Sobrevia, Luis

2018-04-01

l-Arginine is taken up via the cationic amino acid transporters (system y + /CATs) and system y + L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y + /CATs and system y + L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH 4 Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 μmol/L, 10 s, 37 °C) in the absence or presence of 200 μmol/L N-ethylmaleimide (NEM) (system y + /CATs inhibitor) or 2 mmol/L l-leucine (systemy + L substrate) was measured. Protein abundance for eNOS and serine 1177 or threonine 495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y + L but not system y + /CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine 1177 phosphorylation. Thus, system y + L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs. Copyright © 2018 Elsevier B.V. All rights reserved.

Dengue Virus Infection Differentially Regulates Endothelial Barrier Function over Time through Type I Interferon Effects

PubMed Central

Liu, Ping; Woda, Marcia; Ennis, Francis A.; Libraty, Daniel H.

2013-01-01

Background The morbidity and mortality resulting from dengue hemorrhagic fever (DHF) are largely caused by endothelial barrier dysfunction and a unique vascular leakage syndrome. The mechanisms that lead to the location and timing of vascular leakage in DHF are poorly understood. We hypothesized that direct viral effects on endothelial responsiveness to inflammatory and angiogenesis mediators can explain the DHF vascular leakage syndrome. Methods We used an in vitro model of human endothelium to study the combined effects of dengue virus (DENV) type 2 (DENV2) infection and inflammatory mediators on paracellular macromolecule permeability over time. Results Over the initial 72 h after infection, DENV2 suppressed tumor necrosis factor (TNF)–α–mediated hyperpermeability in human umbilical vein endothelial cell (HUVEC) monolayers. This suppressive effect was mediated by type I interferon (IFN). By 1 week, TNF-α stimulation of DENV2-infected HUVECs synergistically increased cell cycling, angiogenic changes, and macromolecule permeability. This late effect could be prevented by the addition of exogenous type I IFN. Conclusions DENV infection of primary human endothelial cells differentially modulates TNF-α–driven angiogenesis and hyperpermeability over time. Type I IFN plays a central role in this process. Our findings suggest a rational model for the DHF vascular leakage syndrome. PMID:19530939

Human umbilical cord blood-derived mesenchymal stromal cells and small intestinal submucosa hydrogel composite promotes combined radiation-wound healing of mice.

PubMed

Lee, Changsun; Shim, Sehwan; Jang, Hyosun; Myung, Hyunwook; Lee, Janet; Bae, Chang-Hwan; Myung, Jae Kyung; Kim, Min-Jung; Lee, Seung Bum; Jang, Won-Suk; Lee, Sun-Joo; Kim, Hwi-Yool; Lee, Seung-Sook; Park, Sunhoo

2017-09-01

Mesenchymal stromal cells (MSCs) are a promising agent for treating impaired wound healing, and their therapeutic potential may be enhanced by employing extracellular matrix scaffolds as cell culture scaffolds or transplant cell carriers. Here, we evaluated the effect of human umbilical cord blood-derived (hUCB)-MSCs and a porcine small intestinal submucosa (SIS)-derived extracellular matrix scaffold in a combined radiation-wound mouse model of impaired wound healing. hUCB-MSCs and SIS hydrogel composite was applied to the excisional wound of whole-body irradiated mice. Assessment of wound closing and histological evaluation were performed in vivo. We also cultured hUCB-MSCs on SIS gel and examined the angiogenic effect of conditioned medium on irradiated human umbilical vein endothelial cells (HUVECs) in vitro. hUCB-MSCs and SIS hydrogel composite treatment enhanced wound healing and angiogenesis in the wound site of mice. Conditioned medium from hUCB-MSCs cultured on SIS hydrogel promoted the chemotaxis of irradiated HUVECs more than their proliferation. The secretion of angiogenic growth factors hepatocyte growth factor, vascular endothelial growth factor-A and angiopoietin-1 from hUCB-MSCs was significantly increased by SIS hydrogel, with HGF being the predominant angiogenic factor of irradiated HUVECs. Our results suggest that the wound healing effect of hUCB-MSCs is enhanced by SIS hydrogel via a paracrine factor-mediated recruitment of vascular endothelial cells in a combined radiation-wound mouse model. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Soy-Leaf Extract Exerts Atheroprotective Effects via Modulation of Krüppel-Like Factor 2 and Adhesion Molecules

PubMed Central

Han, Jong-Min; Li, Hua; Cho, Moon-Hee; Baek, Seung-Hwa; Lee, Chul-Ho; Park, Ho-Yong; Jeong, Tae-Sook

2017-01-01

Soy-leaf extracts exert their cardioprotective effects by inducing endothelium-dependent vasodilation in the arteries, and they favorably modulate the serum lipid profile. In this study, we investigated the atheroprotective effects of an ethanol extract of soy leaf (ESL) in human umbilical vein endothelial cells (HUVECs) and high-cholesterol diet (HCD)-fed low-density lipoprotein receptor deficient (LDLR−/−) mice. ESL induced the expression of Krüppel-like factor 2 (KLF2), an endothelial transcription factor, and endothelial nitric oxide synthase (eNOS), and suppressed the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) through moderate inflammatory signal activation, not only in tumor necrosis factor-α (TNF-α)-stimulated HUVECs but also in 7-ketocholesterol (7-KC)-stimulated HUVECs. ESL supplementation reduced aortic lesion formation in Western diet-fed LDLR−/− mice by 46% (p < 0.01) compared to the HCD group. ESL also markedly decreased the aortic expression levels of VCAM-1, ICAM-1, monocyte chemotactic protein-1 (MCP-1), TNF-α, IL-6, IL-1β, matrix metallopeptidase 9 (MMP-9), and fractalkine, while the expression of KLF2 was significantly increased. These results suggest that ESL supplementation has potential for preventing HCD-induced atherosclerosis effectively. PMID:28208647

Rhamnazin, a novel inhibitor of VEGFR2 signaling with potent antiangiogenic activity and antitumor efficacy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yao; Department of Endocrinology and Metabolism, The Third Hospital of Nanchang, Nanchang Key Laboratory of Diabetes, No.1 Qianjing Road, Xihu District, Nanchang 330009, Jiangxi Province; Cai, Wei

Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer therapy. The identification of new drugs from natural products has a long and successful history. In this study, we described a novel VEGFR2 inhibitor, rhamnazin, which inhibits tumor angiogenesis and growth. Rhamnazin significantly inhibited proliferation, migration and tube formation of human umbilical vascular endothelial cells (HUVECs) in vitro as well as inhibited sprouts formation of rat aorta ring. In addition, it inhibited vascular endothelial growth factor (VEGF)-induced phosphorylation of VEGFR2 and its downstream signaling regulator in HUVECs. Moreover, rhamnazin could directly inhibit proliferation ofmore » breast cancer cells MDA-MB-231 in vitro and in vivo. Oral administration of rhamnazin at a dose of 200 mg/kg/day could markedly inhibited human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that rhamnazin inhibits angiogenesis and may be a promising anticancer drug candidate. - Highlights: • Rhamnazin inhibits the response of HUVECs to VEGF in vitro. • Rhamnazin inhibits VEGFR2 kinase activity and its downstream signaling. • Rhamnazin prevents the growth of MDA-MB-231 tumor and reduces micro-vessel density in vivo.« less

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

PubMed

Du, Xiaohui; Li, Wanming; Du, Guansheng; Cho, Hansang; Yu, Min; Fang, Qun; Lee, Luke P; Fang, Jin

2018-03-06

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling.

PubMed

Giorgetti-Peraldi, S; Murdaca, J; Mas, J C; Van Obberghen, E

2001-07-05

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.

The ADMA/DDAH/NO pathway in human vein endothelial cells exposed to arsenite.

PubMed

Osorio-Yáñez, Citlalli; Chin-Chan, Miguel; Sánchez-Peña, Luz C; Atzatzi-Aguilar, Octavio G; Olivares-Reyes, Jesus A; Segovia, José; Del Razo, Luz M

2017-08-01

Inorganic arsenic (iAs) exposure is related to cardiovascular disease, which is characterized by endothelial dysfunction and nitric oxide (NO) depletion. The mechanisms underlying NO depletion as related to iAs exposure are not fully understood. The endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine (ADMA), might be a molecular target of iAs. ADMA concentrations are regulated by proteins involved in its synthesis (arginine methyl transferase 1 [PRMT-1]) and degradation (dimethylarginine dimethylaminohydrolase [DDAH]). Both, ADMA and NO are susceptible to oxidative stress. We aimed to determine the ADMA/DDAH/NO pathway in human vein endothelial cells (HUVEC-CS) exposed to arsenite. We exposed HUVEC-CS cells to 1, 2.5 and 5μM of arsenite for 24h. We proved that arsenite at 5μM was able to decrease NO levels with an associated increase in ADMA and depletion of l-arginine in HUVEC-CS cells. We also found a decrease in DDAH-1 protein expression with 5μM of arsenite compared to the control group. However, we did not observe significant differences in PRMT-1 protein expression at any of the concentrations of arsenite employed. Finally, arsenite (2.5 and 5μM) increased NADPH oxidase 4 protein levels compared with the control group. We conclude that ADMA, l-arginine and DDAH are involved in NO depletion produced by arsenite, and that the mechanism is related to oxidative stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

Towards cavitation-enhanced permeability in blood vessel on a chip

NASA Astrophysics Data System (ADS)

De Luca, R.; Silvani, G.; Scognamiglio, C.; Sinibaldi, G.; Peruzzi, G.; Chinappi, M.; Kiani, M. F.; Casciola, C. M.

2017-08-01

The development of targeted delivery systems releasing pharmaceutical agents directly at the desired site of action may improve their therapeutic efficiency while minimizing damage to healthy tissues, toxicity to the patient and drug waste. In this context, we have developed a bio-inspired microdevice mimicking the tumour microvasculature which represents a valuable tool for assessing the enhancement of blood vessel permeability due to cavitation. This novel system allows us to investigate the effects of ultrasound-driven microbubbles that temporarily open the endothelial intercellular junctions allowing drug to extravasate blood vessels into tumour tissues. The blood vessel on a chip consists of a tissue chamber and two independent vascular channels (width 200 µm, height 100 µm, length 2762 µm) cultured with endothelial cells placed side-by-side and separated by a series of 3 µm pores. Its geometry and dimensions mimic the three-dimensional morphology, size and flow characteristics of microvessels in vivo. The early stage of this project had a twofold objective: 1. To define the protocol for culturing of Human Umbilical Vein Endothelial Cells (HUVECs) within the vascular channel; 2. To develop a fluorescence based microscopy technique for measuring permeability. We have developed a reliable and reproducible protocol to culture endothelial cells within the artificial vessels in a realistic manner: HUVECs show the typical elongated shape in the direction of flow, exhibit tight junction formation and form a continuous layer with a central lumen that completely covers the channels wall. As expected, the permeability of cell-free device is higher than the one cultured with HUVECs in the vascular channels. The proposed blood vessel on a chip and the permeability measurement protocol have a significant potential to allow for the study of cavitation-enhanced permeability of the endothelium and improve efficiency in screening drug delivery systems.

Progranulin Protects Vascular Endothelium against Atherosclerotic Inflammatory Reaction via Akt/eNOS and Nuclear Factor-κB Pathways

PubMed Central

Hwang, Hwan-Jin; Jung, Tae Woo; Hong, Ho Cheol; Choi, Hae Yoon; Seo, Ji-A; Kim, Sin Gon; Kim, Nan Hee; Choi, Kyung Mook; Choi, Dong Seop; Baik, Sei Hyun; Yoo, Hye Jin

2013-01-01

Objective Atherosclerosis is considered a chronic inflammatory disease, initiated by activation and dysfunction of the endothelium. Recently, progranulin has been regarded as an important modulator of inflammatory processes; however, the role for prgranulin in regulating inflammation in vascular endothelial cells has not been described. Method and Results Signaling pathways mediated by progranulin were analyzed in human umbilical vein endothelial cells (HUVECs) treated with progranulin. Progranulin significantly induced Akt and endothelial nitric oxide synthase (eNOS) phosphorylation in HUVECs, an effect that was blocked with Akt inhibitor. Furthermore, nitric oxide (NO) level, the end product of Akt/eNOS pathway, was significantly upregulated after progranulin treatment. Next, we showed that progranulin efficiently inhibited lipopolysaccharide (LPS)-mediated pro-inflammatory signaling. LPS-induced phosphorylation of IκB and nuclear factor-κB (NF-κB) levels decreased after progranulin treatment. Also, progranulin blocked translocation of NF-κB from the cytosol to the nucleus. In addition, progranulin significantly reduced the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) by inhibiting binding of NF- κB to their promoter regions and blocked attachment of monocytes to HUVECs. Progranulin also significantly reduced the expression of tumor necrosis factor receptor-α (TNF-α) and monocyte chemo-attractant protein-1 (MCP-1), the crucial inflammatory molecules known to aggravate atherosclerosis. Conclusion Progranulin efficiently inhibited LPS-mediated pro-inflammatory signaling in endothelial cells through activation of the Akt/eNOS pathway and attenuation of the NF-κB pathway, suggesting its protective roles in vascular endothelium against inflammatory reaction underlying atherosclerosis. PMID:24098801

Two cyclic hexapeptides from Penicillium sp. FN070315 with antiangiogenic activities.

PubMed

Jang, Jun-Pil; Jung, Hye Jin; Han, Jang Mi; Jung, Narae; Kim, Yonghyo; Kwon, Ho Jeong; Ko, Sung-Kyun; Soung, Nak-Kyun; Jang, Jae-Hyuk; Ahn, Jong Seog

2017-01-01

In the course of searching for angiogenesis inhibitors from microorganisms, two cyclic peptides, PF1171A (1) and PF1171C (2) were isolated from the soil fungus Penicillium sp. FN070315. In the present study, we investigated the antiangiogenic efficacy and associated mechanisms of 1 and 2 in vitro using human umbilical vein endothelial cells (HUVECs). Compounds 1 and 2 inhibited the proliferation of HUVECs at concentrations not exhibiting cytotoxicity. Moreover, 1 and 2 significantly suppressed vascular endothelial growth factor (VEGF)-induced migration, invasion, proliferation and tube formation of HUVECs as well as neovascularization of the chorioallantoic membrane in developing chick embryos. We also identified an association between the antiangiogenic activity of 1 and 2 and the downregulation of both the phosphorylation of VEGF receptor 2 and the expression of hypoxia inducible factor-1α at the protein level. Taken together, these results further suggest that compounds 1 and 2 will be promising angiogenesis inhibitors.

C1q/TNF-Related Protein-9 Ameliorates Ox-LDL-Induced Endothelial Dysfunction via PGC-1α/AMPK-Mediated Antioxidant Enzyme Induction

PubMed Central

Sun, Haijian; Zhu, Xuexue; Zhou, Yuetao; Cai, Weiwei; Qiu, Liying

2017-01-01

Oxidized low-density lipoprotein (ox-LDL) accumulation is one of the critical determinants in endothelial dysfunction in many cardiovascular diseases such as atherosclerosis. C1q/TNF-related protein 9 (CTRP9) is identified to be an adipocytokine with cardioprotective properties. However, the potential roles of CTRP9 in endothelial function remain largely elusive. In the present study, the effects of CTRP9 on the proliferation, apoptosis, migration, angiogenesis, nitric oxide (NO) production and oxidative stress in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL were investigated. We observed that treatment with ox-LDL inhibited the proliferation, migration, angiogenesis and the generation of NO, while stimulated the apoptosis and reactive oxygen species (ROS) production in HUVECs. Incubation of HUVECs with CTRP9 rescued ox-LDL-induced endothelial injury. CTRP9 treatment reversed ox-LDL-evoked decreases in antioxidant enzymes including heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase quinone 1, and glutamate-cysteine ligase (GCL), as well as endothelial nitric oxide synthase (eNOS). Furthermore, CTRP9 induced activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC1-α) and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Of interest, AMPK inhibition or PGC1-α silencing abolished CTRP9-mediated antioxidant enzymes levels, eNOS expressions, and endothelial protective effects. Collectively, we provided the first evidence that CTRP9 attenuated ox-LDL-induced endothelial injury by antioxidant enzyme inductions dependent on PGC-1α/AMPK activation. PMID:28587104

Depolymerized products of lambda-carrageenan as a potent angiogenesis inhibitor.

PubMed

Chen, Haimin; Yan, Xiaojun; Lin, Jing; Wang, Feng; Xu, Weifeng

2007-08-22

Since angiogenesis is involved in initiating and promoting several diseases such as cancer and cardiovascular events, this study was designed to evaluate the anti-angiogenesis of low-molecular-weight (LMW), highly sulfated lambda-carrageenan oligosaccharides (lambda-CO) obtained by carrageenan depolymerization, by CAM (chick chorioallantoic membrane) model and human umbilical vein endothelial cells (HUVECs). Significant inhibition of vessel growth was observed at 200 microg/pellet. A histochemistry assay also revealed a decrease of capillary plexus and connective tissue in lambda-CO treated samples. lambda-CO inhibited the viability of cells at the high concentration of 1 mg/mL, whereas it affected the cell survival slightly (>95%) at a low concentration (<250 microg/mL), and HUVEC is the most sensitive to lambda-CO among three kinds of cells. Furthermore, the inhibitory action of lambda-CO was also observed in the endothelial cell invasion and migration at relatively low concentration (150-300 microg/mL), through down-regulation of intracellular matrix metalloproteinases (MMP-2) expression on endothelial cells. Taken together, these findings demonstrate that lambda-CO is a potential angiogenesis inhibitor with combined effects of inhibiting invasion, migration, and proliferation.

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

PubMed Central

Gakhar, Gunjan; Navarro, Vicente N.; Jurish, Madelyn; Lee, Guang Yu.; Tagawa, Scott T.; Akhtar, Naveed H.; Seandel, Marco; Geng, Yue; Liu, He; Bander, Neil H.; Giannakakou, Paraskevi; Christos, Paul J.; King, Michael R.; Nanus, David M.

2013-01-01

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm2. CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLex) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLex expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis. PMID:24386459

Anti-proliferative and anti-angiogenic effects of CB2R agonist (JWH-133) in non-small lung cancer cells (A549) and human umbilical vein endothelial cells: an in vitro investigation.

PubMed

Vidinský, B; Gál, P; Pilátová, M; Vidová, Z; Solár, P; Varinská, L; Ivanová, L; Mojžíš, J

2012-01-01

Non-small cell lung cancer has one of the highest mortality rates among cancer-suffering patients. It is well known that the unwanted psychotropic effects of cannabinoids (CBs) are mediated via the CB(1) receptor (R), and selective targeting of the CB(2)R would thus avoid side effects in cancer treatment. Therefore, the aim of our study was to evaluate the effect of selective CB(2)R agonist, JWH-133, on A549 cells (non-small lung cancer) and human umbilical vein endothelial cells (HUVECs). Cytotoxicity assay and DNA fragmentation assay were employed to evaluate the influence of JWH-133 (3-(1,1-dimethylbutyl)- 1-deoxy-Δ8-tetrahydrocannabinol) on investigated cancer cells. In addition, migration assay and gelatinase zymography were performed in HUVECs to asses JWH-133 anti-angiogenic activity. Our study showed that JWH-133 exerted cytotoxic effect only at the highest concentration used (10(-4) mol/l), while inhibition of colony formation was also detected at the non-toxic concentrations (10(-5)-10(-8) mol/l). JWH-133 was also found to be able to induce weak DNA fragmentation in A549 cells. Furthermore, JWH-133 at non-toxic concentrations inhibited some steps in the process of angiogenesis. It significantly inhibited endothelial cell migration after 17 h of incubation at concentrations of 10(-4)-10(-6) mol/l. In addition, JWH-133 inhibited MMP-2 secretion as assessed by gelatinase zymography. The present study demonstrates the in vitro anti-proliferative and anti-angiogenic potential of CB(2)R agonist, JWH-133, in nonsmall lung cancer cells and HUVECs. Our results generate a rationale for further in vivo efficacy studies with this compound in preclinical cancer models.

Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting and Affects the Notch Pathway in Human Endothelial Cells

PubMed Central

Pannella, Micaela; Caliceti, Cristiana; Fortini, Francesca; Aquila, Giorgio; Sega, Francesco Vieceli Dalla; Pannuti, Antonio; Fortini, Cinzia; Morelli, Marco Bruno; Fucili, Alessandro; Francolini, Gloria; Voltan, Rebecca; Secchiero, Paola; Dinelli, GiovannI; Leoncini, Emanuela; Ferracin, Manuela; Hrelia, Silvana; Miele, Lucio; Rizzo, Paola

2017-01-01

It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta-like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT-proBNP (N-terminal of the pro-hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera-treated cells (r = −0.526 and r = −0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. PMID:26987674

Effect of PKC-β Signaling Pathway on Expression of MCP-1 and VCAM-1 in Different Cell Models in Response to Advanced Glycation End Products (AGEs)

PubMed Central

Rempel, Lisienny C. T.; Finco, Alessandra B.; Maciel, Rayana A. P.; Bosquetti, Bruna; Alvarenga, Larissa M.; Souza, Wesley M.; Pecoits-Filho, Roberto; Stinghen, Andréa E. M.

2015-01-01

Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs–endothelium interactions through the receptor for AGEs (RAGE) in the PKC-β pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-β inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-β inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-β inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease. PMID:26008233

Effect of PKC-β Signaling Pathway on Expression of MCP-1 and VCAM-1 in Different Cell Models in Response to Advanced Glycation End Products (AGEs).

PubMed

Rempel, Lisienny C T; Finco, Alessandra B; Maciel, Rayana A P; Bosquetti, Bruna; Alvarenga, Larissa M; Souza, Wesley M; Pecoits-Filho, Roberto; Stinghen, Andréa E M

2015-05-14

Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs-endothelium interactions through the receptor for AGEs (RAGE) in the PKC-β pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKC-β inhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKC-β inhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKC-β inhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

PubMed

Kono, Ken; Hiruma, Hitomi; Kobayashi, Shingo; Sato, Yoji; Tanaka, Masaru; Sawada, Rumi; Niimi, Shingo

2016-01-01

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

Omega-3 fatty acids modulate Weibel-Palade body degranulation and actin cytoskeleton rearrangement in PMA-stimulated human umbilical vein endothelial cells.

PubMed

Bürgin-Maunder, Corinna S; Brooks, Peter R; Russell, Fraser D

2013-11-08

Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

SiO2-induced release of sVEGFRs from pulmonary macrophages.

PubMed

Chao, Jie; Lv, Yan; Chen, Jin; Wang, Jing; Yao, Honghong

2018-01-01

The inhalation of silicon dioxide (SiO 2 ) particles causes silicosis, a stubborn pulmonary disease that is characterized by alveolar inflammation during the early stage. Soluble cytokine receptors (SCRs) play important roles in regulating inflammation by either attenuating or promoting cytokine signaling. However, the role of SCRs in silicosis remains unknown. Luminex assays revealed increased soluble vascular endothelial growth factor receptor (sVEGFR) family levels in the plasma of silicosis patients. In an enzyme-linked immunosorbent assay (ELISA), cells from the differentiated human monocytic cell line U937 released sVEGFR family proteins after exposure to SiO 2 (50μg/cm 2 ). Further Western blot experiments revealed that VEGFR expression was also elevated in U937 cells. In contrast, levels of sVEGFR family members did not change in the supernatants of human umbilical vein endothelial cells (HUVECs) after exposure to SiO 2 (50μg/cm 2 ). Interestingly, VEGFR expression in HUVECs decreased after SiO 2 treatment. In a scratch assay, HUVECs exhibited cell migration ability, indicating the acquisition of mesenchymal properties. Our findings highlight the important role of sVEGFRs in both inflammation and fibrosis induced by SiO 2 , suggesting a possible mechanism for the fibrogenic effects observed in pulmonary diseases associated with fibrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Engineering of Surface Functionality onto Polystyrene Microcarriers for the Attachment and Growth of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Xiong, Gordon M.; Foord, John S.; Griffiths, Jon-Paul; Parker, Emily M.; Moloney, Mark G.; Choong, Cleo

2014-08-01

This work reports the effects of introducing diverse chemical functionalities onto the surface of polystyrene microcarrier beads on their ability to function as injectable cell carriers. Cellular adhesion and proliferation, as well as cellular outgrowths from microcarrier surfaces, using human umbilical vein endothelial cells (HUVECs), were examined in detail. It was observed that initial cell adhesion appeared to be most significantly decreased by hydrophobicity, whilst cell proliferation appeared to be improved in most chemical functional groups over unmodified polystyrene. Overall, our study highlights the importance of surface chemistry in directing the growth and function of human endothelial cells.

OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Omar, Hany A.; Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514; Arafa, El-Shaimaa A.

2013-11-01

Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to studymore » the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy. - Highlights: • The antiangiogenic activity of OSU-A9 in HUVECs was explored. • OSU-A9 inhibited HUVECs proliferation, migration, invasion and tube formation. • OSU-A9 targeted signaling pathways mediated by Akt-NF-kB, VEGF, and MMP-2. • The anti-angiogenic activity of OSU-A9 supports its clinical promise.« less

Atorvastatin inhibits the apoptosis of human umbilical vein endothelial cells induced by angiotensin II via the lysosomal-mitochondrial axis.

PubMed

Chang, Ye; Li, Yuan; Ye, Ning; Guo, Xiaofan; Li, Zhao; Sun, Guozhe; Sun, Yingxian

2016-09-01

This study was aimed to evaluate lysosomes-mitochondria cross-signaling in angiotensin II (Ang II)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and whether atorvastatin played a protective role via lysosomal-mitochondrial axis. Apoptosis was detected by flow cytometry, Hoechst 33342 and AO/EB assay. The temporal relationship of lysosomal and mitochondrial permeabilization was established. Activity of Cathepsin D (CTSD) was suppressed by pharmacological and genetic approaches. Proteins production were measured by western blotting. Our study showed that Ang II could induce the apoptosis of HUVECs in a dose-depended and time-depended manner. Exposure to 1 μM Ang II for 24 h resulted in mitochondrial depolarization, cytochrome c release, and increased ROS production. Lysosomal permeabilization and CTSD redistribution into the cytoplasm occurred several hours prior to mitochondrial dysfunction. These effects were all suppressed by atorvastatin. Either pharmacological or genetic inhibition of CTSD preserved mitochondrial function and decreased apoptosis in HUVECs. Most importantly, we found that the protective effect of atorvastatin was significantly greater than pharmacological or genetic inhibition of CTSD. Finally, overexpression of CTSD without exposure to Ang II had no effect on mitochondrial function and apoptosis. Our data strongly suggested that Ang II induced apoptosis through the lysosomal-mitochondrial axis in HUVECs. Furthermore, atorvastatin played an important role in the regulation of lysosomes and mitochondria stability, resulting in an antagonistic role against Ang II on HUVECs.

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin–vascular endothelial growth factor pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhishan; Humphries, Brock; Xiao, Hua

2013-08-15

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchialmore » epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro-angiogenic effect. • EMT in arsenic-transformed cells activates β-catenin. • β-Catenin activation increases VEGF expression in arsenic-transformed cells. • β-Catenin activation is likely independent of canonical Wnt signaling. • EMT in arsenic-transformed cells promotes angiogenesis via β-catenin–VEGF pathway.« less

Gene expression profiling of rubella virus infected primary endothelial cells of fetal and adult origin.

PubMed

Geyer, Henriette; Bauer, Michael; Neumann, Jennifer; Lüdde, Amy; Rennert, Paul; Friedrich, Nicole; Claus, Claudia; Perelygina, Ludmilla; Mankertz, Annette

2016-02-02

Rubella virus (RV) infection is usually a mild illness in children and adults. However, maternal infection during the first trimester of pregnancy can lead to congenital rubella syndrome (CRS) in the infant. Fetuses with CRS show damage to the endothelium of the heart and blood vessels; thus, it has been speculated that the clinical manifestations associated with CRS may be a result of endothelial cells persistently infected with RV. Here, we compared the effects of RV infection on gene expression in primary endothelial cells of fetal (HUVEC) and of adult (HSaVEC) origin by transcriptional profiling. More than 75 % of the genes differentially regulated following RV infection were identical in both cell types. Gene Ontology (GO) analysis of these commonly regulated genes showed an enrichment of terms involved in cytokine production and cytokine regulation. Increased accumulation of inflammatory cytokines following RV infection was verified by protein microarray. Interestingly, the chemokine CCL14, which is implicated in supporting embryo implantation at the fetal-maternal interface, was down-regulated following RV infection only in HUVEC. Most noticeably, when analyzing the uniquely regulated transcripts for each cell type, GO term-based cluster analysis of the down-regulated genes of HUVEC revealed an enrichment of the GO terms "sensory organ development", "ear development" and "eye development". Since impairment in vision and hearing are the most prominent clinical manifestations observed in CRS patients, the here detected down-regulated genes involved in the development of sensory organs sheds light on the molecular mechanisms that may contribute to the teratogenic effect of RV.

