Science.gov

Sample records for affect endothelial cell

  1. Inhibition of cyclooxygenase (COX)-2 affects endothelial progenitor cell proliferation

    SciTech Connect

    Colleselli, Daniela; Bijuklic, Klaudija; Mosheimer, Birgit A.; Kaehler, Christian M. . E-mail: C.M.Kaehler@uibk.ac.at

    2006-09-10

    Growing evidence indicates that inducible cyclooxygenase-2 (COX-2) is involved in the pathogenesis of inflammatory disorders and various types of cancer. Endothelial progenitor cells recruited from the bone marrow have been shown to be involved in the formation of new vessels in malignancies and discussed for being a key point in tumour progression and metastasis. However, until now, nothing is known about an interaction between COX and endothelial progenitor cells (EPC). Expression of COX-1 and COX-2 was detected by semiquantitative RT-PCR and Western blot. Proliferation kinetics, cell cycle distribution and rate of apoptosis were analysed by MTT test and FACS analysis. Further analyses revealed an implication of Akt phosphorylation and caspase-3 activation. Both COX-1 and COX-2 expression can be found in bone-marrow-derived endothelial progenitor cells in vitro. COX-2 inhibition leads to a significant reduction in proliferation of endothelial progenitor cells by an increase in apoptosis and cell cycle arrest. COX-2 inhibition leads further to an increased cleavage of caspase-3 protein and inversely to inhibition of Akt activation. Highly proliferating endothelial progenitor cells can be targeted by selective COX-2 inhibition in vitro. These results indicate that upcoming therapy strategies in cancer patients targeting COX-2 may be effective in inhibiting tumour vasculogenesis as well as angiogenic processes.

  2. Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality.

    PubMed

    Nakagawa, Kiyotaka; Kiko, Takehiro; Kuriwada, Satoko; Miyazawa, Taiki; Kimura, Fumiko; Miyazawa, Teruo

    2011-01-01

    It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.

  3. 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

  4. Lack of ADAM10 in endothelial cells affects osteoclasts at the chondro-osseus junction.

    PubMed

    Zhao, Ren; Wang, Aimin; Hall, Katherine C; Otero, Miguel; Weskamp, Gisela; Zhao, Baohong; Hill, Daniel; Goldring, Mary B; Glomski, Krzysztof; Blobel, Carl P

    2014-02-01

    Mice lacking ADAM10 in endothelial cells (Adam10ΔEC mice) have shorter femurs, tibiae, and humeri than controls, raising questions about how endothelial cells could control long bone growth. We performed a histopathological evaluation of the femur and tibia growth plates at different postnatal stages, and assessed the distribution of TRAP-positive osteoclasts and endothelial cells at the growth plate. The growth plates in Adam10ΔEC mice appeared normal at P7 and P14, but a thickened zone of hypertrophic chondrocytes and increased trabecular bone density were apparent by P21 and later. The number of TRAP+ cells at the COJ was normal at P7 and P14, but was strongly reduced at P21 and later. Moreover, the density of endomucin-stained endothelial cells at the COJ was increased starting at P7. The defects in long bone growth in Adam10ΔEC mice could be caused by a lack of osteoclastogenesis at the COJ. Moreover, ADAM10 appears to regulate endothelial cell organization in the developing bone vasculature, perhaps in a similar manner as in the developing retinal vascular tree, where ADAM10 is thought to control Notch-dependent endothelial cell fate decisions. This study provides evidence for the regulation of osteoclast function by endothelial cells in vivo.