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration.

PubMed

Bilgin, Mehmet; Neuhof, Christiane; Doerr, Oliver; Benscheid, Utz; Andrade, Sheila S; Most, Astrid; Abdallah, Yaser; Parahuleva, Mariana; Guenduez, Dursun; Oliva, Maria L; Erdogan, Ali

2010-12-01

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10-100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10-100 μM) on the membrane potential was measured with the fluorescence dye DiBAC4(3) and the effect on [Ca+2]i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10-100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1±1.8% as compared to control (p≤0.05; n=45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca2+ of 28.4±5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5±0.9% as compared to control (p≤0.05; n=80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.

Up-regulation of Heme Oxygenase-1 by Korean Red Ginseng Water Extract as a Cytoprotective Effect in Human Endothelial Cells

PubMed Central

Yang, Hana; Lee, Seung Eun; Jeong, Seong Il; Park, Cheung-Seog; Jin, Young-Ho; Park, Yong Seek

2011-01-01

Korean red ginseng (KRG) is used worldwide as a popular traditional herbal medicine. KRG has shown beneficial effects on cardiovascular diseases, such as atherosclerosis, diabetes, and hypertension. Up-regulation of a cytoprotective protein, heme oxygenase (HO)-1, is considered to augment the cellular defense against various agents that may induce cytotoxic injury. In the present study, we demonstrate that KRG water extract induces HO-1 expression in human umbilical vein endothelial cells (HUVECs) and possible involvement of the anti-oxidant transcription factor nuclear factor-eythroid 2-related factor 2 (Nrf2). KRG-induced HO-1 expression was examined by western blots, reverse transcriptase polymerase chain reaction and immunofluorescence staining. Specific silencing of Nrf2 genes with Nrf2-siRNA in HUVECs abolished HO-1 expression. In addition, the HO inhibitor zinc protoporphyrin blunted the preventive effect of KRG on H2O2-induced cell death, as demonstrated by terminal transferase dUTP nick end labeling assay. Taken together, these results suggest that KRG may exert a vasculoprotective effect through Nrf2- mediated HO-1 induction in human endothelial cell by inhibition of cell death. PMID:23717080

Total alkaloids of Rubus alceifolius Poir shows anti-angiogenic activity in vivo and in vitro.

PubMed

Zhao, Jinyan; Lin, Wei; Zhuang, Qunchuan; Zhong, Xiaoyong; Cao, Zhiyun; Hong, Zhenfeng; Peng, Jun

2014-11-01

Total alkaloids is an active ingredient of the natural plant Rubus alceifolius Poir, commonly used for the treatment of various cancers. Antitumor effects may be mediated through anti-angiogenic mechanisms. As such, the goal of the present study was to investigate and evaluate the effect of total alkaloids in Rubus alceifolius Poir (TARAP) on tumor angiogenesis and investigate the underlying molecular mechanisms of TARAP action in vivo and in vitro. A chick embryo chorioallantoic membrane (CAM) assay was used to assess angiogenesis in vivo. An MTT assay was performed to determine the viability of human umbilical vein endothelial cells (HUVECs) with and without treatment. Cell cycle progression of HUVECs was examined by FACS analysis with propidium iodide staining. HUVEC migration was determined using a scratch wound method. Tube formation of HUVECs was assessed with an ECMatrix gel system, and mRNA and protein expression of VEGF-A in both HUVECs and HepG2 human hepatocellular carcinoma cells were examined by RT-PCR and ELISA, respectively. Our results showed that TARAP inhibited angiogenesis in the CAM model in vivo and inhibited HUVEC proliferation via blocking cell cycle G1 to S progression in a dose- and time-dependent manners in vitro. Moreover, TARAP inhibited HUVEC migration and tube formation and downregulated mRNA and protein expression of VEGF-A in both HepG2 cells and HUVECs. Our findings suggest that the anti-angiogenic activity of TARAP may partly contribute to its antitumor properties and may be valuable for the treatment of diseases involving pathologic angiogenesis such as cancer. © The Author(s) 2014.

Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells

PubMed Central

Luzak, Boguslawa; Golanski, Jacek; Rozalski, Marek; Krajewska, Urszula; Olas, Beata

2010-01-01

Introduction Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. Material and methods This study encompasses two models of monitoring platelet reactivity: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. Results We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. Conclusions It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases. PMID:22371737

Ascorbic Acid Prevents VEGF-induced Increases in Endothelial Barrier Permeability

PubMed Central

Ulker, Esad; Parker, William H.; Raj, Amita; Qu, Zhi-chao; May, James M.

2015-01-01

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 μM and complete inhibition at 50 μM. Loading cells with 100 μM ascorbate also decreased basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25%, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 μM L-NAME (but not D-NAME) as well as by 30 μM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema. PMID:26590088

[The role of homeobox B2 gene in vascular endothelial proliferation and the protective effects of VEGF on the endothelia against radiation injury].

PubMed

Liu, Xu-sheng; Zhang, Xiao-qi; Liu, Liang; Ming, Jia; Xu, Hui; Ran, Xin-ze; Cheng, Tian-min

2004-10-01

To explore the role of HOXB2 gene in the proliferation of primary human umbilical vein endothelial cells (HUVECs) and the protective effects of VEGF on the endothelia against radiation injury. HUVECs were isolated, cultured, subcultured and identified. (1) Liposome coated oligodeoxynucleotide (odn) and homeoboxB2 antisense oligodeoxyncleotide (HOXB2asodn) were prepared prepared in the concentrations of 0.25, 0.5, 1.0 and 2.5 mg/L for the stimulation of HUVEC. (3)H-TdR incorporation test and MTT method were employed to determine the proliferation activity of HUVECs after activation. The cell cycle analysis of HUVECs was determined by flow cytometry. The expression level of HOXB2mRNA within HUVECs was detected by RT-PCR (reverse transcription polymerase chain reaction). (2) HUVECs were separately treated with the addition of VEGF in concentration of 50 microg/L, by radiation in the dose of 6 Gy or 12 Gy (60)Co gamma gamma ray, or radiation with 12 Gy (60)Co gamma gamma ray followed by the addition of VEGF in dose of 50 microg/L. The cellular morphology was observed and the cellular proliferation activity was determined by MTT method. (1) The proliferation activity of HUVECs could be markedly inhibited by liposome coated HOXB2asodn in comparison to liposome-odn (P < 0.05 or 0.001), and the inhibition effect was positively correlated with the increase in asodn concentration. The cell ratio in S phase and the expression level of the HOXB2mRNA could be lowered by asodn in dose of 2.5 mg/L (P < 0.05 or 0.001). (2) Radiation by (60)Co gamma ray could lead to the nuclear enlargement, vacuolation in the cytoplasm, multiplicity of nucleus and nuclear swelling. The proliferative activity of HUVECs was increased from 0.365 +/- 0.047 and 0.487 +/- 0.022 without radiation to 0.557 +/- 0.042 and 0.648 +/- 0.021 24 and 48 hours after 6 Gy radiation However it was decreased to 0.263 +/- 0.038 and 0.306 +/- 0.024 (P < 0.01) after 12 Gy (60)Co gamma ray radiation. Nevertheless, the cell morphology was obviously improved and the proliferation was enhanced by the addition of VEGF after 12 Gy radiation. HOXB2 gene played important roles in the biological activities of HUVECs. Small dose (6 Gy) gamma-radiation could promote, but large dose (12 Gy) could decrease the mRNA expression of HOXB2 gene in HUVECs. In addition, VEGF could protect HUVECs against radiation injury.

Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In Vitro and In Vivo.

PubMed

Jung, Hong-Ryul; Kang, Hyun Mi; Ryu, Jea-Woon; Kim, Dae-Soo; Noh, Kyung Hee; Kim, Eun-Su; Lee, Ho-Joon; Chung, Kyung-Sook; Cho, Hyun-Soo; Kim, Nam-Soon; Im, Dong-Soo; Lim, Jung Hwa; Jung, Cho-Rok

2017-09-05

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

Cytotoxic 2',5'-dihydroxychalcones with unexpected antiangiogenic activity.

PubMed

Nam, Nguyen-Hai; Kim, Yong; You, Young-Jae; Hong, Dong-Ho; Kim, Hwan-Mook; Ahn, Byung-Zun

2003-02-01

A series of 2',5'-dihydroxychalcones were synthesized and evaluated for cytotoxicity against tumor cell lines and human umbilical venous endothelial cells (HUVEC). It was found that chalcones with electron-withdrawing substituents on the B ring exhibited potent cytotoxicity against a variety of tumor cell lines while compounds with electron-releasing groups were less potent in general. Those compounds with B ring replaced by extended or heteroaromatic rings exhibited significant bioactivity. Several compounds were shown to have marked cytotoxic selectivity towards HUVECs. Especially, among the synthesized compounds, 2-chloro-2',5'-dihydroxychalcone (2-3) showed the highest selectivity index up to 66 in comparison to HCT116 cells. This compound also exhibited strong inhibitory effects on the HUVEC tube formation in an in vitro model. When administered into BDF1 mice bearing Lewis lung carcinoma cells at 50 mg kg(-1) day(-1), 2-3 was found to inhibit the growth of tumor mass by 60.5%.

Characterization of Adsorbents for Cytokine Removal from Blood in an In Vitro Model

PubMed Central

Gabor, Franz; Hartmann, Jens

2015-01-01

Introduction. Cytokines are basic targets that have to be removed effectively in order to improve the patient's health status in treating severe inflammation, sepsis, and septic shock. Although there are different adsorbents commercially available, the success of their clinical use is limited. Here, we tested different adsorbents for their effective removal of cytokines from plasma and the resulting effect on endothelial cell activation. Methods. The three polystyrene divinylbenzene (PS-DVB) based adsorbents Amberchrom CG161c and CG300m and a clinically approved haemoperfusion adsorbent (HAC) were studied with regard to cytokine removal in human blood. To induce cytokine release from leucocytes, human blood cells were stimulated with 1 ng/ml LPS for 4 hours. Plasma was separated and adsorption experiments in a dynamic model were performed. The effect of cytokine removal on endothelial cell activation was evaluated using a HUVEC-based cell culture model. The beneficial outcome was assessed by measuring ICAM-1, E-selectin, and secreted cytokines IL-8 and IL-6. Additionally the threshold concentration for HUVEC activation by TNF-α and IL-1β was determined using this cell culture model. Results. CG161c showed promising results in removing the investigated cytokines. Due to its pore size the adsorbent efficiently removed the key factor TNF-α, outperforming the commercially available adsorbents. The CG161c treatment reduced cytokine secretion and expression of cell adhesion molecules by HUVEC which underlines the importance of effective removal of TNF-α in inflammatory diseases. Conclusion. These results confirm the hypothesis that cytokine removal from the blood should approach physiological levels in order to reduce endothelial cell activation. PMID:26770992

Binding and internalization of NGR-peptide-targeted liposomal doxorubicin (TVT-DOX) in CD13-expressing cells and its antitumor effects.

PubMed

Garde, Seema V; Forté, André J; Ge, Michael; Lepekhin, Eugene A; Panchal, Chandra J; Rabbani, Shafaat A; Wu, Jinzi J

2007-11-01

In an effort to develop new agents and molecular targets for the treatment of cancer, aspargine-glycine-arginine (NGR)-targeted liposomal doxorubicin (TVT-DOX) is being studied. The NGR peptide on the surface of liposomal doxorubicin (DOX) targets an aminopeptidase N (CD13) isoform, specific to the tumor neovasculature, making it a promising strategy. To further understand the molecular mechanisms of action, we investigated cell binding, kinetics of internalization as well as cytotoxicity of TVT-DOX in vitro. We demonstrate the specific binding of TVT-DOX to CD13-expressing endothelial [human umbilical vein endothelial cells (HUVEC) and Kaposi sarcoma-derived endothelial cells (SLK)] and tumor (fibrosarcoma, HT-1080) cells in vitro. Following binding, the drug was shown to internalize through the endosomal pathway, eventually leading to the localization of doxorubicin in cell nuclei. TVT-DOX showed selective toxicity toward CD13-expressing HUVEC, sparing the CD13-negative colon-cancer cells, HT-29. Additionally, the nontargeted counterpart of TVT-DOX, Caelyx, was less cytotoxic to the CD13-positive HUVECs demonstrating the advantages of NGR targeting in vitro. The antitumor activity of TVT-DOX was tested in nude mice bearing human prostate-cancer xenografts (PC3). A significant growth inhibition (up to 60%) of PC3 tumors in vivo was observed. Reduction of tumor vasculature following treatment with TVT-DOX was also apparent. We further compared the efficacies of TVT-DOX and free doxorubicin in the DOX-resistant colon-cancer model, HCT-116, and observed the more pronounced antitumor effects of the TVT-DOX formulation over free DOX. The potential utility of TVT-DOX in a variety of vascularized solid tumors is promising.

Sulphonated Formononetin Induces Angiogenesis through Vascular Endothelial Growth Factor/cAMP Response Element-Binding Protein/Early Growth Response 3/Vascular Cell Adhesion Molecule 1 and Wnt/β-Catenin Signaling Pathway.

PubMed

Dong, Zhaoju; Shi, Yanan; Zhao, Huijuan; Li, Ning; Ye, Liang; Zhang, Shuping; Zhu, Haibo

2018-01-01

Sodium formononetin-3'-sulphonate (Sul-F) is a derivative of the isoflavone formononetin. In this study, we investigated whether Sul-F can regulate angiogenesis and the potential mechanism in vitro. We examined the effects of Sul-F on cell proliferation, cell invasion, and tube formation in the human umbilical vein endothelial cell line (HUVEC). To better understand the mechanism involved, we investigated effects of the following compounds: cAMP response element-binding protein (CREB) inhibitor 2-naphthol-AS-E-phosphate (KG-501), early growth response 3 (Egr-3) siRNA, vascular endothelial growth factor (VEGF) antagonist soluble VEGF receptor 1 (sFlt-1), VEGF receptor 2 blocker SU-1498, Wnt5a antagonist WIF-1 recombinant protein (WIF-1), and inhibitor of Wnt/β-catenin recombinant Dickkopf-1 protein (DKK-1). HUVEC proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). A scratch adhesion test was used to assess cell invasion ability. Matrigel tube formation assay was performed to test capillary tube formation ability. Activation of the VEGF/CREB/Egr-3/Vascular cell adhesion molecule 1 (VCAM-1) pathway in HUVEC was tested by Western blot analysis. Our results suggest that Sul-F induced angiogenesis in vitro by enhancing cell proliferation, invasion, and tube formation. The increase in proliferation and tube formation by Sul-F was counteracted by DKK-1, WIF-1, SU1498, KG-501, sFlt-1, and Egr-3 siRNA. These results may suggest that Sul-F induces angiogenesis in vitro via a programed Wnt/β-catenin pathway and VEGF/CREB/Egr-3/VCAM-1 signaling axis. © 2017 S. Karger AG, Basel.

New Natural Pigment Fraction Isolated from Saw Palmetto: Potential for Adjuvant Therapy of Hepatocellular Carcinoma

PubMed Central

Tan, Hor-Yue; Wang, Ning; Takahashi, Masao; Feng, Yigang; Li, Hongyun; Feng, Yibin

2016-01-01

For the first time, we discovered a small proportion of aqueous fraction from Saw Palmetto apart from the fatty acid-rich fraction exhibited pharmacological activity. Therefore, this study aims to explore the anti-tumor potential of red pigmented aqueous fraction of Saw Palmetto, NYG on human hepatocellular carcinoma and its possible targets. Subcutaneous xenograft and orthotopic implantation models of HCC were used to evaluate the tumor inhibitory effect of NYG. Human hepatocellular carcinoma (HCC) cell lines and human umbilical vein endothelial cells (HUVEC) were used as in vitro model. The mRNA expression was conducted by qPCR. Protein expression was monitored by immunoblotting and immunohistochemistry. Cell migration and blood vessel formation were determined by chamber assay and tube formation assay, respectively. Significant tumor inhibition of NYG in dose-dependent manner was observed on subcutaneous xenograft and orthotopic HCC model. NYG has no direct action on cell viability or VEGF secretion of HCC cells. However, NYG reduced in vitro migration and vessel formation activities of HUVEC cells, as well as in vivo intratumoral neovascularization. NYG attenuated extracellular signal-regulated kinases (ERK) activation in endothelial cells, which may be associated with the suppression of migration and tube formation of HUVEC. NYG suppressed tumor expansion of HCC via inhibiting neovascularization, and may be potential adjuvant treatment for HCC. PMID:27527161

3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh.

PubMed

Kim, Min Sung; Lee, Byungjun; Kim, Hong Nam; Bang, Seokyoung; Yang, Hee Seok; Kang, Seong Min; Suh, Kahp-Yang; Park, Suk-Hee; Jeon, Noo Li

2017-03-23

We present a novel approach for assembling 3D tissue by layer-by-layer stacking of cell sheets formed on aligned nanofiber mesh. A rigid frame was used to repeatedly collect aligned electrospun PCL (polycaprolactone) nanofiber to form a mesh structure with average distance between fibers 6.4 µm. When human umbilical vein endothelial cells (HUVECs), human foreskin dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the nanofiber mesh, they formed confluent monolayers and could be handled as continuous cell sheets with areas 3 × 3 cm 2 or larger. Thicker 3D tissues have been formed by stacking multiple cell sheets collected on frames that can be nested (i.e. Matryoshka dolls) without any special tools. When cultured on the nanofiber mesh, skeletal muscle, C2C12 cells oriented along the direction of the nanofibers and differentiated into uniaxially aligned multinucleated myotube. Myotube cell sheets were stacked (upto 3 layers) in alternating or aligned directions to form thicker tissue with ∼50 µm thickness. Sandwiching HUVEC cell sheets with two dermal fibroblast cell sheets resulted in vascularized 3D tissue. HUVECs formed extensive networks and expressed CD31, a marker of endothelial cells. Cell sheets formed on nanofiber mesh have a number of advantages, including manipulation and stacking of multiple cell sheets for constructing 3D tissue and may find applications in a variety of tissue engineering applications.

Nitride coating enhances endothelialization on biomedical NiTi shape memory alloy.

PubMed

Ion, Raluca; Luculescu, Catalin; Cimpean, Anisoara; Marx, Philippe; Gordin, Doina-Margareta; Gloriant, Thierry

2016-05-01

Surface nitriding was demonstrated to be an effective process for improving the biocompatibility of implantable devices. In this study, we investigated the benefits of nitriding the NiTi shape memory alloy for vascular stent applications. Results from cell experiments indicated that, compared to untreated NiTi, a superficial gas nitriding treatment enhanced the adhesion of human umbilical vein endothelial cells (HUVECs), cell spreading and proliferation. This investigation provides data to demonstrate the possibility of improving the rate of endothelialization on NiTi by means of nitride coating. Copyright © 2016 Elsevier B.V. All rights reserved.

Luteolin suppresses angiogenesis and vasculogenic mimicry formation through inhibiting Notch1-VEGF signaling in gastric cancer.

PubMed

Zang, Mingde; Hu, Lei; Zhang, Baogui; Zhu, Zhenglun; Li, Jianfang; Zhu, Zhenggang; Yan, Min; Liu, Bingya

2017-08-26

Gastric cancer is a great threat to the health of the people worldwide and lacks effective therapeutic regimens. Luteolin is one of Chinese herbs and presents in many fruits and green plants. In our previous study, we observed that luteolin inhibited cell migration and promoted cell apoptosis in gastric cancer. In the present study, luteolin significantly inhibited tube formation of human umbilical vein endothelial cells (HUVECs) through decreasing cell migration and proliferation of HUVECs in a dose-dependent manner. Vasculogenic mimicry (VM) tubes formed by gastric cancer cells were also inhibited with luteolin treatment. To explore how luteolin inhibited tubes formation, ELISA assay for VEGF was performed. Both of the VEGF secretion from Hs-746T cells and HUVECs were significantly decreased subsequent to luteolin treatment. In addition, cell migration was increased with the interaction between gastric cancer cells and HUVECs in co-culture assays. However, the promoting effects were abolished subsequent to luteolin treatment. Furthermore, luteolin inhibited VEGF secretion through suppressing Notch1 expression in gastric cancer. Overexpression of Notch1 in gastric cancer cells partially rescued the effects on cell migration, proliferation, HUVECs tube formation, and VM formation induced by luteolin treatment. In conclusion, luteolin inhibits angiogenesis and VM formation in gastric cancer through suppressing VEGF secretion dependent on Notch1 expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Protective effects of hydrogen-rich medium on lipopolysaccharide-induced monocytic adhesion and vascular endothelial permeability through regulation of vascular endothelial cadherin.

PubMed

Yu, Y; Wang, W N; Han, H Z; Xie, K L; Wang, G L; Yu, Y H

2015-06-11

We observed the effect of hydrogen-rich medium on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs), hyaline leukocyte conglutination, and permeability of the endothelium. Endotheliocytes were inoculated on 6-well plates and randomly divided into 4 groups: control, H2, LPS, LPS+H2, H2, and LPS+H2 in saturated hydrogen-rich medium. We applied Wright's stain-ing to observe conglutination of hyaline leukocytes and HUVECs, flow cytometry to determine the content of vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), enzyme-linked immunosorbent assay to measure the E-selectin concentration in the cell liquor, the transendothelial electrical resistance (TEER) to test the permeability of endothelial cells, and Western blot and immunofluorescence to test the expression and distribution of vascular endothelial (VE)-cadherin. Compared with control cells, there was an increase in endothelium-hyaline leukocyte conglutination, a reduction in VCAM-1, ICAM-1, and E-selectin, and the TEER value increased obviously. Compared with LPS, there was an obvious reduction in the conglutination of LPS+H2 cells, a reduction in VCAM-1, ICAM-1, and E-selectin levels, and a reduction in the TEER-resistance value, while the expression of VE-cadherin increased. Fluorescence results showed that, compared with control cells, the VE-cadherin in LPS cells was in-complete at the cell joints. Compared with LPS cells, the VE-cadherin in LPS+H2 cells was even and complete at the cell joints. Liquid rich in hydrogen could reduce LPS-induced production of adhesion molecules and endothelium-hyaline leukocyte conglutination, and influence the expression and distribution of VE-cadherin to regulate the permeability of the endothelium.

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

PubMed Central

Xie, Liping; Liu, Zhen; Lu, Hui; Zhang, Wen; Mi, Qiongyu; Li, Xiaozhen; Tang, Yan; Chen, Qi; Ferro, Albert; Ji, Yong

2012-01-01

BACKGROUND AND PURPOSE One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O2•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism. EXPERIMENTAL APPROACH HUVECs were incubated with ox-LDL with/without pyridoxine, NG-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O2•− was measured using lucigenin chemiluminescence, and O2•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot. KEY RESULTS Ox-LDL significantly increased O2•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O2•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments. CONCLUSIONS AND IMPLICATIONS Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction. PMID:21797845

Real space flight travel is associated with ultrastructural changes, cytoskeletal disruption and premature senescence of HUVEC.

PubMed

Kapitonova, M Y; Muid, S; Froemming, G R A; Yusoff, W N W; Othman, S; Ali, A M; Nawawi, H M

2012-12-01

Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.

Fabrication of viable and functional pre-vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks.

PubMed

Zhang, Songjie; Zhou, Min; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

2017-08-01

Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre-vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two-stage (proliferative-osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. Collectively, this work established an effective method to fabricate pre-vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RXR agonists inhibit high glucose-induced upregulation of inflammation by suppressing activation of the NADPH oxidase-nuclear factor-κB pathway in human endothelial cells.

PubMed

Ning, R B; Zhu, J; Chai, D J; Xu, C S; Xie, H; Lin, X Y; Zeng, J Z; Lin, J X

2013-12-13

An inflammatory response induced by high glucose is a cause of endothelial dysfunction in diabetes and is an important contributing link to atherosclerosis. Diabetes is an independent risk factor of atherosclerosis and activation of retinoid X receptor (RXR) has been shown to exert anti-atherogenic effects. In the present study, we examined the effects of the RXR ligands 9-cis-retinoic acid (9-cis-RA) and SR11237 on high glucose-induced inflammation in human umbilical endothelial vein endothelial cells (HUVECs) and explored the potential mechanism. Our results showed that the inflammation induced by high-glucose in HUVECs was mainly mediated by the activation of nuclear factor-B (NF- κB). High glucose-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were in comparison, significantly decreased by treatment with RXR. The effect of RXR agonists was mainly due to the inhibition of NF-κB activation. Using pharmacological inhibitors and siRNA, we confirmed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was an upstream activator of NF-κB. Furthermore, RXR agonists significantly inhibited high glucose-induced activation of NADPH oxidase and significantly decreased the production of reactive oxygen species (ROS). To explore whether the rapid inhibitory effects of RXR agonists were in fact mediated by RXR, we examined the effect of RXR downregulation by RXR siRNA. Our results showed that RXR siRNA largely abrogated the effects of RXR agonists, suggesting the requirement of RXR expression. Therefore, we have shown that RXR is involved in the regulation of NADPH oxidase- NF-κB signal pathway, as the RXR ligands antagonized the inflammatory response in HUVECs induced by high glucose.

O-GlcNAc modification of Sp1 mediates hyperglycaemia-induced ICAM-1 up-regulation in endothelial cells.

PubMed

Zhang, Yuan; Qu, Yuan; Niu, Tian; Wang, Haiyan; Liu, Kun

2017-02-26

Intracellular adhesion molecule 1 (ICAM-1) is an important inflammatory factor that causes retinal damage during diabetic retinopathy. Hyperglycaemia can increase ICAM-1 expression in endothelial cells and the ICAM-1 promoter is responsive to the transcription factor specificity protein 1 (Sp1). O-GlcNAc modification is driven by the glucose concentration and has a profound effect on Sp1 activity. In this study, we investigated the underlying mechanism through which hyperglycaemia triggers ICAM-1 expression, which is mediated by O-GlcNAc modification of Sp1 in human umbilical vein endothelial cells (HUVECs) and rat retinal capillary endothelial cells (RRCECs). We showed that hyperglycaemia (30 mM) increased ICAM-1 expression compared to control conditions (5 mM). The addition of an OGT inhibitor decreased ICAM-1 expression and addition of an OGA inhibitor enhanced ICAM-1 expression. Furthermore, cells transduced with siSp1 exhibited dramatically decreased ICAM-1 expression. These results proved that the up-regulation of ICAM-1 with hyperglycaemia is mediated by O-GlcNAc modification of Sp1. It helps to explain the mechanism of ICAM-1 processing in HUVECs and RRCECs. Understanding how this inflammatory factor is modulated during diabetic retinopathy will ultimately help to design novel therapeutics to treat this condition. Copyright © 2017. Published by Elsevier Inc.

FGF21 protects human umbilical vein endothelial cells against high glucose-induced apoptosis via PI3K/Akt/Fox3a signaling pathway.

PubMed

Guo, Dongmin; Xiao, Lele; Hu, Huijun; Liu, Mihua; Yang, Lu; Lin, Xiaolong

2018-05-25

Diabetic macroangiopathy is the main cause of morbidity and mortality in patients with diabetes. Endothelial cell injury is a pathological precondition for diabetic macroangiopathy. Fibroblast growth factor 21 (FGF21) is a key metabolic regulator which has recently been suggested to protect cardiac myocytes and vascular cells against oxidative stress-induced injury in vitro and vivo. In this study, we aimed to investigate the protective capacity of FGF21 in human umbilical vein endothelial cells (HUVECs) against high glucose (HG)-induced apoptosis via phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt)/FoxO3a pathway. The cell viability was examined by CCK-8 assay, Intracellular ROS levels were measured by the detection of the fluorescent product formed by the oxidation of DCFH-DA, Apoptosis was analyzed using Hoechst 33258 nuclear staining and Flow Cytometry Analysis (FCA), the expression of protein were detected by Western blot. Results show that pretreating HUVECs with FGF21 before exposure to HG increases cell viability, while decreasing apoptosis and the generation of reactive oxygen species. Western blot analysis shows that HG reduces the phosphorylation of Akt and FoxO3a, and induces nuclear localization of FoxO3a. The effects were significantly reversed by FGF21 pre-treatment. Furthermore, the protective effects of FGF21 were prevented by PI3K/Akt inhibitor LY294002. Our data demonstrates that FGF21 protects HUVECs from HG-induced oxidative stress and apoptosis via the activation of PI3K/Akt/FoxO3a signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

The Antiangiogenesis Effect of Pirfenidone in Wound Healing In Vitro.