  5. Polyphenolic composition of grape stem extracts affects antioxidant activity in endothelial and muscle cells.

    PubMed

    Goutzourelas, Nikolaos; Stagos, Dimitrios; Spanidis, Ypatios; Liosi, Maria; Apostolou, Anna; Priftis, Alexandros; Haroutounian, Serko; Spandidos, Demetrios A; Tsatsakis, Aristidis M; Kouretas, Demetrios

    2015-10-01

    The aim of the present study was the assessment of the antioxidant effects of polyphenolic extracts derived from the stems of three Greek grape varieties (Moshomayro, Mavrotragano and Mandilaria) in endothelial (EA.hy926) and muscle (C2C12) cells. We also investigated the effects of the polyphenolic composition on the antioxidant effects of the grape stem extracts. For this purpose, the endothelial and muscle cells were treated with low non-cytotoxic concentrations of the extracts for 24 h in order to assess the effects of the extracts on cellular redox status using oxidative stress biomarkers. The oxidative stress markers were thiobarbituric acid reactive substances (TBARS), protein carbonyl (CARB) levels, reactive oxygen species (ROS) levels and glutathione (GSH) levels. The results revealed that treatment of the EA.hy926 cells with Mandilaria extract significantly decreased the TBARS levels by 14.8% and the CARB levels by 25.9 %, while it increased the GSH levels by 15.8% compared to the controls. Moreover, treatment of the EA.hy926 cells with Mavrotragano extract significantly increased the GSH levels by 20.2%, while it significantly decreased the TBARS and CARB levels by 12.5% and 16.6%, respectively. Treatment of the C2C12 cells with Mandilaria extract significantly decreased the TBARS levels by 47.3 %, the CARB levels by 39.0 % and the ROS levels by 21.8%, while it increased the GSH levels by 22.6% compared to the controls. Moreover, treatment of the C2C12 cells with Mavrotragano significantly decreased the TBARS, CARB and ROS levels by 36.2%, 35.9% and 16.5%, respectively. In conclusion, to the best of our knowledgel, our results demonstrate for the first time that treatment with grape stem extracts at low concentrations improves the redox status of endothelial and muscle cells. Thus, grape stem extracts may be used for developing antioxidant food supplements or biofunctional foods. However, it was also found that the polyphenolic composition of grape stem

  6. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells.

    PubMed

    Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex; Krueger, Katharina; Liu, Ying; Baumunk, Daniel; Zakrzewicz, Andreas; Tepel, Martin

    2012-06-01

    The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.

  7. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells.

    PubMed

    Parkinson, Fiona E; Hacking, Cindy

    2005-07-05

    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  8. Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability.

    PubMed

    Godbey, W T; Wu, K K; Mikos, A G

    2001-03-01

    Poly(ethylenimine) (PEI) was used to transfect the endothelial cell line EA.hy 926, and the secreted levels of three gene products, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand Factor (vWF), were assessed via ELISA. We found that the levels of these gene products in cell supernatants increased by factors up to 16.3 (tPA), 8.3 (PAI-1), or 6.7 (vWF) times the levels recorded for untreated cells, and roughly correlated with the percentage of cells that expressed the reporter plasmid. Transfections carried out using promotorless constructs of the same reporter plasmid also yielded increases in tPA, PAI-1, and vWF to similar extents. Additionally, data regarding cell viability were gathered and found to inversely relate to both the effectiveness of the PEI used for transfection and the secreted levels of the three mentioned products. There appeared to be two distinct types of cell death, resulting from the use of either free PEI (which acts within 2 h) or PEI/DNA complexes (which cause death 7-9 h after transfection). Cells were also transfected by poly(L-lysine) and liposomal carriers, and increases in secreted tPA similar to those seen with PEI-mediated transfection were observed for positively transfected cells. The results of these investigations indicate that non-viral gene delivery can induce a state of endothelial cell dysfunction, and that PEI-mediated transfection can lead to two distinct types of cell death.

  9. Hydrostatic pressure and shear stress affect endothelin-1 and nitric oxide release by endothelial cells in bioreactors.

    PubMed

    Vozzi, Federico; Bianchi, Francesca; Ahluwalia, Arti; Domenici, Claudio

    2014-01-01

    Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation.

  10. [Endothelial cell adhesion molecules].

    PubMed

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  11. TGF-beta and TNF-a affect cell surface proteoglycan and sialic acid expression on vascular endothelial cells.