PubMed

Liu, Xiao'an; Yang, Yangfan; Guo, Xiujuan; Liu, Liling; Wu, Kaili; Yu, Minbin

2017-11-01

Abstracts Purpose: Pirfenidone is mostly used in antifibrotic and anti-inflammatory therapies. We have previously demonstrated that pirfenidone had antifibrotic and anti-inflammatory effects on the wound healing process after glaucoma filtration surgery in vitro and in vivo. Since the wound healing and reactive scarring process simultaneously involves inflammation, fibrosis, and angiogenesis, and angiogenesis plays a more important role in chronic or prolonged wound healing, we tried to explore the antiangiogenesis effect in pirfenidone and its potential multitarget function in regulating excessive scarring. The aim of the present study was to investigate the antiangiogenesis effect of pirfenidone. The proliferation of human umbilical vein endothelial cells (HUVECs) and human Tenon's fibroblasts (HTFs) were detected by WST-1 assay. The cell viability of HUVECs was measured by Trypan Blue together with lactate dehydrogenase, Annexin 5 experiment, and Ki-67 immunofluorescence assay. The functions of HUVECs and HTFs were demonstrated using cell migration assay, transwell invasion assay, and tube formation assay. The expression levels of vascular endothelial growth factor-A (VEGF-A), VEGF receptor-2 (VEGFR-2), neuropilin-1(NRP-1), and their downstream signaling proteins p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mechanistic target of rapamycin (mTOR) were indicated by western blot assay. The secretion of VEGF-A was detected by enzyme-linked immunosorbent assay. Pirfenidone inhibited proliferation, migration, invasion, and tube formation of HUVECs in vitro, and had an equivalent antiangiogenesis effect when compared with Ranibizumab in HUVECs and HTFs. Pirfenidone downregulated VEGF-A/VEGFR-2, VEGF-A/NRP-1, and its downstream signaling pathway protein expression. Pirfenidone has an antiangiogenesis effect in the wound healing process and may become an ideal multitarget antiscarring agent after glaucoma filtration surgery.

Melatonin affects the dynamic steady-state equilibrium of estrogen sulfates in human umbilical vein endothelial cells by regulating the balance between estrogen sulfatase and sulfotransferase.

PubMed

González, Alicia; Martínez-Campa, Carlos; Alonso-González, Carolina; Cos, Samuel

2015-12-01

Melatonin is known to reduce the growth of endocrine-responsive breast cancers by interacting with estrogen signaling pathways. Estrogens play an important role in breast cancer, but also in various types of tissues, including vascular tissue. Estrogen sulfatase (STS) converts inactive estrogen sulfates into active estrogens, whereas estrogen sulfotransferase (EST) sulfonates estrogens to estrogen sulfates. Therefore, STS and EST are considered to be involved in the regulation of local estrogen levels in hormone‑dependent tumors and in non-pathologic tissues, such as those of the vascular system. Estrogens have a major impact on the vasculature, influencing vascular function, the expression of adhesion proteins, angiogenesis and the inflammatory state. In this study, we investigated the status of STS and EST in human umbilical vein endothelial cells (HUVECs) and the modulatory effects of melatonin. Both STS and EST were highly expressed in the HUVECs. The enzymatic activity correlated with the expression levels in these cells. Our findings also demonstrated that melatonin, at physiological concentrations, modulated the synthesis and transformation of biologically active estrogens in HUVECs through the inhibition of STS activity and expression, and the stimulation of EST activity and expression. Since melatonin decreased the STS levels and increased the EST levels, it modified the dynamic steady‑state equilibrium of estrogen sulfates by increasing the inactive estrogen levels and decreasing the active estrogen levels. Therefore, melatonin may modulate the known different biological actions of estrogens in endothelial cells, as well as in estrogen-dependent tumors and non-pathologic tissues.

Difference in protective effects of GIP and GLP-1 on endothelial cells according to cyclic adenosine monophosphate response.

PubMed

Lim, Dong-Mee; Park, Keun-Young; Hwang, Won-Min; Kim, Ju-Young; Kim, Byung-Joon

2017-05-01

Receptors for glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are present in vascular endothelial cells. Previous studies investigating euglycemic status have demonstrated that GIP is directly involved in the physiology of blood vessels by controlling the blood flow rate of portal veins and that GLP-1 has a protective effect on blood vessels by acting on endothelial cells. However, to the best of our knowledge, the effects of GIP and GLP-1 on endothelial cells in patients with hyperglycemia remain unknown. Therefore, the present study investigated whether the effect of the incretin hormones GLP-1 and GIP differed with regards to the reversal of endothelial cell dysfunction caused by hyperglycemia. The production of nitric oxide (NO) was measured using the Griess reagent system kit and the expression of cyclic adenosine monophosphate (cAMP) in the cell was measured at a wavelength of 405 nm with the ELISA reader using the cyclic AMP EIA kit. Exposure of human umbilical vein endothelial cells (HUVEC) to a high glucose concentration decreased NO and endothelial nitric oxide synthase (eNOS) levels but increased inducible NOS (iNOS) levels. However, when HUVECs were pretreated with GLP-1, a reduction of iNOS expression was observed and the expression of eNOS and NO were increased, as opposed to pretreatment with GIP. The results differed according to the response of cAMP, the second messenger of incretin hormones: The GIP pretreatment group did not exhibit an increase in cAMP levels while the GLP-1 pretreatment group did. The results of the present study provide evidence that GLP-1, but not GIP, has a protective effect on endothelial function associated with cardiovascular disease, as it is associated with increased eNOS expression and the levels of NO. This effect may be due to an increase in the cAMP concentration during hyperglycemic events.

Effects of gestational hypertension and pre-eclampsia in mRNA expression of fibrinolysis genes in primary cultured human umbilical vein endothelial cells.

PubMed

Poblete-Naredo, Irais; Rodríguez-Yáñez, Yury; Corona-Núñez, Rogelio O; González-Monroy, Stuart; Salinas, Juan E; Albores, Arnulfo

2018-05-17

Hypertension disorders (HD) and pre-eclampsia (PRE) are leading causes of maternal deaths worldwide. PRE is associated with vascular endothelial dysfunction and with deregulation of the fibrinolysis pathway genes. Fibrinolysis is the fibrin clot hydrolysis process catalyzed by plasmin, a proteolytic enzyme formed from plasminogen. Plasminogen is cleaved by tissue-type (tPA) and urokinase-type (uPA) activators and inhibited by the plasminogen activator inhibitors type-1 (PAI-1) and type-2 (PAI-2). The whole process maintains blood hemostasis. This study aims to assess PAI-1, PAI-2, tPA and uPA mRNA expression in primary cultured human umbilical vein endothelial cells (HUVEC) isolated and cultured from healthy, HD and PRE women. Results show that PAI-1 and PAI-2 mRNA decreased in HD-HUVEC, whereas PAI-1 and uPA decreased in PRE-HUVEC cultures compared to control ones. Notably, the expression ratio between pro- and anti-fibrinolytic actors remained unchanged among the studied groups. It seems that newborn's hemostasis is maintained balanced probably by a compensatory mechanism that involves changes in the fibrinolysis gene expression profile. The real impact of these changes in mRNA expression is unknown, however, it is suggested that these changes could be associated with an increased predisposition to vascular disease development in the progeny. Copyright © 2018. Published by Elsevier Ltd.

Brain-derived neurotrophic factor (BDNF) -TrKB signaling modulates cancer-endothelial cells interaction and affects the outcomes of triple negative breast cancer

PubMed Central

Tsai, Yi-Fang; Hsu, Chih-Yi; Yang, Muh-Hwa; Shyr, Yi-Ming

2017-01-01

Aims There is good evidence that the tumor microenvironment plays an important role in cancer metastasis and progression. Our previous studies have shown that brain-derived neurotrophic factor (BDNF) participates in the process of metastasis and in the migration of cancer cells. The aim of this study was to investigate the role of BDNF on the tumor cell microenvironment, namely, the cancer cell-endothelial cell interaction of TNBC cells. Methods We conducted oligoneucleotide microarray analysis of potential biomarkers that are able to differentiate recurrent TNBC from non-recurrent TNBC. The MDA-MB-231 and human endothelial HUVEC lines were used for this study and our approaches included functional studies, such as migration assay, as well as Western blot and real-time PCR analysis of migration and angiogenic signaling. In addition, we analyzed the survival outcome of TNBC breast cancer patients according to their expression level of BDNF using clinical samples. Results The results demonstrated that BDNF was able to bring about autocrinal (MDA-MB-231) and paracrinal (HUVECs) regulation of BDNF-TrkB gene expression and this affected cell migratory activity. The BDNF-induced migratory activity was blocked by inhibitors of ERK, PI3K and TrkB when MDA-MB-231 cells were examined, but only an inhibitor of ERK blocked this activity when HUVEC cells were used. Furthermore, decreased migratory activity was found for △BDNF and △TrkB cell lines. Ingenuity pathway analysis (IPA) of MDA-MB-231 cells showed that BDNF is a key factor that is able to regulate a network made up of metalloproteases and calmodulin. Protein expression levels in a tissue array of tumor slices were found to be correlated with patient prognosis and the results showed that there was significant correlation of TrkB expression, but not of BDNF. expressionwith patient DFS and OS. Conclusion Our study demonstrates that up-regulation of the BDNF signaling pathway seems tobe involved in the mechanism associated with early recurrence in triple negative breast cancer cell. In addition, BDNF can function in either an autocrine or a paracrine manner to increase the migration ability of both MDA-MB-231 cells and HUVEC cells. Finally, overexpression of TrkB, but not of BDNF, is significantly associated with a poor survival outcome for TNBC patients. PMID:28604807

Brain-derived neurotrophic factor (BDNF) -TrKB signaling modulates cancer-endothelial cells interaction and affects the outcomes of triple negative breast cancer.

PubMed

Tsai, Yi-Fang; Tseng, Ling-Ming; Hsu, Chih-Yi; Yang, Muh-Hwa; Chiu, Jen-Hwey; Shyr, Yi-Ming

2017-01-01

There is good evidence that the tumor microenvironment plays an important role in cancer metastasis and progression. Our previous studies have shown that brain-derived neurotrophic factor (BDNF) participates in the process of metastasis and in the migration of cancer cells. The aim of this study was to investigate the role of BDNF on the tumor cell microenvironment, namely, the cancer cell-endothelial cell interaction of TNBC cells. We conducted oligoneucleotide microarray analysis of potential biomarkers that are able to differentiate recurrent TNBC from non-recurrent TNBC. The MDA-MB-231 and human endothelial HUVEC lines were used for this study and our approaches included functional studies, such as migration assay, as well as Western blot and real-time PCR analysis of migration and angiogenic signaling. In addition, we analyzed the survival outcome of TNBC breast cancer patients according to their expression level of BDNF using clinical samples. The results demonstrated that BDNF was able to bring about autocrinal (MDA-MB-231) and paracrinal (HUVECs) regulation of BDNF-TrkB gene expression and this affected cell migratory activity. The BDNF-induced migratory activity was blocked by inhibitors of ERK, PI3K and TrkB when MDA-MB-231 cells were examined, but only an inhibitor of ERK blocked this activity when HUVEC cells were used. Furthermore, decreased migratory activity was found for △BDNF and △TrkB cell lines. Ingenuity pathway analysis (IPA) of MDA-MB-231 cells showed that BDNF is a key factor that is able to regulate a network made up of metalloproteases and calmodulin. Protein expression levels in a tissue array of tumor slices were found to be correlated with patient prognosis and the results showed that there was significant correlation of TrkB expression, but not of BDNF. expressionwith patient DFS and OS. Our study demonstrates that up-regulation of the BDNF signaling pathway seems tobe involved in the mechanism associated with early recurrence in triple negative breast cancer cell. In addition, BDNF can function in either an autocrine or a paracrine manner to increase the migration ability of both MDA-MB-231 cells and HUVEC cells. Finally, overexpression of TrkB, but not of BDNF, is significantly associated with a poor survival outcome for TNBC patients.

Increased leukocyte adhesion to vascular endothelium in preeclampsia is inhibited by antioxidants.

PubMed

Ryu, Seongho; Huppmann, Alison R; Sambangi, Nirmala; Takacs, Peter; Kauma, Scott W

2007-04-01

To test the hypothesis that plasma from women with preeclampsia increases leukocyte adhesion to vascular endothelial cells and that antioxidants inhibit this effect. Plasma from 12 women with severe preeclampsia and 12 with normal pregnancy was tested in an in vitro leukocyte-endothelium adhesion assay in the presence or absence of vitamin E, vitamin C, or N-acetylcysteine. Preeclamptic plasma significantly increased monocyte (U937 cells) and T-cell (Jurkat) adhesion to human umbilical vein (HUVEC) and microvascular endothelial cells, compared with normal pregnant plasma. The antioxidants vitamin E, vitamin C, and N-acetylcysteine significantly inhibited monocyte adhesion to HUVEC in the presence of preeclamptic but not normal pregnant plasma. Increased adhesion in response to preeclamptic plasma was not mediated through a protein kinase C (PKC) mechanism, because the PKC inhibitor bisindolylmaleimide I had no effect on adhesion in the presence of preeclamptic plasma. Severe preeclampsia is associated with increased leukocyte-endothelium adhesion and clinically useful antioxidants can inhibit this effect.

Melittin suppresses cathepsin S-induced invasion and angiogenesis via blocking of the VEGF-A/VEGFR-2/MEK1/ERK1/2 pathway in human hepatocellular carcinoma

PubMed Central

ZHANG, ZHI; ZHANG, HANGUANG; PENG, TAO; LI, DONGDONG; XU, JING

2016-01-01

Melittin, a significant constituent of Apis mellifera (honeybee) venom, is a water-soluble toxic peptide that has traditionally been used as an antitumor agent. However, the underlying mechanisms by which it inhibits tumor cell growth and angiogenesis remain to be elucidated. In the present study, screening for increased cathepsin S (Cat S) expression levels was performed in MHCC97-H cells and various other hepatocellular carcinoma cell lines by reverse transcription-polymerase chain reaction and western blot analysis. A pcDNA3.1-small hairpin RNA (shRNA)-Cat S vector was stably transfected into MHCC97-H cells (shRNA/MHCC97-H) in order to knockdown the expression of Cat S. The effects resulting from the inhibition of Cat S-induced proliferation, invasion and angiogenesis by melittin were examined using cell proliferation, cell viability, flat plate colony formation, migration, wound healing, Transwell migration and ELISA assays. In order to substantiate the evidence for melittin-mediated inhibition of Cat S-induced angiogenesis, Cat S RNA was transfected into primary human umbilical vein endothelial cells (Cat S-HUVECs) to induce overexpression of the Cat S gene. The effects of melittin on HUVECs were examined using Transwell migration and tube formation assays. The findings demonstrated that melittin was able to significantly suppress MHCC97-H cell (Mock/MHCC97-H) proliferation, invasion and angiogenesis, as well as capillary tube formation of Cat S-HUVECs, in a dose-dependent manner. However, proliferation, invasion and angiogenesis in shRNA/MHCC97-H and in native HUVECs (Mock-HUVECs) were unaffected. In addition, melittin specifically decreased the expression of phosphorylated (activated) Cat S, and components of the vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR-2)/mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK)1/2 signaling pathway in Mock/MHCC97-H cells. In conclusion, the inhibition of tumor cell growth and anti-angiogenic activity exerted by melittin may be associated with anti-Cat S actions, via the inhibition of VEGF-A/VEGFR-2/MEK1/ERK1/2 signaling. PMID:26870255

Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.

PubMed

Mason, R Preston; Dawoud, Hazem; Jacob, Robert F; Sherratt, Samuel C R; Malinski, Tadeusz

2018-07-01

The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO - ) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia. Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partiallymore » abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.« less

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

PubMed

Wang, Xiangmin; Pan, Bin; Honda, Goichi; Wang, Xintao; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-06-14

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Here, we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from GPR15 knock-out mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal ECs numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. Use of TME5C may be a promising treatment strategy to prevent or treat lethal complications such as sinusoidal obstruction syndrome whose pathogenesis is based on endothelial insults. Copyright © 2018, Ferrata Storti Foundation.

Anti-inflammatory evaluation of the methanolic extract of Taraxacum officinale in LPS-stimulated human umbilical vein endothelial cells.

PubMed

Jeon, Daun; Kim, Seok Joong; Kim, Hong Seok

2017-11-29

Atherosclerosis is a chronic vascular inflammatory disease. Since even low-level endotoxemia constitutes a powerful and independent risk factor for the development of atherosclerosis, it is important to find therapies directed against the vascular effects of endotoxin to prevent atherosclerosis. Taraxacum officinale (TO) is used for medicinal purposes because of its choleretic, diuretic, antioxidative, anti-inflammatory, and anti-carcinogenic properties, but its anti-inflammatory effect on endothelial cells has not been established. We evaluated the anti-inflammatory activity of TO filtered methanol extracts in LPS-stimulated human umbilical vein endothelial cells (HUVECs) by monocyte adhesion and western blot assays. HUVECs were pretreated with 100 μg/ml TO for 1 h and then incubated with 1 μg/ml LPS for 24 h. The mRNA and protein expression levels of the targets (pro-inflammatory cytokines and adhesion molecules) were analyzed by real-time PCR and western blot assays. We also preformed HPLC analysis to identify the components of the TO methanol extract. The TO filtered methanol extracts dramatically inhibited LPS-induced endothelial cell-monocyte interactions by reducing vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pro-inflammatory cytokine expression. TO suppressed the LPS-induced nuclear translocation of NF-κB, whereas it did not affect MAPK activation. Our findings demonstrated that methanol extracts of TO could attenuate LPS-induced endothelial cell activation by inhibiting the NF-κB pathway. These results indicate the potential clinical benefits and applications of TO for the prevention of vascular inflammation and atherosclerosis.

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

PubMed Central

Colman, R W; Pixley, R A; Najamunnisa, S; Yan, W; Wang, J; Mazar, A; McCrae, K R

1997-01-01

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties. PMID:9294114

Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores

PubMed Central

Sobolewski, Peter; Kandel, Judith; Klinger, Alexandra L.

2011-01-01

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50–150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway. PMID:21633077

Mycophenolate mofetil decreases endothelial prostaglandin E2 in response to allogeneic T cells or cytokines.

PubMed

Blaheta, R A; Nelson, K; Oppermann, E; Leckel, K; Harder, S; Cinatl, J; Weber, S; Shipkova, M; Encke, A; Markus, B H

2000-05-15

Prostaglandin E2 (PGE2) is a powerful endogenous immune suppressant and interferes with various T-cell functions. However, it is not known in detail whether immunosuppressive drugs influence the PGE2-driven immune response in transplant patients. Therefore, we investigated the effect of several immunosuppressive compounds, in particular the novel drug mycophenolate mofetil (MMF), on endothelial PGE2 release. Endothelial cells (HUVEC) were activated by either allogeneic CD4+ or CD8+ T cells, or by the cytokines interleukin-1 or gamma-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HWEC in the presence of MMF, cyclosporine, or tacrolimus. As verapamil and mibefradil also possess immunosuppressive properties, they were included in the study as well. Activation of HUVEC with interleukin-1 or T cells resulted in a drastic accumulation of PGE2 in the supernatant. Cyclosporine or tacrolimus had no effect on PGE2 release. However, Ca2+ channel blockers, when applied at higher dosages, caused a significant increase in PGE2. Interestingly, MMF strongly diminished the PGE2 level in the cell culture supernatant in a concentration-dependent manner. The results demonstrate an inhibitory effect of MMF on PGE2 production, which may lower the benefits of the PGE2-triggered immune response after organ transplantation.

Impaired Expression of Uncoupling Protein 2 (UCP2) Causes Defective Post-ischemic Angiogenesis in Mice Deficient in AMP-activated Protein Kinase α Subunits

PubMed Central

Xu, Ming-Jiang; Song, Ping; Shirwany, Najeeb; Liang, Bin; Xing, Junjie; Viollet, Benoit; Wang, Xian; Zhu, Yi; Zou, Ming-Hui

2011-01-01

Objective The aim of the present study was to determine whether mitochondrial uncoupling protein (UCP)-2 is required for AMPK-dependent angiogenesis in ischemia in vivo. Methods and Results Angiogenesis was assayed by monitoring endothelial tube formation (a surrogate for angiogenesis) in human umbilical vein endothelial cells (HUVECs), isolated mouse aortic endothelial cells (MAECs), and pulmomary microvascular endothelial cells (PMECs), or in ischemic thigh adductor muscles from wild-type (WT) mice or mice deficient in either AMPKα1 or AMPKα2. AMPK inhibition with pharmacological inhibitor (compound C) or genetic means (transfection of AMPKα-specific siRNA) significantly lowered the tube formation in HUVECs. Consistently, compared with WT mice, tube formation in MAECs isolated from either AMPKα1−/− or AMPKα2−/− mice, which exhibited oxidative stress and reduced expression of UCP2, were significantly impaired. In addition, adenoviral overexpression of UCP2, but not adenoviruses encoding green florescence protein (GFP), normalized tube formation in MAECs from either AMPKα1−/− or AMPKα2−/− mice. Similarly, supplementation with sodium nitroprusside (SNP), a nitric oxide (NO) donor, restored tube formation. Furthermore, ischemia significantly increased angiogenesis, serine 1177 phosphorylation of endothelial NO synthase (eNOS), and UCP2 in ischemic thigh adductor muscles from WT mice, but not from either AMPKα1−/− or AMPKα2−/− mice. Conclusion We conclude that AMPK-dependent UCP2 expression in endothelial cells promotes angiogenesis in vivo. PMID:21597006

Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

PubMed

Hu, X Y; Fang, Q; Ma, D; Jiang, L; Yang, Y; Sun, J; Yang, C; Wang, J S

2016-06-10

Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

Dimethyl sulfoxide attenuates nitric oxide generation via modulation of cationic amino acid transporter-1 in human umbilical vein endothelial cells.

PubMed

Bentur, Ohad S; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Schwartz, Idit F

2016-10-01

Dimethyl sulfoxide (DMSO) is a solvent that is commonly used in medicine. Conflicting data exist as to its effects on endothelial function. Endothelial cell dysfunction (ECD) is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1), the specific arginine transporter for eNOS, has been shown to modulate eNOS activity. We hypothesize that DMSO inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. We studied the effect of DMSO on arginine transport, NO2/NO3 generation as an index of NO production, as well as CAT-1 and Protein Kinase C alpha (PKC-α) (CAT-1 inhibitor) protein expression in human umbilical vein endothelial cell cultures (HUVECs). DMSO 2.5% and 3.5% (v/v) significantly attenuated arginine transport, a phenomenon which was prevented by co-incubation with l-arginine (1 mM). The aforementioned findings were accompanied by a decrease in NO2/NO3 generation. DMSO significantly increased the abundance of phosphorylated CAT-1 (the inactive form) and phosphorylated PKC-α protein, an effect that was attenuated by l-arginine. GO 6976 (PKC-α antagonist) prevented the decrease in arginine transport caused by DMSO. DMSO also induced profound transient morphological changes in HUVECs' structure but these were not related to its effect on arginine transport. In conclusion, DMSO inhibits NO generation by endothelial cells through modulation of CAT-1 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of Citral on Lipopolysaccharide-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

PubMed

Song, Yan; Zhao, Hongfeng; Liu, Jinyang; Fang, Chao; Miao, Renying

2016-04-01

Citral is an active compound of lemongrass oil which has been reported to have anti-inflammatory effects. In this study, we investigated the effects of citral on lipopolysaccharide (LPS)-induced inflammatory response in a rat model of peritonitis and human umbilical vein endothelial cells (HUVECs). LPS was intraperitoneally injected into rats to establish a peritonitis model. The HUVECs were treated with citral for 12 h before exposure to LPS. The levels of TNF-α and IL-8 were measured using ELISA. Western blotting was used to detect the expression of VCAM-1, ICAM-1, NF-κB, and PPAR-γ. The results showed that citral had a protective effect against LPS-induced peritonitis. Citral decreased the levels of WBCs and inflammatory cytokines TNF-α and IL-6. Citral also inhibited LPS-induced myeloperoxidase (MPO) activity in the peritoneal tissue. Treatment of HUVECs with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. LPS-induced VCAM-1 and ICAM-1 expression were also suppressed by citral. Meanwhile, we found that citral inhibited LPS-induced NF-κB activation in HUVECs. Furthermore, we found that citral activated PPAR-γ and the anti-inflammatory effects of citral can be reversed by PPAR-γ antagonist GW9662. In conclusion, citral inhibits LPS-induced inflammatory response via activating PPAR-γ which attenuates NF-κB activation and inflammatory mediator production.

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

PubMed

Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

2013-08-20

Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Iron oxide nanoparticles surface coating and cell uptake affect biocompatibility and inflammatory responses of endothelial cells and macrophages

NASA Astrophysics Data System (ADS)

Orlando, Antonina; Colombo, Miriam; Prosperi, Davide; Gregori, Maria; Panariti, Alice; Rivolta, Ilaria; Masserini, Massimo; Cazzaniga, Emanuela

2015-09-01

Engineered iron oxide nanoparticles (IONP) offer the possibility of a wide range of medical uses, from clinical imaging to magnetically based hyperthermia for tumor treatment. These applications require their systemic administration in vivo. An important property of nanoparticles is their stability in biological media. For this purpose, a multicomponent nanoconstruct combining high colloidal stability and improved physical properties was synthesized and characterized. IONP were coated with an amphiphilic polymer (PMA), which confers colloidal stability, and were pegylated in order to obtain the nanoconstruct PEG-IONP-PMA. The aim of this study was to utilize cultured human endothelial cells (HUVEC) and murine macrophages, taken as model of cells exposed to NP after systemic administration, to assess the biocompatibility of PEG-IONP-PMA (23.1 ± 1.4 nm) or IONP-PMA (15.6 ± 3.4 nm). PEG-IONP-PMA, tested at different concentrations as high as 20 μg mL-1, exhibited no cytotoxicity or inflammatory responses. By contrast, IONP-PMA showed a concentration-dependent increase of cytotoxicity and of TNF-α production by macrophages and NO production by HUVECs. Cell uptake analysis suggested that after PEGylation, IONP were less internalized either by macrophages or by HUVEC. These results suggest that the choice of the polymer and the chemistry of surface functionalization are a crucial feature to confer to IONP biocompatibility.

Angiogenic and wound healing potency of fermented virgin coconut oil: in vitro and in vivo studies

PubMed Central

Ibrahim, Ahmad H; Li, Haibo; Al-Rawi, Sawsan S; Majid, Aman Shah Abdul; Al-Habib, Omar AM; Xia, Xiaobo; Majid, Amin MS Abdul; Ji, Dan

2017-01-01

Objective: The process of wound healing involves activation of keratinocytes, fibroblasts, endothelial cells, etc. Angiogenesis is crucial during the process of wound healing. Virgin coconut oil is widely utilized in South Asia for various purposes including food, medicinal and industrial applications. This study aimed to evaluate the potency of fermented virgin coconut oil (FVCO) in angiogenesis and wound healing via both in vitro and in vivo assays. Methods: Human umbilical vein endothelial (HUVEC), fibroblast (CCD-18) and retinal ganglion (RGC-5) cells were cultured in medium containing different concentrations of FVCO. The proliferation, migration and morphological changes of cells were determined. The angiogenic effect of FVCO was evaluated by rat aortic assay. The therapeutic effect of FVCO on wound healing was further assessed in a wound excision model in Sprague Dawley rats. The expression of phospho-VEGFR2 (vascular endothelial growth factor receptor 2) in HUVECs was detected by Western blot. Results: FVCO (6 and 12 µg/mL) significantly improved the proliferation of HUVEC, CCD-18 and RGC-5 cells (P < 0.05 or 0.01). FVCO (25 µg/mL) markedly increased the migration ability of CCD-18 and RGC-5 cells (P < 0.05). FVCO did not affect cell morphology as indicated by fluorescein diacetate (FDA), rhodamine 123 and Hoechst staining. FVCO (25, 50 and 100 µg/mL) significantly stimulated the ex vivo blood vessel formation as compared with negative control (P < 0.05). Rats in FVCO group had significantly smaller wound size, higher wound healing percentage, and shorter wound closure time when compared with control group since day 8 (P < 0.05), suggesting that oral FVCO administration notably promoted the wound healing process. FVCO treatment (6 and 12 µg/mL) significantly enhanced the phospho-VEGFR2 expression in HUVECs (P = 0.006 and 0.000, respectively). Conclusion: Our study confirms a high angiogenic and wound healing potency of FVCO that might be mediated by the regulation of VEGF signing pathway. PMID:29218091

MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Martina; Baker, Meredith B.; Moore, Jeffrey P.