    PubMed

    Doiron, Amber L; Kirkpatrick, Allison P; Rinker, Kristina D

    2004-01-01

    Atherosclerosis is the formation of plaques in the arterial wall brought about by numerous events including the accumulation of oxidized low density lipoprotein (LDL), stimulation of inflammatory responses, the release of cytokines, and the attachment of monocytes to the arterial wall. Proteoglycans are implicated in many aspects of atherosclerosis including the metabolism of lipoproteins, regulation of cytokine activity, cell adhesion, and modification of the extracellular matrix. Due to their complex role in molecular recognition and cellular adhesion, the glycosaminoglycan (GAG) chains attached to the proteoglycan core and sialic acids on the terminal ends of the glycan chains are of interest. This study investigated the effects of exposure to transforming growth factor-beta 1 (TGF-beta 1) and tumor necrosis factor-a (TNF-a) on the expression of cell surface GAGs and sialic acids on human umbilical vein endothelial cells (HUVECs). Initial results show that TGF-beta 1 affected GAG expression compared to a control condition. Results also show that the combination of TGF-beta 1 and TNF-a affected GAG expression differently than does TGF-beta 1 alone. Additionally, TNF-a decreased the number of sialic acid residues per cell and TGF-beta 1 slightly upregulated sialic acid expression as compared to the control. The combination of the two cytokines showed a larger upward trend in this value. These data indicate that TNF-a and TGF-beta 1 play a role in the expression of GAG chains and sialic acids on the cell surface. Further study may clarify the implications of these findings for atherosclerosis.

  12. Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite.

    PubMed

    Jones, Charles I; Han, Zhaosheng; Presley, Tennille; Varadharaj, Saradhadevi; Zweier, Jay L; Ilangovan, Govindasamy; Alevriadou, B Rita

    2008-07-01

    Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

  13. Endothelial cell micropatterning: Methods, effects, and applications

    PubMed Central

    Anderson, Deirdre E.J.; Hinds, Monica T.

    2012-01-01

    The effects of flow on endothelial cells have been widely examined for the ability of fluid shear stress to alter cell morphology and function; however, the effects of endothelial cell morphology without flow have only recently been observed. An increase in lithographic techniques in cell culture spurred a corresponding increase in research aiming to confine cell morphology. These studies lead to a better understanding of how morphology and cytoskeletal configuration affect the structure and function of the cells. This review examines endothelial cell micropatterning research by exploring both the many alternative methods used to alter endothelial cell morphology and the resulting changes in cellular shape and phenotype. Micropatterning induced changes in endothelial cell proliferation, apoptosis, cytoskeletal organization, mechanical properties, and cell functionality. Finally, the ways these cellular manipulation techniques have been applied to biomedical engineering research, including angiogenesis, cell migration, and tissue engineering, is discussed. PMID:21761242

  14. Factors Affecting the Endothelial Retention of Targeted Microbubbles: Influence of Microbubble Shell Design and Cell Surface Projection of the Endothelial Target Molecule

    PubMed Central

    Khanicheh, Elham; Mitterhuber, Martina; Kinslechner, Katharina; Xu, Lifen; Lindner, Jonathan R.; Kaufmann, Beat A.

    2014-01-01

    Background In biologic systems, the arrest of circulating cells is mediated by adhesion molecules projecting their active binding domain above the cell surface to enhance bond formation and tether strength. Similarly, molecular spacers are used for ligands on particle-based molecular imaging agents. The aim of this study was to evaluate the influence of tether length for targeting ligands on ultrasound molecular imaging agents. Methods Microbubbles bearing biotin at the end of variable-length polyethylene glycol spacer arms (MB2000 and MB3400) were prepared. To assess in vivo attachment efficiency to endothelial counterligands that vary in their distance from the endothelial cell surface, contrast-enhanced ultrasound (CEU) molecular imaging of tumor necrosis factor–α–induced P-selectin (long distance) or intercellular adhesion molecule–2 (short distance) was performed with each agent in murine hind limbs. To assess the influence of the glycocalyx on microbubble attachment, CEU molecular imaging of intercellular adhesion molecule–2 was performed after degradation of the glycocalyx. Results CEU molecular imaging targeted to P-selectin showed signal enhancement above control agent for MB2000 and MB3400, the degree of which was significantly higher for MB3400 compared with MB2000. CEU molecular imaging targeted to intercellular adhesion molecule–2 showed low overall signal for all agents and signal enhancement above control for MB3400 only. Glycocalyx degradation increased signal for MB3400 and MB2000. Conclusions Microbubble targeting to endothelial ligands is influenced by (1) the tether length of the ligand, (2) the degree to which the endothelial target is projected from the cell surface, and (3) the status of the glycocalyx. These considerations are important for designing targeted imaging probes and understanding potential obstacles to molecular imaging. PMID:22266330

  15. 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.

  16. Hypoxia Affects the Structure of Breast Cancer Cell-Derived Matrix to Support Angiogenic Responses of Endothelial Cells.

    PubMed

    Hielscher, Abigail; Qiu, Connie; Porterfield, Josh; Smith, Quinton; Gerecht, Sharon

    2013-01-01

    Hypoxia, a common feature of the tumor environment and participant in tumor progression, is known to alter gene and protein expression of several Extracellular Matrix (ECM) proteins, many of which have roles in angiogenesis. Previously, we reported that ECM deposited from co-cultures of Neonatal Fibroblasts (NuFF) with breast cancer cells, supported 3-dimensional vascular morphogenesis. Here, we sought to characterize the hypoxic ECM and to identify whether the deposited ECM induce angiogenic responses in Endothelial Cells (ECs). NuFF and MDA-MB-231 breast cancer cells were co-cultured, subjected to alternating cycles of 24 hours of 1% (hypoxia) and 21% (atmospheric) oxygen and de-cellularized for analyses of deposited ECM. We report differences in mRNA expression profiles of matrix proteins and crosslinking enzymes relevant to angiogenesis in hypoxia-exposed co-cultures. Interestingly, overt differences in the expression of ECM proteins were not detected in the de-cellularized ECM; however, up-regulation of the cell-binding fragment of fibronecin was observed in the conditioned media of hypoxic co-cultures. Ultrastructure analyses of the de-cellularized ECM revealed differences in fiber morphology with hypoxic fibers more compact and aligned, occupying a greater percent area and having larger diameter fibers than atmospheric ECM. Examining the effect of hypoxic ECM on angiogenic responses of ECs, morphological differences in Capillary-Like Structures (CLS) formed atop de-cellularized hypoxic and atmospheric ECM were not evident. Interestingly, we found that hypoxic ECM regulated the expression of angiogenic factors and matrix metalloproteinases in CLS. Overall, we report that in vitro, hypoxia does not alter the composition of the ECM deposited by co-cultures of NuFF/MDA-MB-231, but rather alters fiber morphology, and induces vascular expression of angiogenic growth factors and metalloproteinases. Taken together, these results have important implications for

  17. Chronic endurance exercise affects paracrine action of CD31+ and CD34+ cells on endothelial tube formation

    PubMed Central

    Landers-Ramos, Rian Q.; Sapp, Ryan M.; Jenkins, Nathan T.; Murphy, Anna E.; Cancre, Lucile; Chin, Eva R.; Spangenburg, Espen E.

    2015-01-01

    We aimed to determine if chronic endurance-exercise habits affected redox status and paracrine function of CD34+ and CD34−/CD31+ circulating angiogenic cells (CACs). Subjects were healthy, nonsmoking men and women aged 18–35 yr and categorized by chronic physical activity habits. Blood was drawn from each subject for isolation and culture of CD34+ and CD34−/CD31+ CACs. No differences in redox status were found in any group across either cell type. Conditioned media (CM) was generated from the cultured CACs and used in an in vitro human umbilical vein endothelial cell-based tube assay. CM from CD34+ cells from inactive individuals resulted in tube structures that were 29% shorter in length (P < 0.05) and 45% less complex (P < 0.05) than the endurance-trained group. CD34−/CD31+ CM from inactive subjects resulted in tube structures that were 26% shorter in length (P < 0.05) and 42% less complex (P < 0.05) than endurance-trained individuals. Proteomics analyses identified S100A8 and S100A9 in the CM. S100A9 levels were 103% higher (P < 0.05) and S100A8 was 97% higher in the CD34−/CD31+ CM of inactive subjects compared with their endurance-trained counterparts with no significant differences in either protein in the CM of CD34+ CACs as a function of training status. Recombinant S100A8/A9 treatment at concentrations detected in inactive subjects' CD34−/CD31+ CAC CM also reduced tube formation (P < 0.05). These findings are the first, to our knowledge, to demonstrate a differential paracrine role in CD34+ and CD34−/CD31+ CACs on tube formation as a function of chronic physical activity habits and identifies a differential secretion of S100A9 by CD34−/CD31+ CACs due to habitual exercise. PMID:26055789