Mechanical forces associated with blood flow play an important role in regulating vascular signaling and gene expression in endothelial cells (ECs). MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth, proliferation, and apoptosis. miRNAs are known to have an important role in modulating EC biology, but their expression and functions in cells subjected to shear stress conditions are unknown. We sought to determine the miRNA expression profile in human ECs subjected to unidirectional shear stress and define the role of miR-21 in shear stress-induced changes inmore » EC function. TLDA array and qRT-PCR analysis performed on HUVECs exposed to prolonged unidirectional shear stress (USS, 24 h, 15 dynes/cm{sup 2}) identified 13 miRNAs whose expression was significantly upregulated (p < 0.05). The miRNA with the greatest change was miR-21; it was increased 5.2-fold (p = 0.002) in USS-treated versus control cells. Western analysis demonstrated that PTEN, a known target of miR-21, was downregulated in HUVECs exposed to USS or transfected with pre-miR-21. Importantly, HUVECs overexpressing miR-21 had decreased apoptosis and increased eNOS phosphorylation and nitric oxide (NO{sup {center_dot}}) production. These data demonstrate that shear stress forces regulate the expression of miRNAs in ECs, and that miR-21 influences endothelial biology by decreasing apoptosis and activating the NO{sup {center_dot}} pathway. These studies advance our understanding of the mechanisms by which shear stress forces modulate vascular homeostasis.« less

Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through NO and Akt

PubMed Central

Zhang, Ping; Hu, Xinli; Xu, Xin; Chen, Yingjie; Bache, Robert J.

2011-01-01

Objective Dimethylarginine dimethylaminohydrolase 1 (DDAH1) modulates NO production by degrading the endogenous NO synthase (NOS) inhibitors ADMA and L-NMMA. This study examined whether, in addition to degrading ADMA, DDAH1 exerts ADMA independent effects that influence endothelial function. Methods and Results Using selective gene silencing of DDAH1 with small interfering RNA and overexpression of DDAH1 in HUVEC, we found that DDAH1 acts to promote endothelial cell proliferation, migration and tube formation both by Akt phosphorylation as well as through the traditional role of degrading ADMA. Incubation of HUVEC with the NOS inhibitors L-NAME or ADMA, the soluble guanylyl cyclase inhibitor ODQ, or the cGMP analog 8-pCPT-cGMP had no effect on p-AktSer473, indicating that the increase of p-AktSer473 produced by DDAH1 was independent of the NO-cGMP signaling pathway. DDAH1 formed a protein complex with Ras, and DDAH1 overexpression increased Ras activity. The Ras inhibitor manumycin-A or dominant-negative Ras significantly attenuated the DDAH1-induced increase of p-AktSer473. Furthermore, DDAH1 knockout impaired endothelial sprouting from cultured aortic rings, and overexpression of constitutively active Akt or DDAH1 rescued endothelial sprouting in the aortic rings from these mice. Conclusions DDAH1 exerts a unique role in activating Akt that affects endothelial function independent of degrading endogenous NOS inhibitors. PMID:21212404

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

PubMed

Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

2006-05-01

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment.

PubMed

Versari, Silvia; Longinotti, Giulia; Barenghi, Livia; Maier, Jeanette Anne Marie; Bradamante, Silvia

2013-11-01

Exposure to microgravity generates alterations that are similar to those involved in age-related diseases, such as cardiovascular deconditioning, bone loss, muscle atrophy, and immune response impairment. Endothelial dysfunction is the common denominator. To shed light on the underlying mechanism, we participated in the Progress 40P mission with Spaceflight of Human Umbilical Vein Endothelial Cells (HUVECs): an Integrated Experiment (SPHINX), which consisted of 12 in-flight and 12 ground-based control modules and lasted 10 d. Postflight microarray analysis revealed 1023 significantly modulated genes, the majority of which are involved in cell adhesion, oxidative phosphorylation, stress responses, cell cycle, and apoptosis. Thioredoxin-interacting protein was the most up-regulated (33-fold), heat-shock proteins 70 and 90 the most down-regulated (5.6-fold). Ion channels (TPCN1, KCNG2, KCNJ14, KCNG1, KCNT1, TRPM1, CLCN4, CLCA2), mitochondrial oxidative phosphorylation, and focal adhesion were widely affected. Cytokine detection in the culture media indicated significant increased secretion of interleukin-1α and interleukin-1β. Nitric oxide was found not modulated. Our data suggest that in cultured HUVECs, microgravity affects the same molecular machinery responsible for sensing alterations of flow and generates a prooxidative environment that activates inflammatory responses, alters endothelial behavior, and promotes senescence.

Dickkopf1 destabilizes atherosclerotic plaques and promotes plaque formation by inducing apoptosis of endothelial cells through activation of ER stress

PubMed Central

Di, Mingxue; Wang, Lin; Li, Mengmeng; Zhang, Yu; Liu, Xinxin; Zeng, Renya; Wang, Han; Chen, Yifei; Chen, Weijia; Zhang, Yun; Zhang, Mei

2017-01-01

Several clinical studies reported that Dickkopf1 (DKK1) plasma levels are correlated with atherosclerosis. However, the impact of DKK1 on the formation and vulnerability of atherosclerotic plaques remains elusive. This study investigated DKK1’s effects on enlargement and destabilization of plaques by targeting endothelial cells and assessing the possible cellular mechanisms involved. The effects of DKK1 on atherogenesis and plaque stability were evaluated in ApoE−/− mice using lentivirus injections to knockdown and knock-in the DKK1 gene. The presence of DKK1 resulted in enlarged and destabilized atherosclerotic lesions and increased apoptosis, while silencing of DKK1 alleviated plaque formation and vulnerability in the whole progression of atherosclerosis. DKK1 expression was upregulated in response to ox-LDL treatment in a time- and concentration-dependent manner on human umbilical vein endothelial cell (HUVEC). The interference of DKK1 reversed ox-LDL-induced apoptosis in HUVECs. The mechanism underlying this effect was DKK1’s activation of the JNK signal transduction pathway and inhibition of canonical Wnt signaling, following by activation of the IRE1α and eif2α/CHOP pathways. In conclusion, DKK1 promotes plaque formation and vulnerability partly by inducing apoptosis in endothelial cells, which partly through inducing the JNK-endoplasmic reticulum stress pathway and inhibiting canonical Wnt signaling. PMID:28703797

Proteomic study of endothelial dysfunction induced by AGEs and its possible role in diabetic cardiovascular complications.

PubMed

Banarjee, Reema; Sharma, Akshay; Bai, Shakuntala; Deshmukh, Arati; Kulkarni, Mahesh

2018-06-20

Endothelial dysfunction is one of the primary steps in the development of diabetes associated cardiovascular diseases. Hyperglycemic condition in diabetes promotes accumulation of advanced glycation end products (AGEs) in the plasma, that interact with the receptor for AGEs (RAGE) present on the endothelial cells and negatively affect their function. Using Human umbilical vascular endothelial cells (HUVECs) in culture, the effect of glycated human serum albumin on global proteomic changes was studied by SWATH-MS, a label free quantitative proteomic approach. Out of the 1860 proteins identified, 161 showed higher abundance while 123 showed lesser abundance in cells treated with glycated HSA. Bioinformatic analysis revealed that the differentially regulated proteins were involved in various processes such as apoptosis, oxidative stress etc. that are associated with endothelial dysfunction. Furthermore, the iRegulon analysis and immunofuorescence studies indicated that several of the differentially regulated proteins were transcriptionally regulated by NF-κB, that is downstream to AGE-RAGE axis. Some of the important differentially regulated proteins include ICAM1, vWF, PAI-1that affect important endothelial functions like cell adhesion and blood coagulation. qPCR analysis showed an increase in expression of the AGE receptor RAGE along with other genes involved in endothelial function. AGE treatment to HUVEC cells led to increased oxidative stress and apoptosis. This is the first proteomics study that provides insight into proteomic changes downstream to AGE-RAGE axis leading to endothelial dysfunction and predisposing to cardiovascular complications. Cardiovascular disease (CVD) is a major pathological outcome in diabetic patients and it is important to address ways that target its development before the onset. Elevated plasma AGEs in diabetes can affect endothelial function and can continue to show their effects even after blood glucose levels are back to normal. Since endothelial dysfunction acts as one of the initiating factors for the development of CVD, understanding how AGEs affect the endothelial cell proteome to cause dysfunction will provide insight into the mechanisms involved and aid designing new therapeutic approaches. Copyright © 2018. Published by Elsevier B.V.

Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

PubMed

Heyninck, Karen; Sabbe, Linde; Chirumamilla, Chandra Sekhar; Szarc Vel Szic, Katarzyna; Vander Veken, Pieter; Lemmens, Kristien J A; Lahtela-Kakkonen, Maija; Naulaerts, Stefan; Op de Beeck, Ken; Laukens, Kris; Van Camp, Guy; Weseler, Antje R; Bast, Aalt; Haenen, Guido R M M; Haegeman, Guy; Vanden Berghe, Wim

2016-06-01

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition of STAT3 phosphorylation by sulforaphane reduces adhesion molecule expression in vascular endothelial cell.

PubMed

Cho, Young S; Kim, Chan H; Ha, Tae S; Ahn, Hee Y

2015-11-18

Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) play key roles in the initiation of vascular inflammation. In this study, we explored whether sulforaphane, a dietary phytochemical, can inhibit the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the mechanisms involved. Sulforaphane prevented the LPS-mediated increase in ICAM-1 and VCAM-1 expression, (P < 0.01) in HUVEC. Sulforaphane also prevented the LPS-mediated increase in the phosphorylation of signal transducer and activator of transcription 3 (STAT3) (P < 0.01). Stattic, a STAT3 inhibitor, reduced the LPS-induced expression of ICAM-1 and VCAM-1, and STAT3 phosphorylation (P < 0.01). STAT3 small interfering RNA treatment reduced the LPS-induced expression of ICAM-1, VCAM-1, and STAT3 (P < 0.01). Sulforaphane reduced LPS-mediated THP-1 monocyte adhesion to HUVEC (P < 0.01). In C57BL/6 mice, injection of LPS increased aortic ICAM-1 and VCAM-1 expression, and this effect was prevented by sulforaphane. These data provide insight into the mechanism through which sulforaphane partly reduces the expression of ICAM-1 and VCAM-1 on the vascular wall by inhibiting STAT3 phosphorylation.

Raf-1/CK2 and RhoA/ROCK signaling promote TNF-α-mediated endothelial apoptosis via regulating vimentin cytoskeleton.

PubMed

Yang, Lifeng; Tang, Lian; Dai, Fan; Meng, Guoliang; Yin, Runting; Xu, Xiaole; Yao, Wenjuan

2017-08-15

Both RhoA/ROCK and Raf-1/CK2 pathway play essential roles in cell proliferation, apoptosis, differentiation, and multiple other common cellular functions. We previously reported that vimentin is responsible for TNF-α-induced cell apoptosis. Herein, we investigated the regulation of RhoA/ROCK and Raf-1/CK2 signaling on vimentin filaments and endothelial apoptosis mediated by TNF-α. Treatment with TNF-α significantly induced the activation of RhoA and ROCK, and the expression of ROCK1. RhoA deficiency could obviously inhibit ROCK activation and ROCK1 expression induced by TNF-α. Both RhoA deficiency and ROCK activity inhibition (Y-27632) greatly inhibited endothelial apoptosis and preserved cell viability in TNF-α-induced human umbilical vein endothelial cells (HUVECs). Also vimentin phosphorylation and the remodeling of vimentin or phospho-vimentin induced by TNF-α were obviously attenuated by RhoA suppression and ROCK inhibition. TNF-α-mediated vimentin cleavage was significantly inhibited by RhoA suppression and ROCK inhibition through decreasing the activation of caspase3 and 8. Furthermore, TNF-α treatment greatly enhanced the activation of Raf-1. Suppression of Raf-1 or CK2 by its inhibitor (GW5074 or TBB) blocked vimentin phosphorylation, remodeling and endothelial apoptosis, and preserved cell viability in TNF-α-induced HUVECs. However, Raf-1 inhibition showed no significant effect on TNF-α-induced ROCK expression and activation, suggesting that the regulation of Raf-1/CK2 signaling on vimentin was independent of ROCK. Taken together, these results indicate that both RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-α-mediated endothelial cytotoxicity via regulating vimentin cytoskeleton. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of 7-ketocholesterol on the activity of endothelial poly(ADP-ribose) polymerase and on endothelium-dependent relaxant function.

PubMed

Kiss, Levente; Chen, Min; Gero, Domokos; Módis, Katalin; Lacza, Zsombor; Szabó, Csaba

2006-12-01

Oxidative and nitrosative stress play an important role in the development of endothelial vascular dysfunction during early atherosclerosis. Oxidative stress activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in endothelial cells. In patients with atherosclerosis the level of oxidized LDL in the plasma is elevated. In oxidized LDL various oxysterols have been identified, such as 7-ketocholesterol (7K). 7K has been shown to induce PARP activation in microglial cells. The aim of the current study was to clarify the effects of 7K on the activity of endothelial PARP and on the endothelium-dependent relaxant function of blood vessels. We treated human umbilical vein endothelial (HUVEC) cells with 2-16 microg/ml 7K as well as vascular rings harvested from BALB/c mouse thoracic aorta with 90 microg/ml 7K for 2 h. A group of mice was treated with 7K subcutaneously for 1 week (10 mg/kg/day). We also conducted in vitro and in vivo experiments using pretreatment with buthionine sulphoximine (BSO), a glutathione-lowering agent. The activity of PARP was calculated by measurement of tritiated NAD incorporation. The activity of PARP increased significantly in 7K-treated HUVEC cells. After BSO pretreatment, this increase was higher. Isolated vascular rings demonstrated no change in endothelium-dependent relaxant function after 2 h of incubation with 7K, even after BSO pretreatment. In vivo treatment with 7K for 1 week had no effect on the relaxant function. Our experimental results suggest that although 7-ketocholesterol can activate PARP enzyme in endothelial cells, it is not sufficient on its own to cause impairment in the endothelium-dependent vascular reactivity.

Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells.

PubMed

Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Farkas, Simon; Salingova, Barbara; Kutalkova, Katerina; Vavreckova, Eva; Matula, Pavel; Matula, Petr; Veverkova, Lenka; Koutna, Irena

2018-01-01

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

KSHV MicroRNAs Repress Tropomyosin 1 and Increase Anchorage-Independent Growth and Endothelial Tube Formation

PubMed Central

Kieffer-Kwon, Philippe; Happel, Christine; Uldrick, Thomas S.; Ramalingam, Dhivya; Ziegelbauer, Joseph M.

2015-01-01

Kaposi’s sarcoma (KS) is characterized by highly vascularized spindle-cell tumors induced after infection of endothelial cells by Kaposi’s sarcoma-associated herpesvirus (KSHV). In KS tumors, KSHV expresses only a few latent proteins together with 12 pre-microRNAs. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1) were targets of KSHV microRNAs. Here we show that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3’ untranslated region (UTR) of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells (HUVEC) by inhibiting anoikis (apoptosis after cell detachment), enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis. PMID:26263384

Calcitonin gene-related peptide stimulates proliferation of human endothelial cells.

PubMed Central

Haegerstrand, A; Dalsgaard, C J; Jonzon, B; Larsson, O; Nilsson, J

1990-01-01

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing. PMID:2159144

Calcitonin gene-related peptide stimulates proliferation of human endothelial cells.

PubMed

Haegerstrand, A; Dalsgaard, C J; Jonzon, B; Larsson, O; Nilsson, J

1990-05-01

The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. 125I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cyclic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.

Anthocyanin-rich Aronox extract from Aronia melanocarpa E protects against 7beta-hydroxycholesterol-induced apoptosis of endothelial cells.

PubMed

Zapolska-Downar, Danuta; Nowicka, Grazyna; Sygitowicz, Grazyna; Jarosz, Mirosław

2008-01-01

It has been previously reported that dietary polyphenolic compounds have a positive effect in cardiovascular diseases, although the underlying mechanisms remain unknown. In this study, the effects of Aronox, an anthocyanin-rich extract from Aronia melanocarpa E, on human umbilical vein endothelial cell (HUVEC) apoptosis was investigated. Apoptosis was measured in HUVEC pretreated with Aronox and than treated with 7beta-hydroxycholesterol (20 microg/ml). For the evaluation of apoptosis we used the cell binding of annexin V, the propidium iodide uptake method and the TUNEL assay. We also measured the generation of reactive oxygen species (ROS) in the cells, using fluorescent probe 2',7'-dichlorofluorescein acetate. Moreover, alteration of the mitochondrial membrane potential with subsequent cytochrome c release, activation of caspase-3, and the level of anti-apoptotic Bcl-2 were investigated. Aronox significantly decreased 7beta-hydroxycholesterol-induced apoptosis and ROS generation, and ameliorated the collapse of the mitochondrial transmembrane potential with subsequent inhibition of cytochrome c release. Furthermore, down-regulation of Bcl-2 and up-regulation of caspase-3 activation were reversed by Aronox. The protective action of the extract against apoptosis in endothelial cells may contribute to the potential benefits drawn from consumption of plant polyphenols. Copyright 2009 S. Karger AG, Basel.

Characterisation of human induced pluripotent stem cell-derived endothelial cells under shear stress using an easy-to-use microfluidic cell culture system.

PubMed

Ohtani-Kaneko, Rsituko; Sato, Kenjiro; Tsutiya, Atsuhiro; Nakagawa, Yuka; Hashizume, Kazutoshi; Tazawa, Hidekatsu

2017-10-09

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.

Sildenafil Inhibits the Proliferation of Cultured Human Endothelial Cells

PubMed Central

Erdogan, Ali; Luedders, Doerte Wiebke; Muenz, Benedikt Manuel; Schaefer, Christian Alexander; Tillmanns, Harald; Wiecha, Johannes; Kuhlmann, Christoph Ruediger Wolfram

2007-01-01

The proliferation of endothelial cells plays a crucial role in the development of intraplaque angiogenesis (IPA). IPA is a major source of intraplaque hemorrhage and therefore contributes to the destabilization of atherosclerotic plaques. Therefore, the aim of the present study was to examine, whether sildenafil inhibits endothelial cell growth. The proliferation of human endothelial cells derived from umbilical cord veins (HUVEC) was examined on DNA level by measurements of (3H)-thymidine incorporation. Cell viability was analyzed using trypan blue staining. The proliferation of cultured human endothelial cells was significantly decreased by 1 μmol/l (-48.4%) and 10 μmol/l (-89.6%) sildenafil (n=10, p<0.05). This was not a cytotoxic effect, because cell viability was only reduced at sildenafil concentrations of 50 μmol/l or greater. In addition sildenafil significantly reduced endothelial proliferation induced by bFGF (n=10, p<0.05). The presented results demonstrate an antiangiogenic effect of sildenafil that might be useful in the prevention of atherosclerotic plaque vascularization. PMID:23675029

[The effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell in vitro].

PubMed

Pan, Zhiguo; Shao, Yu; Geng, Yan; Chen, Jinghe; Su, Lei

2015-08-01

To study the effect of heat stress on the cytoskeleton and cell cycle of human umbilical vein endothelial cell ( HUVEC ) in vitro. HUVEC was cultured in vitro in 5%CO(2) medium at 37 centigrade ( control group ) or 43 centigrade ( heat stress group ) for 1 hour. Coomassie brilliant blue R-250 staining was used to determine the effect of heat stress on the cytoskeleton. The cells in heat stress group were subsequently cultured at 37 centigradein 5%CO(2) medium after heat stress for 1 hour, and cell cycle of HUVEC was determined at 0, 6, 12, 18 and 24 hours with flow cytometry. Under light microscopy normal cytoskeleton was observed in control group, but thicker and shorter cytoskeleton was found after a rise of temperature, and stress fibers were found in heat stress group. The DNA content of HUVEC at all time points in G0/G1 stage was 38.07%-55.19% after heat stress. The DNA content in control group was 48.57%, and it was 54.06%, 55.19%, 48.23%, 38.07%, and 41.03% at 0, 6, 12, 18, 24 hours in G0/G1 stage in heat stress group. DNA content in S phase was 35.33%-48.18%. The DNA content in control group was 44.62%, and it was 35.33%, 39.50%, 42.50%, 48.18%, and 47.99% at 0, 6, 12, 18, 24 hours in S stage in heat stress group. DNA content in G2/M phase was 5.31%-13.75%. The DNA content in control group was 6.81, and it was 10.61%, 5.31%, 9.27%,13.75%, and 10.98% at 0, 6, 12, 18, 24 hours in G2/M stage in heat stress group. It was demonstrated that compared with control group, the DNA content in G0/G1 stage was significantly increased when the HUVEC were separated from heat stress within 6 hours, and it recovered at a similar level as control group at 12 hours. Heat stress can change the cytoskeleton of HUVEC, and cause stagnation at G0/G1 stage in cell cycle.

G3139, an Anti-Bcl-2 Antisense Oligomer that Binds Heparin-Binding Growth Factors and Collagen I, Alters In Vitro Endothelial Cell Growth and Tubular Morphogenesis

PubMed Central

Stein, C.A.; Wu, SiJian; Voskresenskiy, Anatoliy M.; Zhou, Jin-Feng; Shin, Joongho; Miller, Paul; Souleimanian, Naira; Benimetskaya, Luba

2009-01-01

Purpose We examined the effects of G3139 on the interaction of heparin-binding proteins (e.g., FGF2 and collagen I) with endothelial cells. G3139 is an 18mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global Phase III trial in advanced melanoma (GM301) has evaluted G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood, as it is unlikely that Bcl-2 silencing is the sole mechanism for chemo-sensitization in melanoma cells. Experimental Design The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high affinity cell surface receptors, and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and HUVEC cells were determined. Results G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and from proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in 3D collagen gels, increases the mitogenesis of HUVEC cells similarly, and induces vessel sprouts in the rat aortic ring model. Conclusions G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with LDH observed clinically. PMID:19351753

Mutant DD genotype of NFKB1 gene is associated with the susceptibility and severity of coronary artery disease.

PubMed

Luo, Jun-Yi; Li, Xiao-Mei; Zhou, Yun; Zhao, Qiang; Chen, Bang-Dang; Liu, Fen; Chen, Xiao-Cui; Zheng, Hong; Ma, Yi-Tong; Gao, Xiao-Ming; Yang, Yi-Ning

2017-02-01

Nuclear factor κappa B (NF-κB) is an important transcription factor in the development and progression of coronary artery disease (CAD). Recent evidence suggests that -94 ATTG ins/del mutant in the promoter of NFKB1 gene is an essential functional mutant. The present study demonstrated the frequencies of the del/del (DD) genotype and del (D) allele were significantly higher in CAD patients than in controls. CAD patients carrying mutant DD genotype had worse stenosis of diseased coronary arteries compared to those carrying ins/ins (II) or ins/del (ID) genotype. Plasma levels of endothelial nitric oxide synthase (eNOS) were lower, while inflammatory cytokine incnterlukin-6 (IL-6) was higher in CAD patients with DD genotype than those with II or ID genotype (both P<0.05). In vitro study showed that mutant human umbilical vein endothelial cells (DD genotype HUVECs) were more susceptible to H 2 O 2 -induced apoptosis, which was accompanied with a decreased Bcl-2 expression. Further, mutant HUVECs had lower eNOS but higher IL-6 mRNA levels and decreased phosphorylation of eNOS under H 2 O 2 -stimulation (both P<0.05). Compared to wild type cells (II genotype), significantly downregulated protein expression of total NF-κB p50 subunit were observed in mutant HUVECs with or without oxidative stress, and a lower expression of unclear p50 was associated with a decreased p50 nuclear translocation in mutant HUVECs versus wild type cells under H 2 O 2 -stimulation (both P<0.05). In conclusion, mutant DD genotype of NFKB1 gene is associated with the risk and severity of CAD. Dwonregulation of NF-κB p50 subunit leads to exacerbated endothelial dysfunction and apoptosis and enhanced inflammatory response that is the potential underlying mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

N-Acetylchitooligosaccharide is a potent angiogenic inhibitor both in vivo and in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zheng; Graduate School of the Chinese Academy of Sciences, Beijing 100039; Zheng, Lanhong

2007-05-25

N-Acetylchitooligosaccharide (N-acetyl-COs) was prepared by N-acetylation of chitooligosaccharide (COs). In vitro study using human umbilical vein endothelial cells (HUVECs) revealed that both N-acetyl-COs and COs inhibited the proliferation of HUVECs by inducing apoptosis. Treatment of HUVECs by N-acetyl-COs resulted in a significant reduction of density of the migration cells and repressed tubulogenesis process. The antiangiogenic effects of the oligosaccharides were further evaluated using in vivo zebrafish angiogenesis model, and the results showed that both oligosaccharides inhibited the growth of subintestinal vessels (SIV) of zebrafish embryos in a dose-dependent manner, as observed by endogenous alkaline phosphatase (EAP) staining assay. In contrast,more » no cytotoxicity was found when treating the NIH3T3 and several other cancer cells with the oligosaccharides. Our results also confirmed the antiangiogenic activity of N-acetyl-COs was significantly stronger than the parent oligosaccharide, COs.« less

Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway.

PubMed

Zhao, Yingshuai; Wang, Liuyi; He, Shanshan; Wang, Xiaoyan; Shi, Weili

2017-05-20

Valsartan (VAL), an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO). In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs). Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L) were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L), VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L), and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L) groups. The NO content in the VAL-treated HUVEC line (EA.hy926) was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05) and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

PubMed

Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

2008-09-15

Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

Anthocyanins and their gut metabolites reduce the adhesion of monocyte to TNFα-activated endothelial cells at physiologically relevant concentrations.

PubMed

Krga, Irena; Monfoulet, Laurent-Emmanuel; Konic-Ristic, Aleksandra; Mercier, Sylvie; Glibetic, Maria; Morand, Christine; Milenkovic, Dragan

2016-06-01

An increasing number of evidence suggests a protective role of dietary anthocyanins against cardiovascular diseases. Anthocyanins' extensive metabolism indicates that their metabolites could be responsible for the protective effects associated with consumption of anthocyanin-rich foods. The aim of this work was to investigate the effect of plasma anthocyanins and their metabolites on the adhesion of monocytes to TNFα-activated endothelial cells and on the expression of genes encoding cell adhesion molecules. Human umbilical vein endothelial cells (HUVECs) were exposed to circulating anthocyanins: cyanidin-3-arabinoside, cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-glucoside, anthocyanin degradation product: 4-hydroxybenzaldehyde, or to their gut metabolites: protocatechuic, vanillic, ferulic and hippuric acid, at physiologically-relevant concentrations (0.1-2 μM) and time of exposure. Both anthocyanins and gut metabolites decreased the adhesion of monocytes to HUVECs, with a magnitude ranging from 18.1% to 47%. The mixture of anthocyanins and that of gut metabolites also reduced monocyte adhesion. However, no significant effect on the expression of genes encoding E-selectin, ICAM1 and VCAM1 was observed, suggesting that other molecular targets are involved in the observed effect. In conclusion, this study showed the potency of anthocyanins and their gut metabolites to modulate the adhesion of monocytes to endothelial cells, the initial step in atherosclerosis development, under physiologically-relevant conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

Low-Energy Ultrasound Treatment Improves Regional Tumor Vessel Infarction by Retargeted Tissue Factor.

PubMed

Brand, Caroline; Dencks, Stefanie; Schmitz, Georg; Mühlmeister, Mareike; Stypmann, Jörg; Ross, Rebecca; Hintelmann, Heike; Schliemann, Christoph; Müller-Tidow, Carsten; Mesters, Rolf M; Berdel, Wolfgang E; Schwöppe, Christian

2015-07-01

To enhance the regional antitumor activity of the vascular-targeting agent truncated tissue factor (tTF)-NGR by combining the therapy with low-energy ultrasound (US) treatment. For the in vitro US exposure of human umbilical vein endothelial cells (HUVECs), cells were put in the focus of a US transducer. For analysis of the US-induced phosphatidylserine (PS) surface concentration on HUVECs, flow cytometry was used. To demonstrate the differences in the procoagulatory efficacy of TF-derivative tTF-NGR on binding to HUVECs with a low versus high surface concentration of PS, we performed factor X activation assays. For low-energy US pretreatment, HT1080 fibrosarcoma xenotransplant-bearing nude mice were treated by tumor-regional US-mediated stimulation (ie, destruction) of microbubbles. The therapy cohorts received the tumor vessel-infarcting tTF-NGR protein with or without US pretreatment (5 minutes after US stimulation via intraperitoneal injection on 3 consecutive days). Combination therapy experiments with xenotransplant-bearing nude mice significantly increased the antitumor activity of tTF-NGR by regional low-energy US destruction of vascular microbubbles in tumor vessels shortly before application of tTF-NGR (P < .05). Mechanistic studies proved the upregulation of anionic PS on the outer leaflet of the lipid bilayer of endothelial cell membranes by low-energy US and a consecutive higher potential of these preapoptotic endothelial cells to activate coagulation via tTF-NGR and coagulation factor X as being a basis for this synergistic activity. Combining retargeted tTF to tumor vessels with proapoptotic stimuli for the tumor vascular endothelium increases the antitumor effects of tumor vascular infarction. Ultrasound treatment may thus be useful in this respect for regional tumor therapy. © 2015 by the American Institute of Ultrasound in Medicine.

Activation of Nrf2-Antioxidant Signaling by 1,25-Dihydroxycholecalciferol Prevents Leptin-Induced Oxidative Stress and Inflammation in Human Endothelial Cells.

PubMed

Teixeira, Thaisa M; da Costa, Danielly C; Resende, Angela C; Soulage, Christophe O; Bezerra, Flavia F; Daleprane, Julio B

2017-04-01

Background: Obesity is associated with hyperleptinemia and endothelial dysfunction. Hyperleptinemia has been reported to induce both oxidative stress and inflammation by increasing reactive oxygen species production. Objective: The objective of this study was to determine the effects of 1,25-dihydroxycholecalciferol [1,25(OH) 2 D 3 ] against leptin-induced oxidative stress and inflammation in human endothelial cells. Methods: Small interfering RNA (siRNA) were used to knock down the expression of vitamin D receptor (VDR) in human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated for 4 h with physiologic (10 -10 M) or supraphysiologic (10 -7 M) concentrations of 1,25(OH) 2 D 3 and exposed to leptin (10 ng/mL). Superoxide anion production and translocation of nuclear factor (erythroid-derived 2)-like 2 (NRF2) and nuclear transcription factor κB (NF-κB) subunit p65 to the nucleus and the activation of their target genes were quantified. Results: Pretreatment of HUVECs with 1,25(OH) 2 D 3 prevented the leptin-induced increase in superoxide anion production ( P < 0.05). Pretreatment with 1,25(OH) 2 D 3 further increased NRF2 translocation to the nucleus (by 3-fold; P < 0.05) and increased mRNA expression of superoxide dismutase 2 ( SOD2 ; by 2-fold), glutathione peroxidase ( GPX ; by 3-fold), NAD(P)H dehydrogenase (quinone) 1 ( NQO1 ; by 4-fold), and heme oxygenase 1 ( HMOX1 ; by 2-fold) ( P < 0.05). Leptin doubled the translocation of NF-κB ( P < 0.05) to the nucleus and increased ( P < 0.05) the upregulation of vascular inflammatory mediators such as monocyte chemoattractant protein 1 ( MCP1 ; by 1-fold), transforming growth factor β ( TGF β by 1-fold), and vascular cell adhesion molecule 1 ( VCAM1 ; by 4-fold) ( P < 0.05), which were prevented ( P < 0.05) by pretreatment with 1,25(OH) 2 D 3 Protective effects of 1,25(OH) 2 D 3 were confirmed to be VDR dependent by using VDR siRNA. Conclusion: Pretreatment with 1,25(OH) 2 D 3 in the presence of a high concentration of leptin has a beneficial effect on HUVECs through the regulation of mediators of antioxidant activity and inflammation. © 2017 American Society for Nutrition.

[Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

PubMed

Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

2012-07-01

To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

An anisotropically and heterogeneously aligned patterned electrospun scaffold with tailored mechanical property and improved bioactivity for vascular tissue engineering.

PubMed

Xu, He; Li, Haiyan; Ke, Qinfei; Chang, Jiang

2015-04-29

The development of vascular scaffolds with controlled mechanical properties and stimulatory effects on biological activities of endothelial cells still remains a significant challenge to vascular tissue engineering. In this work, we reported an innovative approach to prepare a new type of vascular scaffolds with anisotropically and heterogeneously aligned patterns using electrospinning technique with unique wire spring templates, and further investigated the structural effects of the patterned electrospun scaffolds on mechanical properties and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs). Results showed that anisotropically aligned patterned nanofibrous structure was obtained by depositing nanofibers on template in a structurally different manner, one part of nanofibers densely deposited on the embossments of wire spring and formed cylindrical-like structures in the transverse direction, while others loosely suspended and aligned along the longitudinal direction, forming a three-dimensional porous microstructure. We further found that such structures could efficiently control the mechanical properties of electrospun vascular scaffolds in both longitudinal and transverse directions by altering the interval distances between the embossments of patterned scaffolds. When HUVECs were cultured on scaffolds with different microstructures, the patterned scaffolds distinctively promoted adhesion of HUVECs at early stage and proliferation during the culture period. Most importantly, cells experienced a large shape change associated with cell cytoskeleton and nuclei remodeling, leading to a stimulatory effect on angiogenesis differentiation of HUVECs by the patterned microstructures of electrospun scaffolds, and the scaffolds with larger distances of intervals showed a higher stimulatory effect. These results suggest that electrospun scaffolds with the anisotropically and heterogeneously aligned patterns, which could efficiently control the mechanical properties and bioactivities of the scaffolds, might have great potential in vascular tissue engineering application.

Plasmin-clipped beta(2)-glycoprotein-I inhibits endothelial cell growth by down-regulating cyclin A, B and D1 and up-regulating p21 and p27.

PubMed

Beecken, Wolf-Dietrich C; Ringel, Eva Maria; Babica, Jan; Oppermann, Elsie; Jonas, Dietger; Blaheta, Roman A

2010-10-28

beta(2)-Glycoprotein-I (beta(2)gpI), an abundant plasma glycoprotein, functions as a regulator of thrombosis. Previously, we demonstrated that plasmin-clipped beta(2)gpI (cbeta(2)gpI) exerts an anti-angiogenic effect on human umbilical vein endothelial cells (HUVEC). The present study was focused on the molecular background responsible for this phenomenon. cbeta(2)gpI strongly reduced HUVEC growth and proliferation as evidenced by the MTT and BrdU assay and delayed cell cycle progression arresting HUVEC in the S-and G2/M-phase. Western blot analysis indicated that cbeta(2)gpI inhibited cyclin A, B and D1, and enhanced p21 and p27 expression. Activity of p38 was down-regulated independently from the cbeta(2)gpI incubation time. Phosphorylation of ERK1/2 was not changed early (30 and 60 min) but became enhanced later (90 min, 4h). JNK activity was reduced rapidly after cbeta(2)gpI treatment but compared to controls, increased thereafter. Annexin II blockade prevented growth inhibition and cell cycle delay evoked by cbeta(2)gpI. We assume that cbeta(2)gpI's effects on HUVEC growth is mediated via cyclin A, B and D1 suppression, up-regulation of p21 and p27 and coupled to modifications of the mitogen-activated protein (MAP) kinase signalling pathway. cbeta(2)gpI may represent a potential endogenous angiogenesis-targeted compound, opening the possibility of a novel tool to treat cancer. 2010 Elsevier Ireland Ltd. All rights reserved.

Angiogenic effect of the aqueous extract of Cynodon dactylon on human umbilical vein endothelial cells and granulation tissue in rat.

PubMed

Soraya, Hamid; Moloudizargari, Milad; Aghajanshakeri, Shahin; Javaherypour, Soheil; Mokarizadeh, Aram; Hamedeyazdan, Sanaz; Esmaeli Gouvarchin Ghaleh, Hadi; Mikaili, Peyman; Garjani, Alireza

2015-01-29

Cynodon dactylon, a valuable medicinal plant, is widely used in Iranian folk medicine for the treatment of various cardiovascular diseases such as heart failure and atherosclerosis. Moreover, its anti-diabetic, anti-cancer and anti-microbial properties have been also reported. Concerning the critical role of angiogenesis in the incidence and progression of tumors and also its protective role in cardiovascular diseases, we investigated the effects of the aqueous extract prepared from the rhizomes of C. dactylon on vascular endothelial growth factor (VEGF) expressions in Human Umbilical Vein Endothelial Cells (HUVECs) and also on angiogenesis in carrageenan induced air-pouch model in rats. In the air-pouch model, carrageenan was injected into an air-pouch on the back of the rats and following an IV injection of carmine red dye on day 6, granulation tissue was processed for the assessment of the dye content. Furthermore, in an in vitro study, angiogenic property of the extract was assessed through its effect on VEGF expression in HUVECs. Oral administration of 400 mg/kg/day of the extract significantly increased angiogenesis (p<0.05) and markedly decreased neutrophil (p<0.05) and total leukocyte infiltration (p<0.001) into the granulation tissues. Moreover, the extract increased the expression of total VEGF in HUVECs at a concentration of (100 μl/ml). The present study showed that the aqueous extract of C. dactylon promotes angiogenesis probably through stimulating VEGF expression.

In vitro induction of protein complexes between bevacizumab, VEGF-A¹⁶⁵ and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

PubMed

Julien, Sylvie; Biesemeier, Antje; Schraermeyer, Ulrich

2013-04-01

By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion.

PubMed

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-02-05

Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. Copyright © 2016 Elsevier Inc. All rights reserved.

Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

PubMed

Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

2016-08-01

Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

PubMed

Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

2008-11-01

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (P<0.05). The MTT test showed that the proliferation rate of HUVEC transfected with HPVA under hypoxia exceeded that of the control group. We conclude that the HRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer studies targeting endothelial cells.

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation.

PubMed

Bou Khzam, Lara; Bouchereau, Olivier; Boulahya, Rahma; Hachem, Ahmed; Zaid, Younes; Abou-Saleh, Haissam; Merhi, Yahye

2015-11-09

Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation. EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents' nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed. We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)-and inducible (iNOS)-NOS isoforms, and COX-1 and weakly express COX-2, whereas EOCs do not constitutively express these NO and PGI2 producing enzymes. The different morphological, phenotypic and more importantly the release of the anti-aggregating agents PGI2 and NO in each EPC subtype are implicated in their respective roles in platelet function and thus, may be linked to the increased efficiency of ECFCs in inhibiting platelet aggregation as compared to EOCs.

Palmitate-induced endothelial dysfunction is attenuated by cyanidin-3-O-glucoside through modulation of Nrf2/Bach1 and NF-κB pathways.

PubMed

Fratantonio, D; Speciale, A; Ferrari, D; Cristani, M; Saija, A; Cimino, F

2015-12-15

Free fatty acids (FFA), commonly elevated in diabetes and obesity, have been shown to impair endothelial functions and cause oxidative stress, inflammation, and insulin resistance. Anthocyanins represent one of the most important and interesting classes of flavonoids and seem to play a role in preventing cardiovascular diseases. Herein, we investigated the in vitro protective effects of cyanidin-3-O-glucoside (C3G) on cell signaling pathways in human umbilical vein endothelial cells (HUVECs) exposed to palmitic acid (PA), the most prevalent saturated FFA in circulation. Our data reported a significant augmentation of free radicals and oxidative stress in HUVECs exposed to PA for 3h, while C3G pretreatment improved intracellular redox status altered by FFA. Moreover, C3G significantly inhibited NF-κB proinflammatory pathway and adhesion molecules induced by PA, and these effects were attributed to the activation of Nrf2/EpRE pathway. In fact, C3G induced Nrf2 nuclear localization and activation of cellular antioxidant and cytoprotective genes at baseline and after PA exposure in endothelial cells. Our data confirm the hypothesis that natural Nrf2 inducers, such as C3G, might be a potential therapeutic strategy to protect vascular system against various stressors preventing several pathological conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

CCL11 promotes angiogenic activity by activating the PI3K/Akt pathway in HUVECs.

PubMed

Park, Jun Young; Kang, Yeo Wool; Choi, Byung Young; Yang, Young Chul; Cho, Byung Pil; Cho, Won Gil

2017-08-01

CCR3, the receptor for CCL11, is expressed on the surface of immune cells and even on non-immune cells. CCL11-CCR3 interactions can promote cell migration and proliferation. In this study, we investigated the effect of CCL11 on angiogenesis in HUVECs and also examined the molecular mechanisms of this process. We found that CCL11 induced mRNA transcription and protein expression of CCR3 in HUVECs. Moreover, the scratch wound healing assay and MTS proliferation assay both demonstrated that CCL11 promotes endothelial cell migration and induces weak proliferation. CCL11 directly induced microvessel sprouting from the rat aortic ring; these effects occurred earlier and to a greater extent than with VEGF stimulation. Furthermore, CCL11-induced phosphorylation of Akt was abolished by PI3K inhibitors. siRNA-mediated knockdown of CCR3 led to a significant reduction of PI3K phosphorylation. However, the phosphorylation levels of ERK1/2 were not changed, even after CCL11 treatment. Cumulatively, our data suggest that the CCL11-CCR3 interaction mainly activates PI3K/Akt signal transduction pathway in HUVECs.

Diffusion of rhodamine B and bovine serum albumin in fibrin gels seeded with primary endothelial cells.

PubMed

Shkilnyy, Andriy; Proulx, Pierre; Sharp, Jamie; Lepage, Martin; Vermette, Patrick

2012-05-01

Scaffolds with adequate mass transport properties are needed in many tissue engineering applications. Fibrin is considered a good biological material to fabricate such scaffolds. However, very little is known about mass transport in fibrin. Therefore, a method based on the analysis of fluorescence intensity for measuring the apparent diffusion coefficient of rhodamine B and fluorescein-labelled bovine serum albumin (FITC-BSA) is described. The experiments are performed in fibrin gels with and without human umbilical vein endothelial cells (HUVEC). The apparent diffusion coefficients of rhodamine B and FITC-BSA in fibrin (fibrinogen concentration of 4 mg/mL) with different cell densities are reported. A LIVE/DEAD(®) assay is performed to confirm the viability of HUVEC seeded at high densities. Diffusion coefficients for rhodamine B remain more or less constant up to 5×10(5) cells/mL and correlate well with literature values measured by other methods in water systems. This indicates that the presence of HUVEC in the fibrin gels (up to 5×10(5) cells/mL) has almost no effect on the diffusion coefficients. Higher cell densities (>5×10(5) cells/mL) result in a decrease of the diffusion coefficients. Diffusion coefficients of rhodamine B and FITC-BSA obtained by this method agree with diffusion coefficients in water predicted by the Stokes-Einstein equation. The experimental design used in this study can be applied to measure diffusion coefficients in different types of gels seeded or not with living cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Actin cytoskeletal rearrangement and dysfunction due to activation of the receptor for advanced glycation end products is inhibited by thymosin beta 4

PubMed Central

Kim, Sokho; Kwon, Jungkee

2015-01-01

The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tβ4) – which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) – in the response to RAGE. Tβ4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tβ4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tβ4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tβ4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tβ4 plays a pivotal role in the RAGE response to AGEs. PMID:25640761

Hyperglycemia-induced PATZ1 negatively modulates endothelial vasculogenesis via repression of FABP4 signaling.

PubMed

Chen, Ren-An; Sun, Xiao-Mian; Yan, Chang-You; Liu, Li; Hao, Miao-Wang; Liu, Qiang; Jiao, Xi-Ying; Liang, Ying-Min

2016-09-02

Vascular endothelial dysfunction, a central hallmark of diabetes, predisposes diabetic patients to numerous cardiovascular complications. The POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1), is an important transcriptional regulatory factor and regulates divergent pathways depending on the cellular context, but its role in endothelial cells remains poorly understood. Herein, we report for the first time that endothelial PATZ1 expression was abnormally upregulated in diabetic endothelial cells (ECs) regardless of diabetes classification. This stimulatory effect was further confirmed in the high glucose-treated human umbilical vein endothelial cells (HUVECs). From a functional standpoint, transgenic overexpression of PATZ1 in endothelial colony forming cells (ECFCs) blunted angiogenesis in vivo and rendered endothelial cells unresponsive to established angiogenic factors. Mechanistically, PATZ1 acted as a potent transcriptional corepressor of fatty acid-binding protein 4 (FABP4), an essential convergence point for angiogenic and metabolic signaling pathways in ECs. Taken together, endothelial PATZ1 thus potently inhibits endothelial function and angiogenesis via inhibition of FABP4 expression, and abnormal induction of endothelial PATZ1 may contribute to multiple aspects of vascular dysfunction in diabetes. Copyright © 2016. Published by Elsevier Inc.

The effect of two novel amino acid-coated magnetic nanoparticles on survival in vascular endothelial cells, bone marrow stromal cells, and macrophages

NASA Astrophysics Data System (ADS)

Wu, Qinghua; Meng, Ning; Zhang, Yanru; Han, Lei; Su, Le; Zhao, Jing; Zhang, Shangli; Zhang, Yun; Zhao, Baoxiang; Miao, Junying

2014-09-01

Magnetic nanoparticles (MNPs) have been popularly used in many fields. Recently, many kinds of MNPs are modified as new absorbents, which have attracted considerable attention and are promising to be applied in waste water. In our previous study, we synthesized two novel MNPs surface-coated with glycine or lysine, which could efficiently remove many anionic and cationic dyes under severe conditions. It should be considered that MNP residues in water may exert some side effects on human health. In the present study, we evaluated the potential nanotoxicity of MNPs in human endothelial cells, macrophages, and rat bone marrow stromal cells. The results showed that the two kinds of nanoparticles were consistently absorbed into the cell cytoplasm. The concentration of MNPs@Gly that could distinctly decrease survival was 15 μg/ml in human umbilical vascular endothelial cells (HUVECs) or bone marrow stromal cells (BMSCs) and 10 μg/ml in macrophages. While the concentration of MNPs@Lys that obviously reduced viability was 15 μg/ml in HUVECs or macrophages and 50 μg/ml in BMSCs. Furthermore, cell nucleus staining and cell integrity assay indicated that the nanoparticles induced cell apoptosis, but not necrosis even at a high concentration. Altogether, these data suggest that the amino acid-coated magnetic nanoparticles exert relatively high cytotoxicity. By contrast, lysine-coated magnetic nanoparticles are more secure than glycine-coated magnetic nanoparticles.

High glucose derived endothelial microparticles increase active caspase-3 and reduce microRNA-Let-7a expression in endothelial cells.

PubMed

Bammert, Tyler D; Hijmans, Jamie G; Reiakvam, Whitney R; Levy, Ma'ayan V; Brewster, Lillian M; Goldthwaite, Zoe A; Greiner, Jared J; Stockelman, Kelly A; DeSouza, Christopher A

2017-11-18

The experimental aim of this study was to determine the effects of high glucose-induced endothelial microparticles (EMPs) on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 10 5  cells/condition. Cells were incubated with media containing 25 mM d-glucose (concentration representing a diabetic glycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144 + expression) and concentration was determined by flow cytometry. HUVECs (3 × 10 6  cells/condition) were treated with EMPs generated from either the normal or high glucose conditions for 24 h. Intracellular concentration of active caspase-3 was determined by enzyme immunoassay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. High glucose-derived EMPs significantly increased both basal (1.5 ± 0.1 vs 1.0 ± 0.1 ng/mL) and staurosporine-stimulated (2.2 ± 0.2 vs 1.4 ± 0.1 ng/mL) active caspase-3 compared with normal glucose EMPs. Additionally, the expression of miR-Let-7a was markedly reduced (∼140%) by high glucose EMPs (0.43 ± 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell active caspase-3. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let-7a expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo

PubMed Central

Xiao, Qi; Zhou, Yachun; Wei, Yingjie; Gong, Zhunan

2018-01-01

Angiogenesis plays a vital role in many physiological and pathological processes and several diseases are connected with its dysregulation. Asiatic acid (AA) has demonstrated anticancer properties and we suspect this might be attributable to an effect on angio-genesis. A modified derivative of AA, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester (AA-PMe), has improved efficacy over its parent compound, but its effect on blood vessel development remains unclear. Methods In this study, we investigated the antiangiogenic activity of AA and AA-PMe in zebrafish embryos and human umbilical vein endothelial cells (HUVECs). First of all, we treated HUVECs with increasing concentrations of AA-PMe or AA, with or without vascular endothelial growth factor (VEGF) present, and assessed cell viability, tube formation, and cell migration and invasion. Quantitative real-time polymerase chain reaction and Western blot analysis were later used to determine the role of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling in AA-PMe inhibition of angiogenesis. We extended these studies to follow angiogenesis using Tg(fli:EGFP) transgenic zebrafish embryos. For these experiments, embryos were treated with varying concentrations of AA-PMe or AA from 24 to 72 hours postfertilization prior to morphological observation, angiogenesis assessment, and endogenous alkaline phosphatase assay. VEGFR2 expression in whole embryos following AA-PMe treatment was also determined. Results We found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling. Conclusion For the first time, this study reveals that AA-PMe acts as a potent VEGFR2 kinase inhibitor and exerts powerful antiangiogenic activity, suggesting it to be a promising therapeutic candidate for further research. PMID:29670362

Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo.

PubMed

Jing, Yue; Wang, Gang; Xiao, Qi; Zhou, Yachun; Wei, Yingjie; Gong, Zhunan

2018-01-01

Angiogenesis plays a vital role in many physiological and pathological processes and several diseases are connected with its dysregulation. Asiatic acid (AA) has demonstrated anticancer properties and we suspect this might be attributable to an effect on angio-genesis. A modified derivative of AA, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester (AA-PMe), has improved efficacy over its parent compound, but its effect on blood vessel development remains unclear. In this study, we investigated the antiangiogenic activity of AA and AA-PMe in zebrafish embryos and human umbilical vein endothelial cells (HUVECs). First of all, we treated HUVECs with increasing concentrations of AA-PMe or AA, with or without vascular endothelial growth factor (VEGF) present, and assessed cell viability, tube formation, and cell migration and invasion. Quantitative real-time polymerase chain reaction and Western blot analysis were later used to determine the role of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling in AA-PMe inhibition of angiogenesis. We extended these studies to follow angiogenesis using Tg(fli:EGFP) transgenic zebrafish embryos. For these experiments, embryos were treated with varying concentrations of AA-PMe or AA from 24 to 72 hours postfertilization prior to morphological observation, angiogenesis assessment, and endogenous alkaline phosphatase assay. VEGFR2 expression in whole embryos following AA-PMe treatment was also determined. We found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling. For the first time, this study reveals that AA-PMe acts as a potent VEGFR2 kinase inhibitor and exerts powerful antiangiogenic activity, suggesting it to be a promising therapeutic candidate for further research.

Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samarzija, Ivana; Sini, Patrizia; Schlange, Thomas

2009-08-28

Untangling the signaling pathways involved in endothelial cell biology is of central interest for the development of antiangiogenesis based therapies. Here we report that Wnt3a induces the proliferation and migration of HUVECs, but does not affect their survival. Wnt3a-induced proliferation was VEGFR signaling independent, but reduced upon CamKII inhibition. In a search for the downstream mediators of Wnt3a's effects on HUVEC biology, we found that Wnt3a treatment leads to phosphorylation of DVL3 and stabilization of {beta}-catenin. Moreover, under the same conditions we observed an upregulation in c-MYC, TIE-2 and GJA1 mRNA transcripts. Although treatment of HUVECs with Wnt5a induced DVL3more » phosphorylation, we did not observe any of the other effects seen upon Wnt3a stimulation. Taken together, our data indicate that Wnt3a induces canonical and non-canonical Wnt signaling in HUVECs, and stimulates their proliferation and migration.« less

Inhibitory effects of myricitrin on oxidative stress-induced endothelial damage and early atherosclerosis in ApoE −/− mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Gui-bo; Qin, Meng; Ye, Jing-xue

Atherosclerosis (AS) is a state of heightened oxidative stress characterized by lipid and protein oxidation in vascular walls. Oxidative stress-induced vascular endothelial cell (VEC) injury is a major factor in the pathogenesis of AS. Myricitrin, a natural flavonoid isolated from the root bark of Myrica cerifera, was recently found to have a strong antioxidative effect. However, its use for treating cardiovascular diseases, especially AS is still unreported. Consequently, we evaluated the cytoprotective effect of myricitrin on AS by assessing oxidative stress-induced VEC damage. The in vivo study using an ApoE −/− mouse model of AS demonstrated that myricitrin treatment protectsmore » against VEC damage and inhibits early AS plaque formation. This effect is associated with the antioxidative effect of myricitrin, as observed in a hydrogen peroxide (H{sub 2}O{sub 2})-induced rat model of artery endothelial injury and primary cultured human VECs. Myricitrin treatment also prevents and attenuates H{sub 2}O{sub 2}-induced endothelial injury. Further investigation of the cytoprotective effects of myricitrin demonstrated that myricitrin exerts its function by scavenging for reactive oxygen species, as well as reducing lipid peroxidation, blocking NO release, and maintaining mitochondrial transmembrane potential. Myricitrin treatment also significantly decreased H{sub 2}O{sub 2}-induced apoptosis in VECs, which was associated with significant inhibition of p53 gene expression, activation of caspase-3 and the MAPK signaling pathway, and alteration of the patterns of pro-apoptotic and anti-apoptotic gene expression. The resulting significantly increased bcl-2/bax ratio indicates that myricitrin may prevent the apoptosis induced by oxidative stress injury. - Highlights: • Myricitrin prevents early atherosclerosis in ApoE−/− mice. • Myricitrin protects endothelial cell from H{sub 2}O{sub 2} induced injury in rat and HUVECs. • Myricitrin enhanced NO release and up regulates eNOS activity in HUVECs. • Myricitrin down regulates P53 expression and MAPKs phosphorylation in HUVECs.« less

Cytocompatibility and early inflammatory response of human endothelial cells in direct culture with Mg-Zn-Sr alloys

PubMed Central

Cipriano, Aaron F.; Sallee, Amy; Tayoba, Myla; Cortez Alcaraz, Mayra C.; Lin, Alan; Guan, Ren-Guo; Zhao, Zhan-Yong; Liu, Huinan

2018-01-01

Crystalline Mg-Zinc (Zn)-Strontium (Sr) ternary alloys consist of elements naturally present in the human body and provide attractive mechanical and biodegradable properties for a variety of biomedical applications. The first objective of this study was to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt%; designated as ZSr41A, B, C, and D respectively) in the direct culture with human umbilical vein endothelial cells (HUVEC) in vitro. The second objective was to investigate, for the first time, the early-stage inflammatory response in cultured HUVECs as indicated by the induction of vascular cellular adhesion molecule-1 (VCAM-1). The results showed that the 24-h in vitro degradation of the ZSr41 alloys containing a β-phase with a Zn/Sr at% ratio ~1.5 was significantly faster than the ZSr41 alloys with Zn/Sr at% ~1. Additionally, the adhesion density of HUVECs in the direct culture but not in direct contact with the ZSr41 alloys for up to 24 h was not adversely affected by the degradation of the alloys. Importantly, neither culture media supplemented with up to 27.6 mM Mg2+ ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on HUVEC responses. In contrast, the significantly higher, yet non-cytotoxic, Zn2+ ion concentration from the degradation of ZSr41D alloy was likely the cause for the initially higher VCAM-1 expression on cultured HUVECs. Lastly, analysis of the HUVEC-ZSr41 interface showed near-complete absence of cell adhesion directly on the sample surface, most likely caused by either a high local alkalinity, change in surface topography, and/or surface composition. The direct culture method used in this study was proposed as a valuable tool for studying the design aspects of Zn-containing Mg-based biomaterials in vitro, in order to engineer solutions to address current shortcomings of Mg alloys for vascular device applications. PMID:27746360

DOE Office of Scientific and Technical Information (OSTI.GOV)

Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko

Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenousmore » PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.« less

Modified rice bran hemicellulose inhibits vascular endothelial growth factor-induced angiogenesis in vitro via VEGFR2 and its downstream signaling pathways

PubMed Central

ZHU, Xia; OKUBO, Aya; IGARI, Naoki; NINOMIYA, Kentaro; EGASHIRA, Yukari

2016-01-01

Angiogenesis is implicated in diverse pathological conditions such as cancer, rheumatoid arthritis, psoriasis, atherosclerosis, and retinal neovascularization. In the present study, we investigated the effects of modified rice bran hemicellulose (MRBH), a water-soluble hemicellulose preparation from rice bran treated with shiitake enzymes, on vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and its mechanism. We found that MRBH significantly inhibited VEGF-induced tube formation in human umbilical vein endothelial cells (HUVECs) co-cultured with human dermal fibroblasts. We also observed that MRBH dose-dependently suppressed the VEGF-induced proliferation and migration of HUVECs. Furthermore, examination of the anti-angiogenic mechanism indicated that MRBH reduced not only VEGF-induced activation of VEGF receptor 2 but also of the downstream signaling proteins Akt, extracellular signal-regulated protein kinase 1/2, and p38 mitogen-activated protein kinase. These findings suggest that MRBH has in vitro anti-angiogenic effects that are partially mediated through the inhibition of VEGF signaling. PMID:28439487

Characterization and biocompatibility studies of new degradable poly(urea)urethanes prepared with arginine, glycine or aspartic acid as chain extenders.