  18. p-Cresol Affects Reactive Oxygen Species Generation, Cell Cycle Arrest, Cytotoxicity and Inflammation/Atherosclerosis-Related Modulators Production in Endothelial Cells and Mononuclear Cells

    PubMed Central

    Chan, Chiu-Po; Yeung, Sin-Yuet; Hsien, Hsiang-Chi; Lin, Bor-Ru; Yeh, Chien-Yang; Tseng, Wan-Yu; Tseng, Shui-Kuan; Jeng, Jiiang-Huei

    2014-01-01

    Aims Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells. Methods EA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2′,7′-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F2α (PGF2α), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA). Results Exposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF2α, and uPA production in EAHY cells. Conclusions p-Cresol may contribute to atherosclerosis and thrombosis in patients with

  19. 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.

  20. Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-{beta}1

    SciTech Connect

    Schmid, Stephan A. . E-mail: leoni.kunz-schughart@oncoray.de; Gaumann, Andreas; Wondrak, Marit; Eckermann, Christoph; Schulte, Stephanie; Mueller-Klieser, Wolfgang; Wheatley, Denys N.; Kunz-Schughart, Leoni A.

    2007-07-15

    When lactate accumulation in a tumor microenvironment reaches an average concentration of 10-20 mM, it tends to reflect a high degree of malignancy. However, the hypothesis that tumor-derived lactate has a number of partially adverse biological effects on malignant and tumor-associated host cells requires further evidence. The present study attempted to evaluate the impact of lactate on the process of angiogenesis, in particular on the formation of tubular structures. The endothelial cell (EC) network in desmoplastic breast tumors is primarily located in areas of reactive fibroblastic stroma. We employed a fibroblast-endothelial cell co-culture model as in vitro angiogenesis system normally producing florid in vitro tubule formation to analyze this situation. In contrast to previous studies, we found that lactate significantly reduces EC network formation in a dose-dependent manner as quantified by semi-automated morphometric analyses following immunohistochemical staining. The decrease in CD31-positive tubular structures and the number of intersections was independent of VEGF supplementation and became more pronounced in the presence of protons. The number of cells, primarily of the fibroblast population, was reduced but cell loss could not be attributed to a decrease in proliferative activity or pronounced apoptotic cell death. Treatment with 10 mM lactate was accompanied by enhanced mRNA expression and release of TGF-{beta}1, which also shows anti-angiogenic activity in the model. Both TGF-{beta}1 and lactate induced myofibroblastic differentiation adjacent to the EC tubular structures. The lactate response on the EC network was diminished by TGF-{beta}1 neutralization, indicating a causal relationship between lactate and TGF-{beta}1 in the finely tuned processes of vessel formation and maturation which may also occur in vivo within tumor tissue.

  1. Stress-Induced Premature Senescence of Endothelial and Endothelial Progenitor Cells

    PubMed Central

    Goligorsky, M.S.; Hirschi, K.

    2016-01-01

    This brief overview of premature senescence of dysfunctional endothelial and endothelial progenitor cells provides information on endothelial cell differentiation and specialization, their ontogeny, and controversies related to endothelial stem and progenitor cells. Stressors responsible for the dysfunction of endothelial and endothelial progenitor cells, as well as cellular mechanisms and consequences of endothelial cell dysfunction are presented. Metabolic signatures of dysfunctional endothelial cells and senescence pathways are described. Emerging strategies to rejuvenate endothelial and endothelial progenitor cells conclude the review. PMID:27451101

  2. Interaction between the Stress Phase Angle (SPA) and the Oscillatory Shear Index (OSI) Affects Endothelial Cell Gene Expression

    PubMed Central

    Amaya, Ronny; Cancel, Limary M.; Tarbell, John M.