PubMed

Chan-Chan, L H; Tkaczyk, C; Vargas-Coronado, R F; Cervantes-Uc, J M; Tabrizian, M; Cauich-Rodriguez, J V

2013-07-01

Polyurethanes are very often used in the cardiovascular field due to their tunable physicochemical properties and acceptable hemocompatibility although they suffer from poor endothelialization. With this in mind, we proposed the synthesis of a family of degradable segmented poly(urea)urethanes (SPUUs) using amino acids (L-arginine, glycine and L-aspartic acid) as chain extenders. These polymers degraded slowly in PBS (pH 7.4) after 24 weeks via a gradual decrease in molecular weight. In contrast, accelerated degradation showed higher mass loss under acidic, alkaline and oxidative media. MTT tests on polyurethanes with L-arginine as chain extenders showed no adverse effect on the metabolism of human umbilical vein endothelial cells (HUVECs) indicating the leachables did not provoke any toxic responses. In addition, SPUUs containing L-arginine promoted higher levels of HUVECs adhesion, spreading and viability after 7 days compared to the commonly used Tecoflex(®) polyurethane. The biodegradability and HUVEC proliferation on L-arginine-based SPUUs suggests that they can be used in the design of vascular grafts for tissue engineering.

AGE/RAGE-Induced EMP Release via the NOX-Derived ROS Pathway

PubMed Central

Chen, Ying-Hua; Chen, Zhang-Wei; Li, Hong-Mei

2018-01-01

Objective Diabetes is associated with accelerated formation of advanced glycation end products (AGEs) that are extensively found in circulating endothelial microparticles (EMPs). This study aimed to investigate whether AGEs have a direct effect on EMP formation and the possible underlying mechanism. Methods In vitro, cultured human umbilical vein endothelial cells (HUVECs) were incubated with AGEs (200 and 400 μg/ml) for 24 hours with or without pretreatment with anti-RAGE antibody, NOX inhibitor, or ROS scavenger. The number of CD31-positive EMPs was assessed by flow cytometry. Results The number of EMPs was significantly increased in HUVECs stimulated by AGEs in a dose-dependent manner. In addition, receptors for AGEs (RAGE), NAD(P)H oxidase (NOX), and reactive oxygen species (ROS) were increased by AGEs as compared to the control group. These changes could be reversed when HUVECs were pretreated with anti-RAGE antibody. Moreover, inhibition of NOX as well as antioxidant treatment reduced the release of EMPs induced by AGEs. Conclusion Our study suggested that AGEs increased EMP generation, which was mediated by RAGE signaling through NOX-derived ROS. PMID:29744367

AGE/RAGE-Induced EMP Release via the NOX-Derived ROS Pathway.

PubMed

Chen, Ying-Hua; Chen, Zhang-Wei; Li, Hong-Mei; Yan, Xin-Feng; Feng, Bo

2018-01-01

Diabetes is associated with accelerated formation of advanced glycation end products (AGEs) that are extensively found in circulating endothelial microparticles (EMPs). This study aimed to investigate whether AGEs have a direct effect on EMP formation and the possible underlying mechanism. In vitro, cultured human umbilical vein endothelial cells (HUVECs) were incubated with AGEs (200 and 400  μ g/ml) for 24 hours with or without pretreatment with anti-RAGE antibody, NOX inhibitor, or ROS scavenger. The number of CD31-positive EMPs was assessed by flow cytometry. The number of EMPs was significantly increased in HUVECs stimulated by AGEs in a dose-dependent manner. In addition, receptors for AGEs (RAGE), NAD(P)H oxidase (NOX), and reactive oxygen species (ROS) were increased by AGEs as compared to the control group. These changes could be reversed when HUVECs were pretreated with anti-RAGE antibody. Moreover, inhibition of NOX as well as antioxidant treatment reduced the release of EMPs induced by AGEs. Our study suggested that AGEs increased EMP generation, which was mediated by RAGE signaling through NOX-derived ROS.

Endothelial glucocorticoid receptor promoter methylation according to dexamethasone sensitivity

PubMed Central

Mata-Greenwood, Eugenia; Jackson, P Naomi; Pearce, William J; Zhang, Lubo

2016-01-01

We have previously shown that in vitro sensitivity to dexamethasone (DEX) stimulation in human endothelial cells is positively regulated by the glucocorticoid receptor (NR3C1, GR). The present study determined the role of differential GR transcriptional regulation in glucocorticoid sensitivity. We studied 25 human umbilical vein endothelial cells (HUVECs) that had been previously characterized as DEX-sensitive (n = 15), or resistant (n = 10). Real-time PCR analysis of GR 5′UTR mRNA isoforms showed that all HUVECs expressed isoforms 1B, 1C, 1D, 1F, and 1H, and isoforms 1B and 1C were predominantly expressed. DEX-resistant cells expressed higher basal levels of the 5′UTR mRNA isoforms 1C and 1D, but lower levels of the 5′UTR mRNA isoform 1F than DEX-sensitive cells. DEX treatment significantly decreased GRα and GR-1C mRNA isoform expression in DEX-resistant cells only. Reporter luciferase assays indicated that differential GR mRNA isoform expression was not due to differential promoter usage between DEX-sensitive and DEX-resistant cells. Analysis of promoter methylation, however, showed that DEX-sensitive cells have higher methylation levels of promoter 1D and lower methylation levels of promoter 1F than DEX-resistant cells. Treatment with 5-aza-2-deoxycytidine abolished the differential 5′UTR mRNA isoform expression between DEX-sensitive and DEX-resistant cells. Finally, both GRα overexpression and 5-aza-2-deoxycytidine treatment eliminated the differences between sensitivity groups to DEX-mediated downregulation of endothelial nitric oxide synthase (NOS3), and upregulation of plasminogen activator inhibitor 1 (SERPINE1). In sum, human endothelial GR 5′UTR mRNA expression is regulated by promoter methylation with DEX-sensitive and DEX-resistant cells having different GR promoter methylation patterns. PMID:26242202

Cytosolic phospholipaseA2 inhibition with PLA-695 radiosensitizes tumors in lung cancer animal models.

PubMed

Thotala, Dinesh; Craft, Jeffrey M; Ferraro, Daniel J; Kotipatruni, Rama P; Bhave, Sandeep R; Jaboin, Jerry J; Hallahan, Dennis E

2013-01-01

Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted.

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

PubMed

Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

2014-04-01

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

APOC3 induces endothelial dysfunction through TNF-α and JAM-1.

PubMed

Tao, Yun; Xiong, Yisong; Wang, Huimin; Chu, Shaopeng; Zhong, Renqian; Wang, Jianxin; Wang, Guihua; Ren, Xiumei; Yu, Juan

2016-09-13

The fatality rate for cardiovascular disease (CVD) has increased in recent years and higher levels of triglyceride have been shown to be an independent risk factor for atherosclerotic CVD. Dysfunction of endothelial cells (ECs) is also a key factor of CVD. APOC3 is an important molecule in lipid metabolism that is closely associated with hyperlipidemia and an increased risk of developing CVD. But the direct effects of APOC3 on ECs were still unknown. This study was aimed at determining the effects of APOC3 on inflammation, chemotaxis and exudation in ECs. ELISA, qRT-PCR, immunofluorescence, flow cytometry and transwell assays were used to investigate the effects of APOC3 on human umbilical vein endothelial cells (HUVECs). SiRNA-induced TNF-α and JAM-1 silencing were used to observe how APOC3 influenced the inflammatory process in the ECs. Our results showed that APOC3 was closely associated with the inflammatory process in ECs, and that this process was characterized by the increased expression of TNF-α. Inflammatory processes further disrupted the tight junctions (TJs) between HUVECs by causing increased expression of JAM-1. JAM-1 was involved in maintaining the integrity of TJs, and it promoted the assembly of platelets and the exudation of leukocytes. Changes in its expression promoted chemotaxis and the exudation of ECs, which contributed to atherosclerosis. While the integrity of the TJs was disrupted, the adhesion of THP-1 cells to HUVECs was also increased by APOC3. In this study, we describe the mechanism by which APOC3 causes inflammation, chemotaxis and the exudation of ECs, and we suggest that controlling the inflammatory reactions that are caused by APOC3 may be a new method to treat CVD.

Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

PubMed

Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

2017-01-01

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the α v β 3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1 H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin α v β 3 . In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization

PubMed Central

Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

2017-01-01

Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice. PMID:28115846

Low-density lipoproteins modulate endothelial cells to secrete endothelin-1 in a polarized pattern: a study using a culture model system simulating arterial intima.

PubMed

Unoki, H; Fan, J; Watanabe, T

1999-01-01

We investigated the structural and functional properties of human umbilical vein endothelial cells (HUVECs) cultured on a two-chamber culture model system using an amnion membrane. Compared to HUVECs cultured on a plastic dish, HUVECs cultured on the model system exhibited several features similar to those of in vivo vessels, including formation of the intercellular junctional devices and expression of tight junction-associated protein ZO-1 and adherence junction-associated protein alpha-catenin. Furthermore, we found that HUVECs had a property of polar secretion of endothelin-1 (ET-1). About 90% of the total amount of synthesized ET-1 was found in the lower well, designated as the basal side. When HUVECs were incubated with either native low-density lipoproteins (nLDLs) or oxidized LDLs (oxLDLs) at a concentration of 100 microgram/ml, ET-1 secretion was significantly increased, dependent on the cell side (apical vs basal) on which the nLDLs or oxLDLs were loaded. When the LDLs were loaded on the apical side, the secretion of ET-1 from HUVECs on the apical side was increased by 48% (nLDL) and 61% (oxLDL), whereas it was accompanied by a concomitant decrease of ET-1 on the basal side (45% by nLDLs and 38% by oxLDLs). When loaded on the basal side, however, ET-1 was increased by 23% (nLDLs) and 53% (oxLDLs) on the basal side, with a 26% simultaneous decrease of ET-1 on the opposite side for both nLDLs and oxLDLs. On the contrary, high-density lipoproteins (HDLs) inhibited ET-1 secretion from HUVECs on the opposite side of the well on which HDLs were loaded; there was a 57% decrease on the basal side when HDLs were loaded on the apical side, and a 46% decrease on the apical side when loaded on the basal side. These results indicate that modulation of ET-1 secretion from ECs by lipoproteins is virtually dependent on the place (apical vs basal) where these proteins are present. The finding that nLDLs and oxLDLs enhance ET-1 secretion by ECs in a polarized pattern suggests that ET-1 may be involved in pathophysiological processes such as atherogenesis.

Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells.

PubMed

Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

2017-01-14

BACKGROUND Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. MATERIAL AND METHODS We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). RESULTS We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. CONCLUSIONS Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer.

Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells

PubMed Central

Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

2017-01-01

Background Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. Material/Methods We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). Results We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. Conclusions Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer. PMID:28087861

β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.

PubMed

Raschi, Elena; Chighizola, Cecilia B; Grossi, Claudia; Ronda, Nicoletta; Gatti, Rita; Meroni, Pier Luigi; Borghi, M Orietta

2014-12-01

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protection of kinsenoside against AGEs-induced endothelial dysfunction in human umbilical vein endothelial cells.

PubMed

Liu, Qing; Qiao, Ai-Min; Yi, Li-Tao; Liu, Zhen-Ling; Sheng, Shi-Mei

2016-10-01

Kinsenoside is the major ingredient of Anoectochilus roxburghii which is a traditional Chinese herb using for the treatment of diabetes. The present study investigated the safety and vascular protection of kinsenoside related to advanced glycation end products (AGEs) in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. HUVECs were pre-incubated with AGEs (200μg/mL) for 1h, and then co-treated with different concentrations of kinsenoside (10-30μg/mL) for another 48h. After the supernatant was collected, the contents of nitric oxide (NO), the levels of reactive oxygen species (ROS) and inflammatory cytokines, and the expressions of AGEs receptor (RAGE) and nuclear factor kappa B (NF-κB) were measured. No significant changes in cell viability were found in kinsenoside-treated cells at the range of 10-70μg/mL. Pretreatment with kinsenoside induced a significant increase in NO production in AGEs-induced cells. In addition, kinsenoside not only inhibited the expression of RAGE but also decreased intracellular ROS generation induced by AGEs. Furthermore, kinsenoside suppressed the protein and gene expression of NF-κB, and reduced the release of intercellular adhesion molecule-1 (ICAM-1) and human monocyte chemoattractant protein-1 (MCP-1) in a dose-dependent manner remarkably. These results indicated that kinsenoside might attenuate AGEs-induced endothelial dysfunction via AGEs-RAGE-NF-κB pathway. Considering the relatively low toxicity of kinsenoside, it might be a promising agent for treatment of vasculopathy in diabetic patients. Copyright © 2016 Elsevier Inc. All rights reserved.

Estradiol, acting through ERα, induces endothelial non-classic renin-angiotensin system increasing angiotensin 1-7 production.

PubMed

Mompeón, Ana; Lázaro-Franco, Macarena; Bueno-Betí, Carlos; Pérez-Cremades, Daniel; Vidal-Gómez, Xavier; Monsalve, Elena; Gironacci, Mariela M; Hermenegildo, Carlos; Novella, Susana

2016-02-15

Intracellular renin-angiotensin system (RAS) can operate independently of the circulating RAS. Estrogens provide protective effects by modulating the RAS. Our aim was to investigate the effect of estradiol (E2) on angiotensin converting enzymes (ACE) 1 and ACE2 expression and activities in human endothelial cells (HUVEC), and the role of estrogen receptors (ER). The results confirmed the presence of active intracellular RAS in HUVEC. Physiological concentrations of E2 induced a concentration-dependent increase of ACE1 and ACE2 mRNA expression and ACE1, but not ACE2, protein levels. ACE1 and ACE2 enzymatic activities were also induced with E2. These effects were mediated through ERα activation, since ER antagonists ICI 182780 and MPP completely abolished the effect of E2. Moreover, the ERα agonist PPT mirrored the E2 effects on ACE1 and ACE2 protein expression and activity. Exposure of endothelial cells to E2 significantly increased Ang-(1-7) production. In conclusion, E2 increases Ang-(1-7) production, through ERα, involving increased ACE1 and ACE2 mRNA expression and activity and ACE1 protein levels. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells.

PubMed

Feng, Bo; Zhang, Qian; Wang, Jianfang; Dong, Hong; Mu, Xiang; Hu, Ge; Zhang, Tao

2018-04-30

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells

PubMed Central

Feng, Bo; Zhang, Qian; Wang, Jianfang; Dong, Hong; Mu, Xiang; Hu, Ge; Zhang, Tao

2018-01-01

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection. PMID:29629559

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells

PubMed Central

Wei, Yujie; Lai, Bin; Liu, Huiliang; Li, Yi; Zhen, Wang; Fu, Ling

2018-01-01

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5–5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences. PMID:29257196

Aspirin prevents TNF-α-induced endothelial cell dysfunction by regulating the NF-κB-dependent miR-155/eNOS pathway: Role of a miR-155/eNOS axis in preeclampsia.

PubMed

Kim, Joohwan; Lee, Kyu-Sun; Kim, Ji-Hee; Lee, Dong-Keon; Park, Minsik; Choi, Seunghwan; Park, Wonjin; Kim, Suji; Choi, Yoon Kyung; Hwang, Jong Yun; Choe, Jongseon; Won, Moo-Ho; Jeoung, Dooil; Lee, Hansoo; Ryoo, Sungwoo; Ha, Kwon-Soo; Kwon, Young-Guen; Kim, Young-Myeong

2017-03-01

Preeclampsia is an inflammatory disease with endothelial cell dysfunction that occurs via decreased endothelial nitric oxide synthase/nitric oxide (eNOS/NO) activity. Aspirin reduces the incidence of hypertensive pregnancy complications. However, the underlying mechanism has not been clearly explained. Here, we found that tumor necrosis factor (TNF)-α, microRNA (miR)-155, and eNOS levels as well as endothelial redox phenotype were differentially regulated in preeclamptic patients, implying the involvement of TNF-α- and redox signal-mediated miR-155 biogenesis and eNOS downregulation in the pathogenesis of preeclampsia. Aspirin prevented the TNF-α-mediated increase in miR-155 biogenesis and decreases in eNOS expression and NO/cGMP production in cultured human umbilical vein endothelial cells (HUVECs). Similar effects of aspirin were also observed in HUVECs treated with H 2 O 2 . The preventive effects of aspirin was associated with the inhibition of nuclear factor-κB (NF-κB)-dependent MIR155HG (miR-155 host gene) expression. Aspirin recovered the TNF-α-mediated decrease in wild-type, but not mutant, eNOS 3'-untranslated region reporter activity, whose effect was blocked by miR-155 mimic. Moreover, aspirin prevented TNF-α-mediated endothelial cell dysfunction associated with impaired vasorelaxation, angiogenesis, and trophoblast invasion, and the preventive effects were blocked by miR-155 mimic or an eNOS inhibitor. Aspirin rescued TNF-α-mediated eNOS downregulation coupled with endothelial dysfunction by inhibiting NF-κB-dependent transcriptional miR-155 biogenesis. Thus, the redox-sensitive NF-κB/miR-155/eNOS axis may be crucial in the pathogenesis of vascular disorders including preeclampsia. Copyright © 2017 Elsevier Inc. All rights reserved.

Tonic inhibition by G protein-coupled receptor kinase 2 of Akt/endothelial nitric-oxide synthase signaling in human vascular endothelial cells under conditions of hyperglycemia with high insulin levels.

PubMed

Taguchi, Kumiko; Sakata, Kimimasa; Ohashi, Wakana; Imaizumi, Takahiro; Imura, Joji; Hattori, Yuichi

2014-05-01

G protein-coupled receptor kinase 2 (GRK2) participates together with β-arrestins in the regulation of G protein-coupled receptor signaling, but emerging evidence suggests that GRK2 can interact with a growing number of proteins involved in signaling mediated by other membrane receptor families under various pathologic conditions. We tested the hypothesis that GRK2 may be an important contributor to vascular endothelial dysfunction in diabetes. Human umbilical venous endothelial cells (HUVECs) were exposed to high glucose and high insulin (HG/HI) to mimic insulin-resistant diabetic conditions. GRK2 expression and membrane translocation were up-regulated under HG/HI conditions. HG/HI did not modify activation of Akt or endothelial nitric-oxide synthase (eNOS), but GRK2 inhibitor or small interfering RNA (siRNA) resulted in an increase in Akt and eNOS activation in HUVECs exposed to HG/HI. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation was increased after exposure to HG/HI, which was prevented by GRK2 inhibitor or siRNA. ERK1/2-mediated GRK2 phosphorylation at Ser-670 confirmed that ERK1/2 participated in a negative feedback regulatory loop. In human embryonic kidney 293T cells that overexpressed GRK2, Akt activity was unchanged, whereas ERK1/2 activity was raised. The effect of GRK inhibitor treatment on Akt/eNOS signaling was associated with membrane translocation of β-arrestin 2. The experiments with β-arrestin 2 siRNA showed that β-arrestin 2 may act as a positive modulator of Akt/eNOS signaling. Our studies reveal that GRK2, which is up-regulated by HG/HI, leads to a tonic inhibition of the insulin Akt/eNOS pathway in endothelial cells. We provide new insights into the pathogenesis of diabetes-associated vascular endothelial dysfunction.

Diesel Exhaust Particles Contribute to Endothelia Apoptosis via Autophagy Pathway.

PubMed

Wang, Jhih-Syuan; Tseng, Chia-Yi; Chao, Ming-Wei

2017-03-01

Epidemiological studies suggest that an increase of PM2.5 diesel exhaust particles (DEP) in ambient air corresponds to increased myocardial infarctions and atherosclerosis. When exposed to DEP, endothelial cells exhibit increases in oxidative stress and apoptosis, but the role of autophagy in this DEP-induced cell death remains unclear. Here, we suggest that acute DEP exposure produces intracellular reactive oxygen species (ROS) leading to induction of DEP internalization, endothelial dysfunction, and pro-inflammation in an in vitro human umbilical vein endothelial cells (HUVEC) model. This study found that increases in intracellular oxidative stress and cellular internalization of DEP occurred within 2 h of exposure to DEP. After 2 h of DEP exposure, Mdm2 expression was increased, which triggered cellular autophagy after 4 h of DEP exposure and suppressed cellular senescence. Unfortunately, phagocytized DEP could not be eliminated by cellular autophagy, which led to a continuous buildup of ROS, an increased release of cytokines, and an increased expression of anchoring molecules. After 12 h of DEP exposure, HUVEC reduced Mdm2 expression leading to increased p53 expression, which triggered apoptosis and ultimately resulted in endothelial dysfunction. On the other hand, when cells lacked the ability to induce autophagy, DEP was unable to induce cell senescence and most of the cells survived with only a small percentage of the cells undergoing necrosis. The results presented in this study clearly demonstrate the role cellular autophagy plays in DEP-induced atherosclerosis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Aldolase B knockdown prevents high glucose-induced methylglyoxal overproduction and cellular dysfunction in endothelial cells.

PubMed

Liu, Jianghai; Mak, Timothy Chun-Ping; Banigesh, Ali; Desai, Kaushik; Wang, Rui; Wu, Lingyun

2012-01-01

We used cultured endothelial cells as a model to examine whether up-regulation of aldolase B and enhanced methylglyoxal (MG) formation play an important role in high glucose-induced overproduction of advanced glycosylation endproducts (AGEs), oxidative stress and cellular dysfunction. High glucose (25 mM) incubation up-regulated mRNA levels of aldose reductase (an enzyme converting glucose to fructose) and aldolase B (a key enzyme that catalyzes MG formation from fructose) and enhanced MG formation in human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA. hy926 cells. High glucose-increased MG production in EA. hy926 cells was completely prevented by siRNA knockdown of aldolase B, but unaffected by siRNA knockdown of aldolase A, an enzyme responsible for MG formation during glycolysis. In addition, inhibition of cytochrome P450 2E1 or semicarbazide-sensitive amine oxidase which produces MG during the metabolism of lipid and proteins, respectively, did not alter MG production. Both high glucose (25 mM) and MG (30, 100 µM) increased the formation of N(ε)-carboxyethyl-lysine (CEL, a MG-induced AGE), oxidative stress (determined by the generation of oxidized DCF, H(2)O(2), protein carbonyls and 8-oxo-dG), O-GlcNAc modification (product of the hexosamine pathway), membrane protein kinase C activity and nuclear translocation of NF-κB in EA. hy926 cells. However, the above metabolic and signaling alterations induced by high glucose were completely prevented by knockdown of aldolase B and partially by application of aminoguanidine (a MG scavenger) or alagebrium (an AGEs breaker). In conclusion, efficient inhibition of aldolase B can prevent high glucose-induced overproduction of MG and related cellular dysfunction in endothelial cells.

C-reactive protein induces matrix metalloproteinase-1 and -10 in human endothelial cells: implications for clinical and subclinical atherosclerosis.

PubMed

Montero, Ines; Orbe, Josune; Varo, Nerea; Beloqui, Oscar; Monreal, José I; Rodríguez, José A; Díez, Javier; Libby, Peter; Páramo, José A

2006-04-04

We examined the effect of C-reactive protein (CRP) on matrix metalloproteinase (MMP) and inhibitor expression in endothelial cells and in patients with clinical and subclinical atherosclerosis. In addition to predicting atherosclerotic vascular disease, CRP may directly promote a proinflammatory/proatherosclerotic phenotype. Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were incubated in the presence or absence of CRP (50 mug/ml). Microarray analysis, real-time polymerase chain reaction, immunological and activity assays for MMPs were performed. Specific inhibitors of mitogen-activated protein kinase pathway were used. The MMP-1 and -10 plasma levels were measured in apparently healthy subjects (n = 70). Immunolocalization of CRP, MMP-1, and MMP-10 was performed in human mammary arteries and carotid endarterectomy specimens. C-reactive protein augmented MMP-1 and -10 messenger ribonucleic acid expression in HUVEC (p < 0.05) and HAEC (p < 0.01). C-reactive protein stimulation also increased MMP-1 and -10 protein in conditioned culture medium (p < 0.001), as well as MMP activity (p = 0.001). Specific inhibition of p38 or MEK abolished the CRP induction of the MMP-1, whereas MMP-10 induction blockade required the simultaneous inhibition of p38 and Jun N-terminal kinase pathways. Subjects with CRP values >3 mg/l (n = 37) had increased plasma MMP-1 and -10 (p < 0.05), the association being significant after adjustment for confounding variables (p = 0.04 and p = 0.008, respectively). The MMP-10 levels were elevated in subjects with higher carotid intima-media thickness (p = 0.009). Increased CRP and MMP-10 colocalized in endothelial layer and macrophage-rich areas in advanced atherosclerotic plaques. Increased local and systemic CRP-related MMP activation might provide a link between inflammation and plaque vulnerability.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukamoto, Ikuko, E-mail: tukamoto@med.kagawa-u.ac.jp; Sakakibara, Norikazu; Maruyama, Tokumi

Research highlights: {yields} A novel nucleic acid analogue (2Cl-C.OXT-A, m.w. 284) showed angiogenic potency. {yields} It stimulated the tube formation, proliferation and migration of HUVEC in vitro. {yields} 2Cl-C.OXT-A induced the activation of ERK1/2 and MEK in HUVEC. {yields} Angiogenic potency in vivo was confirmed in CAM assay and rabbit cornea assay. {yields} A synthesized small angiogenic agent would have great clinical therapeutic value. -- Abstract: A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100 {mu}M was stronger than that of vascular endothelial growth factor (VEGF), a positivemore » control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.« less

Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation

PubMed Central

Devalliere, Julie; Chang, William G.; Andrejecsk, Jillian W.; Abrahimi, Parwiz; Cheng, Christopher J.; Jane-wit, Dan; Saltzman, W. Mark; Pober, Jordan S.

2014-01-01

Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin αvβ3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (≥6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.—Devalliere, J., Chang, W. G., Andrejecsk, J. W., Abrahimi, P., Cheng, C. J., Jane-wit, D., Saltzman, W. M., Pober, J. S. Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation. PMID:24221087

The Increased Endogenous Sulfur Dioxide Acts as a Compensatory Mechanism for the Downregulated Endogenous Hydrogen Sulfide Pathway in the Endothelial Cell Inflammation

PubMed Central

Zhang, Da; Wang, Xiuli; Tian, Xiaoyu; Zhang, Lulu; Yang, Guosheng; Tao, Yinghong; Liang, Chen; Li, Kun; Yu, Xiaoqi; Tang, Xinjing; Tang, Chaoshu; Zhou, Jing; Kong, Wei; Du, Junbao; Huang, Yaqian; Jin, Hongfang

2018-01-01

Endogenous hydrogen sulfide (H2S) and sulfur dioxide (SO2) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for in vivo experiments. We found that endogenous H2S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO2 level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO2 synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H2S donor could reverse the CSE knockdown-induced increase in the endogenous SO2 level and AAT activity. Moreover, H2S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H2S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartate-β-hydroxamate (HDX), which blocked the upregulation of endogenous SO2/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In in vivo experiment, H2S donor restored the deficiency of endogenous H2S production induced by MCT, and reversed the upregulation of endogenous SO2/AAT pathway via sulfhydrating AAT1 and AAT2. In accordance with the results of the in vitro experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H2S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H2S inhibited endogenous SO2 generation by inactivating AAT via the sulfhydration of AAT1/2; and the increased endogenous SO2 generation might play a compensatory role when H2S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses in vitro and in vivo. PMID:29760703

[Effect of haw leaf extract and its preparation on polymorphonuclear leucocyte adhesion during HUVEC anoxia/reoxygenation injury].

PubMed

Li, Peng; Fu, Jian-hua; Li, Xin-zhi

2008-08-01

To study the effect and molecular mechanism of two haw leaf extracts, Vitexin-rhamnoside (VR) and Vitexin-glucoside (VG), and their preparation, Aoshaen injection (AI), on the polymorphonuclear leucocyte (PMN) adhesion during human umbilical vein endothelial cell (HUVEC) anoxia/reoxygenation (A/R) injury. The cell model of A/R injury duplicated by breaking off the oxygen supplying of HUVEC for 60 min followed with reoxygenating for 30 min (phase 1) or 240 min (phase 2) was taken as the experimental objective. The effects of testing drugs (VR, VG and AI) on PMN adhesion in the model cells were measured by enzyme immunoassay, and their effects on PMN superficial adhesion molecule CD11/CD18 expression were measured by flow cytometer respectively. After 60 min of anoxia, HUVEC was shrunk and deformed. The adhesion between PMN and HUVEC significantly revealed at phase 1 in the model group, but it was fewer in the normal cell group, and also lesser in the groups treated with various drugs. The condition of cell adhesion revealed at phase 2 was the similar to that at phase 1. All testing drugs, VR, VG and AI, showed inhibitory effect on the cell adhesion at either phase 1 or phase 2, showing a certain dose-effect relationship. The expression of CD11/ CD18 was also inhibited by the testing drugs, and a good dose-effect relation was shown by VG and AI. At the resting condition, there are almost no expression of CD11/CD18 molecule, but it could be enhanced by incubating PMN with supernate of A/R injured HUVEC culture, and more marked at phase 1. Adding the test drugs into the supernate could inhibit the enhancing of CD11/CD18 molecule expression and reduce the PMN-HUVEC adhesion, which may be one of the molecular mechanisms of haw leaf extracts and their preparation in protecting heart against A/R injury.

Synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK inhibitors with anti-angiogenic activity.

PubMed

Dao, Pascal; Jarray, Rafika; Le Coq, Johanne; Lietha, Daniel; Loukaci, Ali; Lepelletier, Yves; Hadj-Slimane, Réda; Garbay, Christiane; Raynaud, Françoise; Chen, Huixiong

2013-08-15

We report herein the synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK (focal adhesion kinase) inhibitors and the evaluation of their anti-angiogenic activity on HUVEC cells. Generally, the effects of these compounds on endothelial cells could be correlated with their kinase inhibitory activity. The most efficient compounds displayed inhibition of viability against HUVEC cells in the micromolar range, as observed with TAE-226, which was designed by Novartis Pharma AG. X-ray crystallographic analysis of the co-crystal structure for compound 34 revealed that the mode of interaction with the FAK kinase domain is highly similar to that observed in the complex of TAE-226. Copyright © 2013 Elsevier Ltd. All rights reserved.

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

PubMed Central

Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

2014-01-01

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10–50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24 hours showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 hours, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2Kb 5′. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 minutes in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions. PMID:24518819

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.

PubMed

Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

2014-03-01

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions. Copyright © 2014 Elsevier Inc. All rights reserved.

[Regulative effects of hydrogen-rich medium on monocytic adhesion and vascular endothelial permeability].

PubMed

Wang, Wei-na; Xie, Ke-liang; Chen, Hong-guang; Han, Huan-zhi; Wang, Guo-lin; Yu, Yong-hao

2013-11-19

To explore the regulative effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced monocytes adhesion to human umbilical vein endothelial cells (HUVEC) and vascular endothelial permeability in vitro. Endothelial cells were seeded in 6-well plates and randomly divided into 4 groups (n = 42 each):control (A), hydrogen-rich medium (B), LPS (C) and LPS+hydrogen-rich medium (D). Cells were cultured in plain culture medium in groups A and C or in hydrogen-saturated culture medium in groups B and D.LPS 1 µg/ml was added into groups C and D.When forming a monolayer, monocytes were added into each group after 6, 12 and 24 h respectively. After a 90-minute co-culturing, adhesion status was detected by Wright-Giemsa stain.Supernatants were collected to detect the concentrations of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by enzyme-linked immunosorbent assay (ELISA). The expression of VE-cadherin was measured by Western blot. Cells were stained with immunofluorescence to show the distribution of VE-cadherin after a 24-hour incubation. Compared with group A, the adhesion of monocytes to endothelial cells increased (P < 0.05) in group C, the levels of E-selectin and VCAM-1 became elevated (P < 0.05) while the expression of VE-cadherin decreased significantly (P < 0.05). Compared with group C, adhesion decreased in group D (P < 0.05), the levels of E-selectin and VCAM-1 decreased (P < 0.05) while there was an increased expression of VE-cadherin (P < 0.05). Three timepoints showed the same tendency. The results of 24 h fluorescence indicated that, compared with group A, VE-cadherin was incomplete in cell-cell connections in group C.However it was complete and well-distributed in group D versus group C. Hydrogen-rich medium may reduce the LPS-induced release of adhesion molecules, lessen monocytic adhesion to HUVEC and regulate the expression of VE-cadherin to protect vascular permeability.

Glycyrrhetinic acid inhibits ICAM-1 expression via blocking JNK and NF-κB pathways in TNF-α-activated endothelial cells

PubMed Central

Chang, Ying-ling; Chen, Chien-lin; Kuo, Chao-Lin; Chen, Bor-chyuan; You, Jyh-sheng

2010-01-01

Aim: To investigate the effects of glycyrrhetinic acid (GA), an active component extracted from the root of Glycyrrhizae glabra, on the expression of intercellular adhesion molecule-1 (ICAM-1) in tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVEC). Methods: ICAM-1 mRNA and protein levels were detected using RT-PCR and cell enzyme-linked immunosorbent assays. The adherence of human monocytic THP-1 cells labeled with [3H]thymidine to HUVEC was determined by counting radioactivity with a scintillation counter. The activation of mitogen-activated protein kinases as well as the degradation of IκB and nuclear factor-κB (NF-κB) or phospho-c-Jun in the nucleus were detected by western blots. NF-κB binding activity was detected using electrophoretic mobility shift assay. Results: GA (50 and 100 μmol/L) significantly inhibits TNF-α-induced ICAM-1 mRNA and protein expressions, as well as THP-1 cell adhesiveness in HUVEC. GA selectively inhibited TNF-α-activated signal pathway of c-Jun N-terminal kinase (JNK), without affecting extracellular signal-regulated kinase 1/2 and p38. Furthermore, GA apparently inhibited IκB/NF-κB signaling system by preventing IκB degradation, NF-κB translocation, and NF-κB/DNA binding activity. Finally, pretreatment with GA or the inhibitors of NF-κB, JNK, and p38 reduced the ICAM-1 protein expression induced by TNF-α. Conclusion: GA inhibits TNF-α-stimulated ICAM-1 expression, leading to a decrease in adherent monocytes to HUVEC. This inhibition is attributed to GA interruption of both JNK/c-Jun and IκB/NF-κB signaling pathways, which decrease activator protein-1 (AP-1) and NF-κB mediated ICAM-1 expressions. The results suggest that GA may provide a beneficial effect in treating vascular diseases associated with inflammation, such as atherosclerosis. PMID:20418897

A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells.

PubMed

Castellino, F; Scaglione, N; Grosso, S B; Sategna-Guidetti, C

2000-01-01

Although tissue transglutaminase was recently identified as the main autoantigen recognized by endomysial antibodies in coeliac patients, anti-endomysium antibody detection still persists as the gold standard for coeliac disease screening and diagnosis. (1) To evaluate human umbilical vein cells (HUVEC) as an alternative source of endomysial antigen and to assess their suitability in the diagnosis of coeliac disease. (2) To verify whether tissue transglutaminase is one target antigen eliciting the endomysial antibody fraction of coeliac serum IgA. University teaching hospital. Sera from 123 untreated adults with biopsy-proven coeliac disease and 84 controls (40 healthy and 44 diseased) were assessed by indirect immunofluorescence, using HUVEC on glass slides prepared by cytocentrifugation and permeabilized by using Triton X (0.5%). Indirect immunofluorescence was performed: (1) using coeliac disease serum samples on HUVEC with or without prior incubation with tissue transglutaminase; and (2) incubating both HUVEC and monkey oesophagus with goat anti-guinea pig tissue transglutaminase antibody. All the coeliac patients, who were also positive on monkey oesophagus, showed the typical fluorescent homogeneous cytoplasmic stain on HUVEC. All control sera were negative both on HUVEC and on monkey oesophagus. IgA antibodies did not react with non-permeabilized cells, with intact membrane. Preincubation of coeliac sera with tissue transglutaminase abolished the typical fluorescent pattern. The incubation of anti-tissue transglutaminase antibody with monkey oesophagus and HUVEC resulted in an immunofluorescence staining pattern identical to that obtained with positive coeliac sera. (1) As a substrate for anti-endomysial antibody, HUVEC may provide the same diagnostic accuracy as monkey oesophagus, thus bypassing economical and ethical problems. The HUVEC antigen reacting with IgA from coeliac disease sera is an intracellular rather than a cell-surface antigen, as IgA antibodies reacted only with permeabilized cells. (2) Pretreatment of untreated coeliac sera with tissue transglutaminase abolished almost completely the specific staining; incubation with anti-tissue transglutaminase antibody elicited the characteristic fluorescent pattern, thus confirming that tissue transglutaminase represents the prominent autoantigen in coeliac disease.

Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

PubMed

Bakopoulou, Athina; Kritis, Aristeidis; Andreadis, Dimitrios; Papachristou, Eleni; Leyhausen, Gabriele; Koidis, Petros; Geurtsen, Werner; Tsiftsoglou, Asterios

2015-11-01

Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial transdifferentiation of SCAP, uncovering the molecular mechanisms regulating their fate. They also validate the angiogenic properties of their secretome giving insights into preconditioning strategies enhancing their therapeutic potential.

Thermoresponsive Polymers with Lower Critical Solution Temperature- or Upper Critical Solution Temperature-Type Phase Behaviour Do Not Induce Toxicity to Human Endothelial Cells.

PubMed

Ji, Yuejia; Zhu, Mengxiang; Gong, Yu; Tang, Haoyu; Li, Juan; Cao, Yi

2017-01-01

Thermoresponsive polymers have gained extensive attention as biomedical materials especially for targeted drug delivery systems. We have recently developed water-soluble polypeptide-based thermoresponsive polymers that exhibit lower critical solution temperature (LCST)- or upper critical solution temperature (UCST)-type phase behaviours. In this study, the toxicity of these polymers to human umbilical vein endothelial cells (HUVECs) was investigated to assess the safety and biocompatibility. Up to 100 μg/ml, thermoresponsive polymers did not induce cytotoxicity to HUVECs, showing as unaltered mitochondrial viability assessed as cell counting kit-8 (CCK-8) assay and membrane integrity assessed as lactate dehydrogenase (LDH) assay. Inflammatory response, assessed as the release of chemokine-soluble monocyte chemotactic protein 1 (sMCP-1) and interleukin-8 (IL-8) as well as cytokine IL-6, was not significantly affected by the polymers. In addition, 1 μM thapsigargin (TG), an endoplasmic reticulum (ER) stress inducer, significantly decreased mitochondrial viability, but did not affect membrane integrity or inflammatory response. The presence of thermoresponsive polymers with LCST-type phase behaviour did not further affect the effects of TG. In conclusion, the thermoresponsive polymers used in this study are not toxic to endothelial cells and therefore could be further considered as safe materials for biomedical applications. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

On the Normal Force Mechanotransduction of Human Umbilical Vein Endothelial Cells

NASA Astrophysics Data System (ADS)

Vahabikashi, Amir; Wang, Qiuyun; Wilson, James; Wu, Qianhong; Vucbmss Team

2016-11-01

In this paper, we report a cellular biomechanics study to examine the normal force mechanotransduction of Human Umbilical Vein Endothelial Cells (HUVECs) with their implications on hypertension. Endothelial cells sense mechanical forces and adjust their structure and function accordingly. The mechanotransduction of normal forces plays a vital role in hypertension due to the higher pressure buildup inside blood vessels. Herein, HUVECs were cultured to full confluency and then exposed to different mechanical loadings using a novel microfluidic flow chamber. One various pressure levels while keeps the shear stress constant inside the flow chamber. Three groups of cells were examined, the control group (neither shear nor normal stresses), the normal pressure group (10 dyne/cm2 of shear stress and 95 mmHg of pressure), and the hypertensive group (10 dyne/cm2 of shear stress and 142 mmHg of pressure). Cellular response characterized by RT-PCR method indicates that, COX-2 expressed under normal pressure but not high pressure; Mn-SOD expressed under both normal and high pressure while this response was stronger for normal pressure; FOS and e-NOS did not respond under any condition. The differential behavior of COX-2 and Mn-SOD in response to changes in pressure, is instrumental for better understanding the pathogenesis of hypertensive cardiovascular diseases. This research was supported by the National Science Foundation under Award #1511096.

Tentacle extract from the jellyfish Cyanea capillata increases proliferation and migration of human umbilical vein endothelial cells through the ERK1/2 signaling pathway

PubMed Central

Wang, Qianqian; Zhang, Hui; Liu, Guoyan; He, Qian; Zhang, Liming

2017-01-01

Wound healing is a complex biological process, and current research finds that jellyfish have a great capacity for promoting growth and healing. However, the underlying mechanisms remain unclear. Thus, this study was conducted to investigate the molecular mechanisms and effects of a tentacle extract (TE) from the jellyfish Cyanea capillata (C. capillata) on cell proliferation and migration in human umbilical vein endothelial cells (HUVECs). First, our results showed that TE at the concentration of 1 μg/ml could promote cell proliferation over various durations, induce a transition of the cells from the G1-phase to the S/G2-phase of the cell cycle, and increase the expression of cell cycle proteins (CyclinB1 and CyclinD1). Second, we found that TE could activate the PI3K/Akt, ERK1/2 and JNK MAPK signaling pathways but not the NF-κB signaling pathway or the apoptosis signaling cascade. Finally, we demonstrated that the TE-induced expression of cell cycle proteins was decreased by ERK1/2 inhibitor PD98059 but not by PI3K inhibitor LY294002 or JNK inhibitor SP600125. Similarly, the TE-enhanced migration ability of HUVECs was also markedly attenuated by PD98059. Taken together, our findings indicate that TE-induced proliferation and migration in HUVECs mainly occurred through the ERK1/2 MAPK signaling pathway. These results are instructively important for further research on the isolation and purification of growth-promoting factors from C. capillata and are hopeful as a means to improve human wound repair in unfavorable conditions. PMID:29261770

Tentacle extract from the jellyfish Cyanea capillata increases proliferation and migration of human umbilical vein endothelial cells through the ERK1/2 signaling pathway.

PubMed

Wang, Beilei; Liu, Dan; Wang, Chao; Wang, Qianqian; Zhang, Hui; Liu, Guoyan; He, Qian; Zhang, Liming

2017-01-01

Wound healing is a complex biological process, and current research finds that jellyfish have a great capacity for promoting growth and healing. However, the underlying mechanisms remain unclear. Thus, this study was conducted to investigate the molecular mechanisms and effects of a tentacle extract (TE) from the jellyfish Cyanea capillata (C. capillata) on cell proliferation and migration in human umbilical vein endothelial cells (HUVECs). First, our results showed that TE at the concentration of 1 μg/ml could promote cell proliferation over various durations, induce a transition of the cells from the G1-phase to the S/G2-phase of the cell cycle, and increase the expression of cell cycle proteins (CyclinB1 and CyclinD1). Second, we found that TE could activate the PI3K/Akt, ERK1/2 and JNK MAPK signaling pathways but not the NF-κB signaling pathway or the apoptosis signaling cascade. Finally, we demonstrated that the TE-induced expression of cell cycle proteins was decreased by ERK1/2 inhibitor PD98059 but not by PI3K inhibitor LY294002 or JNK inhibitor SP600125. Similarly, the TE-enhanced migration ability of HUVECs was also markedly attenuated by PD98059. Taken together, our findings indicate that TE-induced proliferation and migration in HUVECs mainly occurred through the ERK1/2 MAPK signaling pathway. These results are instructively important for further research on the isolation and purification of growth-promoting factors from C. capillata and are hopeful as a means to improve human wound repair in unfavorable conditions.

A biological approach to assembling tissue modules through endothelial capillary network formation.

PubMed

Riesberg, Jeremiah J; Shen, Wei

2015-09-01

To create functional tissues having complex structures, bottom-up approaches to assembling small tissue modules into larger constructs have been emerging. Most of these approaches are based on chemical reactions or physical interactions at the interface between tissue modules. Here we report a biological assembly approach to integrate small tissue modules through endothelial capillary network formation. When adjacent tissue modules contain appropriate extracellular matrix materials and cell types that support robust endothelial capillary network formation, capillary tubules form and grow across the interface, resulting in assembly of the modules into a single, larger construct. It was shown that capillary networks formed in modules of dense fibrin gels seeded with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs); adjacent modules were firmly assembled into an integrated construct having a strain to failure of 117 ± 26%, a tensile strength of 2208 ± 83 Pa and a Young's modulus of 2548 ± 574 Pa. Under the same culture conditions, capillary networks were absent in modules of dense fibrin gels seeded with either HUVECs or MSCs alone; adjacent modules disconnected even when handled gently. This biological assembly approach eliminates the need for chemical reactions or physical interactions and their associated limitations. In addition, the integrated constructs are prevascularized, and therefore this bottom-up assembly approach may also help address the issue of vascularization, another key challenge in tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Lemon grass (Cymbopogon citratus (D.C) Stapf) polyphenols protect human umbilical vein endothelial cell (HUVECs) from oxidative damage induced by high glucose, hydrogen peroxide and oxidised low-density lipoprotein.

PubMed

Campos, J; Schmeda-Hirschmann, G; Leiva, E; Guzmán, L; Orrego, R; Fernández, P; González, M; Radojkovic, C; Zuñiga, F A; Lamperti, L; Pastene, E; Aguayo, C

2014-05-15

The aromatic herb Cymbopogon citratus Stapf is widely used in tropical and subtropical countries in cooking, as a herbal tea, and in traditional medicine for hypertension and diabetes. Some of its properties have been associated with the in vitro antioxidant effect of polyphenols isolated from their aerial parts. However, little is known about C. citratus effects on endothelial cells oxidative injury. Using chromatographic procedures, a polyphenol-rich fraction was obtained from C. citratus (CCF) and their antioxidant properties were assessed by cooper-induced LDL oxidation assay. The main constituents of the active CCF, identified by high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD-MS), were chlorogenic acid, isoorientin and swertiajaponin. CCF 10 and 100 μg/ml diminishes reactive oxidative species (ROS) production in human umbilical vein endothelial cell (HUVECs), challenged with high D-glucose (60% inhibition), hydrogen peroxide (80% inhibition) or oxidised low-density lipoprotein (55% inhibition). CCF 10 or 100 μg/ml did not change nitric oxide (NO) production. However, CCF was able to inhibit vasoconstriction induced by the thromboxane A2 receptor agonist U46619, which suggest a NO-independent vasodilatador effect on blood vessels. Our results suggest that lemon grass antioxidant properties might prevent endothelial dysfunction associated to an oxidative imbalance promoted by different oxidative stimuli. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular action mechanism against apoptosis by aqueous extract from guava budding leaves elucidated with human umbilical vein endothelial cell (HUVEC) model.

PubMed

Hsieh, Chiu-Lan; Huang, Chien-Ning; Lin, Yuh-Charn; Peng, Robert Y

2007-10-17

Chronic cardiovascular and neurodegenerative complications induced by hyperglycemia have been considered to be associated most relevantly with endothelial cell damages (ECD). The protective effects of the aqueous extract of Psidium guajava L. budding leaves (PE) on the ECD in human umbilical vein endothelial cell (HUVEC) model were investigated. Results revealed that glyoxal (GO) and methylglyoxal (MGO) resulting from the glycative and autoxidative reactions of the high blood sugar glucose (G) evoked a huge production of ROS and NO, which in turn increased the production of peroxynitrite, combined with the activation of the nuclear factor kappaB (NFkappaB), leading to cell apoptosis. High plasma glucose activated p38-MAPK, and high GO increased the expressions of p38-MAPK and JNK-MAPK, whereas high MGO levels induced the activity of ERK-MAPK. Glucose and dicarbonyl compounds were all found to be good inducers of intracellular PKCs, which together with MAPK acted as the upstream triggering factor to activate NFkappaB. Conclusively, high plasma glucose together with dicarbonyl compounds can trigger the signaling pathways of MAPK and PKC and induce cell apoptosis through ROS and peroxynitrite stimulation and finally by NFkappaB activation. Such effects of PE were ascribed to its high plant polyphenolic (PPP) contents, the latter being potent ROS inhibitors capable of blocking the glycation of proteins, which otherwise could have brought forth severe detrimental effects to the cells.

MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Bing; Xiao, Bo; Liang, Desheng

Highlights: {yields} We evaluated the role of miRNAs in ox-LDL induced apoptosis in ECs. {yields} We found 4 up-regulated and 11 down-regulated miRNAs in apoptotic ECs. {yields} Target genes of the dysregulated miRNAs regulate ECs apoptosis and atherosclerosis. {yields} MiR-365 promotes ECs apoptosis via suppressing Bcl-2 expression. {yields} MiR-365 inhibitor alleviates ECs apoptosis induced by ox-LDL. -- Abstract: Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth,more » proliferation, and apoptosis. However, whether miRNAs are associated with ox-LDL induced apoptosis and their effect on ECs is still unknown. Therefore, this study evaluated potential miRNAs and their involvement in ECs apoptosis in response to ox-LDL stimulation. Microarray and qRT-PCR analysis performed on human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL identified 15 differentially expressed (4 up- and 11 down-regulated) miRNAs. Web-based query tools were utilized to predict the target genes of the differentially expressed miRNAs, and the potential target genes were classified into different function categories with the gene ontology (GO) term and KEGG pathway annotation. In particular, bioinformatics analysis suggested that anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) is a target gene of miR-365, an apoptomir up-regulated by ox-LDL stimulation in HUVECs. We further showed that transfection of miR-365 inhibitor partly restored Bcl-2 expression at both mRNA and protein levels, leading to a reduction of ox-LDL-mediated apoptosis in HUVECs. Taken together, our findings indicate that miRNAs participate in ox-LDL-mediated apoptosis in HUVECs. MiR-365 potentiates ox-LDL-induced ECs apoptosis by regulating the expression of Bcl-2, suggesting potential novel therapeutic targets for atherosclerosis.« less

MicroRNA-34a regulation of endothelial senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ito, Takashi; Yagi, Shusuke; Yamakuchi, Munekazu, E-mail: munekazu_yamakuchi@urmc.rochester.edu

2010-08-06

Research highlights: {yields} MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. {yields} MiR-34a expression increases during endothelial cell senescence and in older mice. {yields} SIRT1 is a miR-34a target gene in endothelial cells. {yields} SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells. -- Abstract: Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelialmore » cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.« less

The expression of miR-125b regulates angiogenesis during the recovery of heat-denatured HUVECs.

PubMed

Zhou, Situo; Zhang, Pihong; Liang, Pengfei; Huang, Xiaoyuan

2015-06-01

In previous studies we found that miR-125b was down-regulated in denatured dermis of deep partial thickness burn patients. Moreover, miR-125b inhibited tumor-angiogenesis associated with the decrease of ERBB2 and VEGF expression in ovarian cancer cells and breast cancer cells, etc. In this study, we investigated the expression patterns and roles of miR-125b during the recovery of denatured dermis and heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burns in Sprague-Dawley rats and the heat-denatured cells (52°C, 35 s) were used for analysis. Western blot analysis and real-time PCR were applied to evaluate the expression of miR-125b and ERBB2 and VEGF. The ability of angiogenesis in heat-denatured HUVECs was analyzed by scratch wound healing and tube formation assay after pri-miR-125b or anti-miR-125b transfection. miR-125b expression was time-dependent during the recovery of heat-denatured dermis and HUVECs. Moreover, miR-125b regulated ERBB2 mRNA and Protein Expression and regulated angiogenesis association with regulating the expression of VEGF in heat-denatured HUVECs. Taken together our results show that the expression of miR-125b is time-dependent and miR-125b plays a regulatory role of angiogenesis during wound healing after burns. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Suppression of endothelial cell adhesion by XJP-1, a new phenolic compound derived from banana peel.

PubMed

Fu, Rong; Yan, Tianhua; Wang, Qiujuan; Guo, Qinglong; Yao, Hequan; Wu, Xiaoming; Li, Yang

2012-01-01

The adhesion of monocytes to activated vascular endothelial cells is a critical event in the initiation of atherosclerosis. Adhesion is mediated by oxidized low-density lipoprotein (ox-LDL) which up-regulates inflammatory markers on endothelial cells. Here we report that (±) 7, 8-dihydroxy-3-methyl-isochromanone-4 (XJP-1), an inhibitor of ox-LDL-induced adhesion of monocytes to endothelial cells blocks cellular functions which are associated with adhesion. We show that XJP-1 down-regulates ox-LDL-induced over-expression of adhesion molecules (ICAM-1 and VCAM-1) in a dose-dependent manner in human umbilical vein endothelial cells (HUVECs), attenuates ox-LDL-induced up-regulation of low-density lipoprotein receptor (LOX)-1, decreases generation of reactive oxygen species (ROS), blocks translocation of nuclear factor-kappa B (NF-κB) activity, and prevents activation of c-Jun N-terminal kinase (JNK)/p38 pathways in endothelial cells. These findings suggest that XJP-1 may attenuate ox-LDL-induced endothelial adhesion of monocytes by blocking expression of adhesion molecules through suppressing ROS/NF-κB, JNK and p38 pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

The effects of small interfering RNA–targeting tissue factor on an in vitro model of neovascularization

PubMed Central

Peng, Wenyan; Yu, Ying; Li, Tiejun; Zhu, Yuanyuan

2013-01-01

Purpose Tissue factor (TF) plays an important role in neovascularization (NV). This study aimed to determine whether small interfering RNA–targeting TF (TF-siRNA) could knock down TF expression and inhibit cell proliferation, cell migration, and tube formation in an in vitro model of NV. Methods Lipopolysaccharide (LPS) was used to stimulate human umbilical vein endothelial cell (HUVEC) lines to express TF and mimic certain phenotypes of NV in vitro. HUVECs were transfected with TF-siRNAs and control siRNAs using LipofectamineTM 2000. The inhibitory effect of the siRNAs on the expression of TF mRNA and protein was evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and western blot analysis. The effects on the cell viability, migration, and tube formation of siRNA-treated cells were examined by MTT assay, wound-healing assay, and Matrigel-induced capillary tube formation. Results Lipopolysaccharide treatment increased the expression of TF. TF-siRNAs effectively knocked down TF expression, with the most efficient TF-siRNA reducing 78.9% of TF expression. TF protein was also notably curtailed by TF-siRNA. The MTT and wound-healing assays showed that the TF-siRNA substantially inhibited the proliferation and migration of HUVECs. Tube formation was decreased by 47.4% and 59.4% in cells treated with the TF-siRNA and vascular endothelial growth factor–siRNA, respectively, compared with the blank control. Conclusions TF-siRNA can knockdown TF expression and inhibit cell proliferation, migration, and tube formation in vitro. TF-siRNA may provide a novel therapeutic candidate for NV-related diseases. PMID:23805036

ET-1 Stimulates Superoxide Production by eNOS Following Exposure of Vascular Endothelial Cells to Endotoxin.