    2016-01-01

    Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS) and solid circumferential stress (CS). Due to variations in impedance (global factors) and geometric complexities (local factors) in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle–SPA). Asynchronous flows (SPA close to -180°) that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI) that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between non-reversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics

  3. Interaction between the Stress Phase Angle (SPA) and the Oscillatory Shear Index (OSI) Affects Endothelial Cell Gene Expression.

    PubMed

    Amaya, Ronny; Cancel, Limary M; Tarbell, John M

    2016-01-01

    Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS) and solid circumferential stress (CS). Due to variations in impedance (global factors) and geometric complexities (local factors) in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle-SPA). Asynchronous flows (SPA close to -180°) that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI) that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between non-reversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without

  4. [Endothelial cell apoptosis in erectile dysfunction].

    PubMed

    Jiang, Rui

    2012-10-01

    Erectile dysfunction (ED) is one of the most common male diseases, which seriously affects the patient's quality of life. The risk factors of ED include aging, diabetes, hypertension, hyperlipidemia, and unhealthy lifestyle, and its exact mechanism remains unclear. The apoptosis of endothelial cells in the corpus cavernosum penis may reduce NOS activity, block NO synthesis, and affect penile erection, and the mechanisms of their apoptosis vary with different causes of ED. This article updates the relationship between the apoptosis of endothelial cells and the development of ED.

  5. Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

    PubMed Central

    2013-01-01

    Introduction Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs. Methods Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ3 integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis. Results Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvβ3 integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and αvβ3 integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed

  6. Endothelial-regenerating cells: an expanding universe.

    PubMed

    Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

    2010-03-01

    Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

  7. Exposure to GSM RF fields does not affect calcium homeostasis in human endothelial cells, rat pheocromocytoma cells or rat hippocampal neurons.

    PubMed

    O'Connor, Rodney P; Madison, Steve D; Leveque, Philippe; Roderick, H Llewelyn; Bootman, Martin D

    2010-07-27

    In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an "electromagnetic smog", with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca2+) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012-2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP(3)-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca2+ signal, recovery rates) were analysed to explore potential impact of radiofrequency field

  8. Exposure to GSM RF Fields Does Not Affect Calcium Homeostasis in Human Endothelial Cells, Rat Pheocromocytoma Cells or Rat Hippocampal Neurons

    PubMed Central

    O'Connor, Rodney P.; Madison, Steve D.; Leveque, Philippe; Roderick, H. Llewelyn; Bootman, Martin D.

    2010-01-01

    In the course of modern daily life, individuals are exposed to numerous sources of electromagnetic radiation that are not present in the natural environment. The strength of the electromagnetic fields from sources such as hairdryers, computer display units and other electrical devices is modest. However, in many home and office environments, individuals can experience perpetual exposure to an “electromagnetic smog”, with occasional peaks of relatively high electromagnetic field intensity. This has led to concerns that such radiation can affect health. In particular, emissions from mobile phones or mobile phone masts have been invoked as a potential source of pathological electromagnetic radiation. Previous reports have suggested that cellular calcium (Ca2+) homeostasis is affected by the types of radiofrequency fields emitted by mobile phones. In the present study, we used a high-throughput imaging platform to monitor putative changes in cellular Ca2+ during exposure of cells to 900 MHz GSM fields of differing power (specific absorption rate 0.012–2 W/Kg), thus mimicking the type of radiation emitted by current mobile phone handsets. Data from cells experiencing the 900 Mhz GSM fields were compared with data obtained from paired experiments using continuous wave fields or no field. We employed three cell types (human endothelial cells, PC-12 neuroblastoma and primary hippocampal neurons) that have previously been suggested to be sensitive to radiofrequency fields. Experiments were designed to examine putative effects of radiofrequency fields on resting Ca2+, in addition to Ca2+ signals evoked by an InsP3-generating agonist. Furthermore, we examined putative effects of radiofrequency field exposure on Ca2+ store emptying and store-operated Ca2+ entry following application of the Ca2+ATPase inhibitor thapsigargin. Multiple parameters (e.g., peak amplitude, integrated Ca2+ signal, recovery rates) were analysed to explore potential impact of radiofrequency field

  9. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells

    PubMed Central

    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. PMID:27348615

  10. Origins of circulating endothelial cells and endothelial outgrowth from blood

    PubMed Central

    Lin, Yi; Weisdorf, Daniel J.; Solovey, Anna; Hebbel, Robert P.