PubMed

Gopalakrishna, Deepak; Pennington, Samantha; Karaa, Amel; Clemens, Mark G

2016-07-01

It has been shown that microcirculation is hypersensitized to endothelin1 (ET-1) following endotoxin (lipopolysaccharide [LPS]) treatment leading to an increased vasopressor response. This may be related in part to decreased activation of endothelial nitric oxide synthase (eNOS) by ET-1. eNOS can also be uncoupled to produce superoxide (O2). This aberrant eNOS activity could further contribute to the hyperconstriction and injury caused by ET-1 following LPS. We therefore tested whether LPS affects ROS production by vascular endothelial cells and whether and how this effect is altered by ET-1. Human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMEC) were subjected to a 6-h treatment with LPS (250 ng/mL) or LPS and sepiapterin (100 μM) followed by a 30-min treatment with 100 μM L-Iminoethyl Ornithine (L-NIO) an irreversible eNOS inhibitor and 30-min treatment with ET-1 (10 nM). Conversion of [H]L-arginine to [H]L-citrulline was used to measure eNOS activity. Superoxide dismutase (SOD) inhibitable reduction of Cytochrome-C, dihydro carboxy fluorescein (DCF), and Mitosox was used to estimate ROS. LT-SDS PAGE was used to assess the degree of monomerization of the eNOS homodimer. Stimulation of HUVECs with ET-1 significantly increased NO synthesis by 1.4-fold (P < 0.05). ET-1 stimulation of LPS-treated HUVECs failed to increase NO production. Western blot for eNOS protein showed no change in eNOS protein levels. LPS alone resulted in an insignificant increase in ROS production as measured by cytochrome C that was increased 4.6-fold by ET-1 stimulation (P < 0.05). L-NIO significantly decreased ET-1-induced ROS production (P < 0.05). Sepiapterin significantly decreased ROS production in both; unstimulated and ET-1-stimulated LPS-treated groups, but did not restore NO production. DCF experiments confirmed intracellular ROS while Mitosox suggested a non-mitochondrial source. ET-1 treatment following a chronic LPS stress significantly monomerized the eNOS homodimer that was inhibited by sepiapterin loading. The two concomitant phenomena of decreased NO production and increased ROS formation seem to be multifactorial in nature with ROS production dependent upon pterin availability.

Rapid Anastomosis of Endothelial Progenitor Cell–Derived Vessels with Host Vasculature Is Promoted by a High Density of Cotransplanted Fibroblasts

PubMed Central

Chen, Xiaofang; Aledia, Anna S.; Popson, Stephanie A.; Him, Linda; Hughes, Christopher C.W.

2010-01-01

To ensure survival of engineered implantable tissues thicker than approximately 2–3 mm, convection of nutrients and waste products to enhance the rate of transport will be required. Creating a network of vessels in vitro, before implantation (prevascularization), is one potential strategy to achieve this aim. In this study, we developed three-dimensional engineered vessel networks in vitro by coculture of endothelial cells (ECs) and fibroblasts in a fibrin gel for 7 days. Vessels formed by cord blood endothelial progenitor cell–derived ECs (EPC-ECs) in the presence of a high density of fibroblasts created an interconnected tubular network within 4 days, compared with 5–7 days in the presence of a low density of fibroblasts. Vessels derived from human umbilical vein ECs (HUVECs) in vitro showed similar kinetics. Implantation of the prevascularized tissues into immune-compromised mice, however, revealed a dramatic difference in the ability of EPC-ECs and HUVECs to form anastomoses with the host vasculature. Vascular beds derived from EPC-ECs were perfused within 1 day of implantation, whereas no HUVEC vessels were perfused at day 1. Further, while almost 90% of EPC-EC–derived vascular beds were perfused at day 3, only one-third of HUVEC-derived vascular beds were perfused. In both cases, a high density of fibroblasts accelerated anastomosis by 2–3 days. We conclude that both EPC-ECs and a high density of fibroblasts significantly accelerate the rate of functional anastomosis, and that prevascularizing an engineered tissue may be an effective strategy to enhance convective transport of nutrients in vivo. PMID:19737050

Caffeic acid phenethyl ester suppresses monocyte adhesion to the endothelium by inhibiting NF-κB/NOX2-derived ROS signaling

PubMed Central

Nakahara, Risa; Makino, Junya; Kamiya, Tetsuro; Hara, Hirokazu; Adachi, Tetsuo

2016-01-01

Caffeic acid phenethyl ester (CAPE), one of the major polyphenols, exhibits anti-oxidative, anti-bacterial, and anti-cancer properties. Atherosclerosis is a chronic inflammatory disease, the progression of which is closely related to the accumulated adhesion of inflammatory monocytes/macrophages to the endothelium. We herein determined whether CAPE and its derivatives suppressed THP-1 cell adhesion to human umbilical vein endothelial cells (HUVEC). Of the four polyphenols tested, CAPE significantly suppressed the 12-O-tetradecanoylphorbol 13-acetate (TPA)-elicited expression of cluster for differentiation (CD) 11b, 14, and 36, and this was accompanied by the inhibition of THP-1 cell adhesion to HUVEC. CAPE also suppressed the activation of TPA-elicited nuclear factor-κB (NF-κB) and accumulation of NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS), but did not affect extracellular signal-regulated kinase (ERK) phosphorylation. Taken together, these results demonstrated that CAPE suppressed THP-1 cell adhesion to HUVEC through, at least in part, the NF-κB, NOX2, and ROS-derived signaling axis. PMID:27257341

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

PubMed

Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

2008-01-01

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

PubMed

Ohtsuki, Sumio; Ikeda, Chiemi; Uchida, Yasuo; Sakamoto, Yumi; Miller, Florence; Glacial, Fabienne; Decleves, Xavier; Scherrmann, Jean-Michel; Couraud, Pierre-Olivier; Kubo, Yoshiyuki; Tachikawa, Masanori; Terasaki, Tetsuya

2013-01-07

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.

An HCG-rich microenvironment contributes to ovarian cancer cell differentiation into endothelioid cells in a three-dimensional culture system.

PubMed

Su, Min; Fan, Chao; Gao, Sainan; Shen, Aiguo; Wang, Xiaoying; Zhang, Yuquan

2015-11-01

We investigated the expression of human chorionic gonadotropin (HCG) and its effects on vasculogenic mimicry (VM) formation in ovarian cancer cells under normoxic and hypoxic conditions in three-dimensional matrices preconditioned by an endothelial-trophoblast cell co-culture system. The co-culture model was established using human umbilical vein endothelial cells (HUVECs) and HTR-8 trophoblast cells in a three-dimensional culture system. The co-cultured cells were removed with NH4OH, and ovarian cancer cells were implanted into the preconditioned matrix. VM was identified morphologically and by detecting vascular markers expressed by cancer cells. The specificity of the effects of exogenous HCG in the microenvironment was assessed by inhibition with a neutralizing anti-HCG antibody. HCG siRNA was used to knock down endogenous HCG expression in OVCAR-3 ovarian cancer cells. HTR-8 cells 'fingerprinted' HUVECs to form capillary-like tube structures in co-cultures. In the preconditioned HCG-rich microenvironment, the number of vessel-like network structures formed by HCG receptor-positive OVCAR-3 cells and the expression levels of CD31, VEGF and factor VIII were significantly increased. The preconditioned HCG-rich microenvironment significantly increased the expression of hypoxia inducible factor-1α (HIF‑1α) and VM formation in OVCAR-3 cells under hypoxic conditions. Treatment with a neutralizing anti-HCG antibody but not HCG siRNA significantly inhibited the formation of vessel-like network structures. HCG in the microenvironment contributes to OVCAR-3 differentiation into endothelioid cells in three-dimensional matrices preconditioned with an endothelial-trophoblast cell co-culture system. HCG may synergistically enhance hypoxia-induced vascular markers and HIF-1α expression. These findings would provide perspectives on new therapeutic targets for ovarian cancer.

Protective Effects of Hydrogen Sulfide in Hypoxic Human Umbilical Vein Endothelial Cells: A Possible Mitochondria-Dependent Pathway

PubMed Central

Shen, Yaqi; Guo, Wei; Wang, Zhijun; Zhang, Yuchen; Zhong, Liangjie; Zhu, Yizhun

2013-01-01

The aim of the study was to investigate the protective effects of sodium hydrosulfide (NaHS), a H2S donor, against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs) and also to look into the possible mechanisms by which H2S exerts this protective effect. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and scratch wound healing assay were chosen to measure the cell viability and migration-promoting effects. The fluorescent probe, DCFH-DA and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) were applied to detect the reactive oxygen species (ROS) level and mitochondrial membrane potential (ΔΨm). Furthermore, western blots were used to measure the expressions of the apoptosis-related proteins. Under hypoxic conditions, 300 μM and 600 μM of H2S could protect HUVECs against hypoxia-induced injury, as determined by MTT assay. Following the treatment of 60 μM NaHS for 18 h, scratch wound healing assays indicated that the scratch became much narrower than control group. After treatment with 60 μM, 120 μM, and 600 μM NaHS, and hypoxia for 30 min, flow cytometry demonstrated that the ROS concentrations decreased to 95.08% ± 5.52%, 73.14% ± 3.36%, and 73.51% ± 3.05%, respectively, compared with the control group. In addition, the JC-1 assay showed NaHS had a protective effect on mitochondria damage. Additionally, NaHS increased Bcl-2 expression and decreased the expression of Bax, Caspase-3 and Caspase-9 in a dose-dependent way. Our results suggest that H2S can protect endothelial cells and promote migration under hypoxic condition in HUVECs. These effects are partially associated with the preservation of mitochondrial function mediated by regulating the mitochondrial-dependent apoptotic pathway. PMID:23799362

Suppression of proatherogenic leukocyte interactions by MCS-18--Impact on advanced atherosclerosis in ApoE-deficient mice.

PubMed

Kuehn, Constanze; Tauchi, Miyuki; Stumpf, Christian; Daniel, Christoph; Bäuerle, Tobias; Schwarz, Marc; Kerek, Franz; Steinkasserer, Alexander; Zinser, Elisabeth; Achenbach, Stephan; Dietel, Barbara

2016-02-01

Atherosclerosis is associated with chronic inflammatory responses of the arterial blood vessels. The previously observed protective effect of the MCS-18 substance against the initiation of atherosclerosis in a murine model was explained by its pronounced anti-inflammatory activity. Here, we investigated its impact on murine plaque progression in advanced atherosclerosis and on proatherogenic processes. ApoE-deficient mice were fed a high-fat diet for 12 weeks to induce atherosclerosis, followed by normal chow and intraperitoneal injections of either MCS-18 (500 μg, n = 10) or saline (n = 10) twice a week for another 12 weeks. Plaque size was reduced in MCS-18 treated mice compared to controls (p = 0.001), which was associated with a reduced size of the lipid core (p = 0.01). There was a decrease in apoptotic cells (p = 0.02), endothelial ICAM-1 expression (p < 0.001), and macrophage density (p = 0.01) in the MCS-18 group. In addition, human and murine dendritic cells (DCs) and human umbilical vein endothelial cells (HUVECs) were treated with MCS-18 (50-200 μg/ml) to analyze cell migration and adhesion under flow conditions. MCS-18 reduced human (p = 0.01) and murine (p = 0.006) DC migration. Furthermore, adhesion of MCS-18-treated DCs to a HUVEC monolayer was decreased (p < 0.001). Compared to controls, CD209 (p < 0.001) and CCR7 (p = 0.003) expression was decreased in MCS-18-treated DCs, while in HUVECs lower levels of ICAM-1 (p < 0.001) and of phosphorylated NF-κB-p65 (p = 0.002) were observed. Blocking of ICAM-1 reduced DC adhesion (p < 0.001). MCS-18 exhibits interesting therapeutic effects when applied in advanced murine atherosclerosis. Its antiatherogenic impact might be associated with a suppressed adhesion to the endothelium due to down-regulation of endothelial ICAM-1 expression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Combining 2D angiogenesis and 3D osteosarcoma microtissues to improve vascularization.

PubMed

Chaddad, Hassan; Kuchler-Bopp, Sabine; Fuhrmann, Guy; Gegout, Hervé; Ubeaud-Sequier, Geneviève; Schwinté, Pascale; Bornert, Fabien; Benkirane-Jessel, Nadia; Idoux-Gillet, Ysia

2017-11-15

Angiogenesis is now well known for being involved in tumor progression, aggressiveness, emergence of metastases, and also resistance to cancer therapies. In this study, to better mimic tumor angiogenesis encountered in vivo, we used 3D culture of osteosarcoma cells (MG-63) that we deposited on 2D endothelial cells (HUVEC) grown in monolayer. We report that endothelial cells combined with tumor cells were able to form a well-organized network, and that tubule-like structures corresponding to new vessels infiltrate tumor spheroids. These vessels presented a lumen and expressed specific markers as CD31 and collagen IV. The combination of 2D endothelial cells and 3D microtissues of tumor cells also increased expression of angiogenic factors as VEGF, CXCR4 and ICAM1. The cell environment is the key point to develop tumor vascularization in vitro and to be closer to tumor encountered in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytosolic PhospholipaseA2 Inhibition with PLA-695 Radiosensitizes Tumors in Lung Cancer Animal Models

PubMed Central

Ferraro, Daniel J.; Kotipatruni, Rama P.; Bhave, Sandeep R.; Jaboin, Jerry J.; Hallahan, Dennis E.

2013-01-01

Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted. PMID:23894523

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

Transport and release of colloidal 3-mercaptopropionic acid-coated CdSe–CdS/ZnS core-multishell quantum dots in human umbilical vein endothelial cells

PubMed Central

Fontana, Jacopo M; Yin, Huijuan; Chen, Yun; Florez, Ricardo; Brismar, Hjalmar; Fu, Ying

2017-01-01

Colloidal semiconductor quantum dots (QDs) have been extensively researched and developed for biomedical applications, including drug delivery and biosensing assays. Hence, it is pivotal to understand their behavior in terms of intracellular transport and toxicological effects. In this study, we focused on 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots (3MPA-QDs) converted from the as-grown octadecylamine-coated quantum dots (ODA-QDs) and their direct and dynamic interactions with human umbilical vein endothelial cells (HUVECs). Live cell imaging using confocal fluorescence microscopy showed that 3MPA-QDs first attached to and subsequently aggregated on HUVEC plasma membrane ~25 min after QD deposition. The aggregated QDs started being internalized at ~2 h and reached their highest internalization degree at ~24 h. They were released from HUVECs after ~48 h. During the 48 h period, the HUVECs responded normally to external stimulations, grew, proliferated and wound healed without any perceptible apoptosis. Furthermore, 1) 3MPA-QDs were internalized in newly formed LysoTracker-stained early endosomes; 2) adenosine 5′-triphosphate-induced [Ca2+]i modulation caused a transient decrease in the fluorescence of 3MPA-QDs that were attached to the plasma membrane but a transient increase in the internalized 3MPA-QDs; and 3) fluorescence signal modulations of co-stained LysoTracker and QDs induced by the lysosomotropic agent Gly-Phe-β-naphthylamide were spatially co-localized and temporally synchronized. Our findings suggest that 3MPA-QDs converted from ODA-QDs are a potential nontoxic fluorescent probe for future use in clinical applications. Moreover, the photophysical strategy and techniques reported in this work are easily applicable to study of direct interactions between other nanoparticles and live cells; contributing to awareness and implementation of the safe applications of nanoparticles. PMID:29270011

Transport and release of colloidal 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots in human umbilical vein endothelial cells.

PubMed

Fontana, Jacopo M; Yin, Huijuan; Chen, Yun; Florez, Ricardo; Brismar, Hjalmar; Fu, Ying

2017-01-01

Colloidal semiconductor quantum dots (QDs) have been extensively researched and developed for biomedical applications, including drug delivery and biosensing assays. Hence, it is pivotal to understand their behavior in terms of intracellular transport and toxicological effects. In this study, we focused on 3-mercaptopropionic acid-coated CdSe-CdS/ZnS core-multishell quantum dots (3MPA-QDs) converted from the as-grown octadecylamine-coated quantum dots (ODA-QDs) and their direct and dynamic interactions with human umbilical vein endothelial cells (HUVECs). Live cell imaging using confocal fluorescence microscopy showed that 3MPA-QDs first attached to and subsequently aggregated on HUVEC plasma membrane ~25 min after QD deposition. The aggregated QDs started being internalized at ~2 h and reached their highest internalization degree at ~24 h. They were released from HUVECs after ~48 h. During the 48 h period, the HUVECs responded normally to external stimulations, grew, proliferated and wound healed without any perceptible apoptosis. Furthermore, 1) 3MPA-QDs were internalized in newly formed LysoTracker-stained early endosomes; 2) adenosine 5'-triphosphate-induced [Ca 2+ ] i modulation caused a transient decrease in the fluorescence of 3MPA-QDs that were attached to the plasma membrane but a transient increase in the internalized 3MPA-QDs; and 3) fluorescence signal modulations of co-stained LysoTracker and QDs induced by the lysosomotropic agent Gly-Phe-β-naphthylamide were spatially co-localized and temporally synchronized. Our findings suggest that 3MPA-QDs converted from ODA-QDs are a potential nontoxic fluorescent probe for future use in clinical applications. Moreover, the photophysical strategy and techniques reported in this work are easily applicable to study of direct interactions between other nanoparticles and live cells; contributing to awareness and implementation of the safe applications of nanoparticles.

[Interaction of FABP4 with plasma membrane proteins of endothelial cells].

PubMed

Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

2015-01-01

Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

Flavonoids from artichoke (Cynara scolymus L.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells.

PubMed

Li, Huige; Xia, Ning; Brausch, Isolde; Yao, Ying; Förstermann, Ulrich

2004-09-01

Nitric oxide (NO) produced by endothelial nitric-oxide synthase (eNOS) represents an antithrombotic and anti-atherosclerotic principle in the vasculature. Hence, an enhanced expression of eNOS in response to pharmacological interventions could provide protection against cardiovascular diseases. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVECs), an artichoke leaf extract (ALE) increased the activity of the human eNOS promoter (determined by luciferase reporter gene assay). An organic subfraction from ALE was more potent in this respect than the crude extract, whereas an aqueous subfraction of ALE was without effect. ALE and the organic subfraction thereof also increased eNOS mRNA expression (measured by an RNase protection assay) and eNOS protein expression (determined by Western blot) both in EA.hy 926 cells and in native HUVECs. NO production (measured by NO-ozone chemiluminescence) was increased by both extracts. In organ chamber experiments, ex vivo incubation (18 h) of rat aortic rings with the organic subfraction of ALE enhanced the NO-mediated vasodilator response to acetylcholine, indicating that the up-regulated eNOS remained functional. Caffeoylquinic acids and flavonoids are two major groups of constituents of ALE. Interestingly, the flavonoids luteolin and cynaroside increased eNOS promoter activity and eNOS mRNA expression, whereas the caffeoylquinic acids cynarin and chlorogenic acid were without effect. Thus, in addition to the lipid-lowering and antioxidant properties of artichoke, an increase in eNOS gene transcription may also contribute to its beneficial cardiovascular profile. Artichoke flavonoids are likely to represent the active ingredients mediating eNOS up-regulation.

Vascular Endothelial Growth Factor Augments Arginine Transport and Nitric Oxide Generation via a KDR Receptor Signaling Pathway.

PubMed

Shashar, Moshe; Chernichovski, Tamara; Pasvolsky, Oren; Levi, Sharon; Grupper, Ayelet; Hershkovitz, Rami; Weinstein, Talia; Schwartz, Idit F

2017-01-01

Vascular endothelial growth factor (VEGF) is an endothelium-specific peptide that stimulates angiogenesis via two receptor tyrosine kinases, Flt-1 and KDR. Endothelial nitric oxide synthase (eNOS) plays a major role in VEGF signaling. Delivery of arginine to membrane bound eNOS by the cationic amino acid transporter-1 (CAT-1) has been shown to modulate eNOS activity. The current studies were designed to test the hypothesis that VEGF enhances eNOS activity via modulation of arginine transport by CAT-1. Using radio-labeled arginine, {[3H] L-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with VEGF with and without silencing the VEGF receptors Flt-1 or KDR. Subsequently, western blotting for CAT-1, PKCα, ERK 1/2, JNK, and their phosphorylated forms were performed. NO generation was measured by the Griess reaction. VEGF (50 and 100 ng/ml) significantly augmented endothelial arginine transport in a time dependent manner, an effect which was prevented by Sunitinib (2 µM), a multi targeted receptor tyrosine kinase inhibitor. The increase in arginine transport velocities by VEGF was not affected by silencing Flt-1 while silencing KDR abrogated VEGF effect. Furthermore, incubating cells with 50 and 100 ng of VEGF for 30 minutes significantly augmented CAT-1 abundance. The expression of PKC-α, JNK, and ERK1/2 and their phosphorylated forms were unchanged following incubation of HUVEC with VEGF. The concentration of NO2/NO3 following incubation with VEGF was significantly higher than from untreated cells. This increase was significantly attenuated by silencing KDR. VEGF increases arginine transport via modulation of CAT-1 in endothelial cells. This effect is exclusively dependent on KDR rather than Flt-1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Cholesteryl butyrate solid lipid nanoparticles inhibit adhesion of human neutrophils to endothelial cells

PubMed Central

Dianzani, Chiara; Cavalli, Roberta; Zara, Gian Paolo; Gallicchio, Margherita; Lombardi, Grazia; Gasco, Maria Rosa; Panzanelli, Patrizia; Fantozzi, Roberto

2006-01-01

Adhesion of polymorphonuclear cells (PMNs) to vascular endothelial cells (EC) is a critical step in recruitment and infiltration of leukocytes into tissues during inflammation. High doses of butyric acid have been shown to ameliorate inflammation in inflammatory bowel diseases (IBD). Cholesteryl-butyrate solid lipid nanoparticles (chol-but SLN) as prodrug are a possible delivery system for butyric acid. Sodium butyrate or chol-but SLN were coincubated with human PMNs and human umbilical vein EC (HUVEC); adhesion was quantified by computerized microimaging fluorescence analysis. Both chol-but SLN and sodium butyrate displayed antiadhesive effects on FMLP- and IL-1β-stimulated cells in a concentration–response curve (10−8–10−5 M), but chol-but SLN were in all cases more active. Moreover, chol-but SLN inhibited FMLP-induced adhesion of PMNs to FCS-coated plastic wells, thus showing a direct effect on PMNs, while sodium butyrate had little effect. Confocal microscopy showed that fluorescent SLN entered PMNs and HUVEC after 10 min incubation. Chol-but SLN acted either on activated PMN or HUVEC. Chol-but SLN inhibited O2−· production and myeloperoxidase release by PMNs evoked by FMLP, in a dose-dependent, but not time-dependent, manner and were more active than sodium butyrate. In conclusion, in all tests chol-but SLN were more active than sodium butyrate. Thus, chol-but SLN might be a valid alternative to sodium butyrate in the anti-inflammatory therapy of ulcerative colitis, avoiding complications related to the administration of sodium butyrate. PMID:16702992

Bis-pyridinium quadrupolar derivatives. High Stokes shift selective probes for bio-imaging

NASA Astrophysics Data System (ADS)

Salice, Patrizio; Versari, Silvia; Bradamante, Silvia; Meinardi, Francesco; Macchi, Giorgio; Pagani, Giorgio A.; Beverina, Luca

2013-11-01

We describe the design, synthesis and characterization of five high Stokes shift quadrupolar heteroaryl compounds suitable as fluorescent probes in bio-imaging. In particular, we characterize the photophysical properties and the intracellular localization in Human Umbilical Vein Endothelial Cells (HUVEC) and Human Mesenchymal Stem Cells (HMSCs) for each dye. We show that, amongst all of the investigated derivatives, the 2,5-bis[1-(4-N-methylpyridinium)ethen-2-yl)]- N-methylpyrrole salt is the best candidates as selective mitochondrial tracker. Finally, we recorded the full emission spectrum of the most performing - exclusively mitochondrial selective - fluorescent probe directly from HUVEC stained cells. The emission spectrum collected from the stained mitochondria shows a remarkably more pronounced vibronic structure with respect to the emission of the free fluorophore in solution.

Dual effects of ouabain on the regulation of proliferation and apoptosis in human umbilical vein endothelial cells: involvement of Na(+)-K(+)-ATPase α-subunits and NF-κB.

PubMed

Ren, Yan-Ping; Zhang, Ming-Juan; Zhang, Ting; Huang, Ruo-Wen

2014-01-01

To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB. HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence. 0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment. Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.

The effects of endoplasmic reticulum stress inducer thapsigargin on the toxicity of ZnO or TiO2 nanoparticles to human endothelial cells.

PubMed

Gu, Yuxiu; Cheng, Shanshan; Chen, Gui; Shen, Yuexin; Li, Xiyue; Jiang, Qin; Li, Juan; Cao, Yi

2017-03-01

It was recently shown that ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress in human umbilical vein endothelial cells (HUVECs). If ER stress is associated the toxicity of ZnO NPs, the presence of ER stress inducer thapsigargin (TG) should alter the response of HUVECs to ZnO NP exposure. In this study, we addressed this issue by assessing cytotoxicity, oxidative stress and inflammatory responses in ZnO NP exposed HUVECs with or without the presence of TG. Moreover, TiO 2 NPs were used to compare the effects. Exposure to 32 μg/mL ZnO NPs (p < 0.05), but not TiO 2 NPs (p > 0.05), significantly induced cytotoxicity as assessed by WST-1 and neutral red uptake assay, as well as intracellular ROS. ZnO NPs dose-dependently increased the accumulation of intracellular Zn ions, and ZnSO 4 induced similar cytotoxic effects as ZnO NPs, which indicated a role of Zn ions. The release of inflammatory proteins tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) or the adhesion of THP-1 monocytes to HUVECs was not significantly affected by ZnO or TiO 2 NP exposure (p > 0.05). The presence of 250 nM TG significantly induced cytotoxicity, release of IL-6 and THP-1 monocyte adhesion (p < 0.01), but did not significantly affect intracellular ROS or release of TNFα (p > 0.05). ANOVA analysis indicated no interaction between exposure to ZnO NPs and the presence of TG on almost all the endpoints (p > 0.05) except neutral red uptake assay (p < 0.01). We concluded ER stress is probably not associated with ZnO NP exposure induced oxidative stress and inflammatory responses in HUVECs.

Exogenous hydrogen sulfide protects human umbilical vein endothelial cells against high glucose‑induced injury by inhibiting the necroptosis pathway.

PubMed

Lin, Jiaqiong; Chen, Meiji; Liu, Donghong; Guo, Ruixian; Lin, Kai; Deng, Haiou; Zhi, Ximei; Zhang, Weijie; Feng, Jianqiang; Wu, Wen

2018-03-01

Hyperglycemia is a key factor in the development of diabetic complications, including the processes of atherosclerosis. Receptor‑interacting protein 3 (RIP3), a mediator of necroptosis, is implicated in atherosclerosis development. Additionally, hydrogen sulfide (H2S) protects the vascular endothelium against hyperglycemia‑induced injury and attenuates atherosclerosis. On the basis of these findings, the present study aimed to confirm the hypothesis that necroptosis mediates high glucose (HG)‑induced injury in human umbilical vein endothelial cells (HUVECs), and that the inhibition of necroptosis contributes to the protective effect of exogenous H2S against this injury. The results revealed that exposure of HUVECs to 40 mM HG markedly enhanced the expression level of RIP3, along with multiple injuries, including a decrease in cell viability, an increase in the number of apoptotic cells, an increase in the expression level of cleaved caspase‑3, generation of reactive oxygen species (ROS), as well as dissipation of the mitochondrial membrane potential (MMP). Treatment of the cells with sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to HG significantly attenuated the increased RIP3 expression and the aforementioned injuries by HG. Notably, treatment of cells with necrostatin‑1 (Nec‑1), an inhibitor of necroptosis, prior to exposure to HG ameliorated the HG‑induced injuries, leading to a decrease in ROS generation and a loss of MMP. However, pre‑treatment of the cells with Nec‑1 enhanced the HG‑induced increase in the expression levels of cleaved caspases‑3 and ‑9. By contrast, pre‑treatment with Z‑VAD‑FMK, a pan ‑caspase inhibitor, promoted the increased expression of RIP3 by HG. Taken together, the findings of the present study have demonstrated, to the best of our knowledge for the first time, that exogenous H2S protects HUVECs against HG‑induced injury through inhibiting necroptosis. The present study has also provided novel evidence that there is a negative interaction between necroptosis and apoptosis in the HG‑treated HUVECs.