    2000-01-01

    Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5–20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after ∼1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only ∼20-fold over this period, whereas donor-genotype endothelial cells expanded ∼1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts. PMID:10619863

  11. Tumor Endothelial Cells

    PubMed Central

    Dudley, Andrew C.

    2012-01-01

    The vascular endothelium is a dynamic cellular “organ” that controls passage of nutrients into tissues, maintains the flow of blood, and regulates the trafficking of leukocytes. In tumors, factors such as hypoxia and chronic growth factor stimulation result in endothelial dysfunction. For example, tumor blood vessels have irregular diameters; they are fragile, leaky, and blood flow is abnormal. There is now good evidence that these abnormalities in the tumor endothelium contribute to tumor growth and metastasis. Thus, determining the biological basis underlying these abnormalities is critical for understanding the pathophysiology of tumor progression and facilitating the design and delivery of effective antiangiogenic therapies. PMID:22393533

  12. A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells

    PubMed Central

    Pagenstecher, Axel; Stahl, Sonja; Sure, Ulrich; Felbor, Ute

    2009-01-01

    Cavernous vascular malformations occur with a frequency of 1:200 and can cause recurrent headaches, seizures and hemorrhagic stroke if located in the brain. Familial cerebral cavernous malformations (CCMs) have been associated with germline mutations in CCM1/KRIT1, CCM2 or CCM3/PDCD10. For each of the three CCM genes, we here show complete localized loss of either CCM1, CCM2 or CCM3 protein expression depending on the inherited mutation. Cavernous but not adjacent normal or reactive endothelial cells of known germline mutation carriers displayed immunohistochemical negativity only for the corresponding CCM protein but not for the two others. In addition to proving loss of function at the protein level, our data are the first to demonstrate endothelial cell mosaicism within cavernous tissues and provide clear pathogenetic evidence that the endothelial cell is the cell of disease origin. PMID:19088124

  13. A two-hit mechanism causes cerebral cavernous malformations: complete inactivation of CCM1, CCM2 or CCM3 in affected endothelial cells.

    PubMed

    Pagenstecher, Axel; Stahl, Sonja; Sure, Ulrich; Felbor, Ute

    2009-03-01

    Cavernous vascular malformations occur with a frequency of 1:200 and can cause recurrent headaches, seizures and hemorrhagic stroke if located in the brain. Familial cerebral cavernous malformations (CCMs) have been associated with germline mutations in CCM1/KRIT1, CCM2 or CCM3/PDCD10. For each of the three CCM genes, we here show complete localized loss of either CCM1, CCM2 or CCM3 protein expression depending on the inherited mutation. Cavernous but not adjacent normal or reactive endothelial cells of known germline mutation carriers displayed immunohistochemical negativity only for the corresponding CCM protein but not for the two others. In addition to proving loss of function at the protein level, our data are the first to demonstrate endothelial cell mosaicism within cavernous tissues and provide clear pathogenetic evidence that the endothelial cell is the cell of disease origin.

  14. Interferon-alpha and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells

    PubMed Central

    Eguchi, K.; Kawakami, A.; Nakashima, M.; Ida, H.; Sakito, S.; Matsuoka, N.; Terada, K.; Sakai, M.; Kawabe, Y.; Fukuda, T.; Ishimaru, T.; Kurouji, K.; Fujita, N.; Aoyagi, T.; Maeda, K.; Nagataki, S.

    1992-01-01

    We investigated whether interferon-gamma (IFN-γ), interferon-alpha (IFN-α) and glucocorticoids affected the adhesion of T cells to human umbilical endothelial cells or human synovial cells. About 30% of peripheral blood T cells could bind to unstimulated endothelial cells, but only a few T cells could bind to unstimulated synovial cells. When both endothelial cells and synovial cells were cultured with recombinant IFN-γ (rIFN-γ), the percentage of T cell binding to both types of cells increased in a dose-dependent manner. rIFN-α and dexamethasone blocked the T cell binding to unstimulated endothelial cells. Furthermore, rIFN-α and dexamethasone suppressed T cell binding to both endothelial cells and synovial cells stimulated by IFN-γ, and also inhibited intercellular adhesion molecule-1 (ICAM-1) expression on both endothelial cells and synovial cells stimulated by IFN-γ. These results suggest that IFN-α and glucocorticoids may inhibit T cell binding to endothelial cells or synovial cells by modulating adhesion molecule expression on these cells. PMID:1606729

  15. Progenitor endothelial cell involvement in Alzheimer's disease

    SciTech Connect

    Budinger, Thomas F.

    2003-05-01

    There is compelling evidence that endothelial cells of the brain and periphery are dysfunctional in Alzheimer's Disease. There is evidence for a fundamental defect in, or abnormal aging of, endothelial progenitor cells in atherosclerosis. The possibility that endothelial cell defects are a primary cause for Alzheimer's Disease or other dementias can be researched by molecular and cell biology studies as well as cell trafficking studies using recently demonstrated molecular imaging methods. The evidence for abnormal endothelial function and the methods to explore this hypothesis are presented.

  16. 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.

  17. Autocrine VEGF Isoforms Differentially Regulate Endothelial Cell Behavior

    PubMed Central

    Yamamoto, Hideki; Rundqvist, Helene; Branco, Cristina; Johnson, Randall S.

    2016-01-01

    Vascular endothelial growth factor A (VEGF) is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration, and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2). We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO) synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell homeostasis in

  18. CXCR4 pos circulating progenitor cells coexpressing monocytic and endothelial markers correlating with fibrotic clinical features are present in the peripheral blood of patients affected by systemic sclerosis.

    PubMed

    Campioni, Diana; Lo Monaco, Andrea; Lanza, Francesco; Moretti, Sabrina; Ferrari, Luisa; Fotinidi, Maria; La Corte, Renato; Cuneo, Antonio; Trotta, Francesco

    2008-08-01

    There is still controversy regarding the role of circulating endothelial and progenitor cells (CECs/CEPs) in the pathogenesis of systemic sclerosis (SSc). Using a sequential Boolean gating strategy based on a 4-color flow cytometric protocol, an increased number of CD31(pos)/CD184(pos)(CXCR4)/CD34(pos)/CD45(pos) and CD31(pos)/CD117(pos) (c-kit-R) /CD34(pos)/ CD45(pos) hematopoietic circulating progenitor cells (HCPCs) was detected in SSc patients compared with healthy subjects. In SSc, no circulating mature and progenitor endothelial cells were observed, while an enhanced generation of erythroid progenitor cells was found to be correlated with the presence of CD117+ HCPCs. The presence of freshly detected CXCR4posHCPC was correlated either to the in vitro cultured spindle-shaped endothelial like cells (SELC) with an endo/myelomonocytic profile or to SDF-1 and VEGF serum level. These data are related to more fibrotic clinical features of the disease, thus supporting a possible role of these cells in fibrosis.

  19. [Transplantation of corneal endothelial cells].

    PubMed

    Amano, Shiro

    2002-12-01

    Though conventional corneal transplantation has achieved great success, it still has several drawbacks including limited availability of donor corneas, recurrent allograft rejection, and subsequent graft failure in certain cases. Reconstructing clinically usable corneas by applying the technology of regenerative medicine can offer a solution to these problems, as well as making corneal transplantation a non-emergency surgery and enabling the usage of banked corneal cells. In the present study, we focused on corneal endothelium that is critical for corneal transparency and investigated the reconstruction of cornea utilizing cultured human corneal endothelial cells (HCECs). We succeeded in steadily culturing HCECs by using culture dishes pre-coated with extracellular matrix produced by calf corneal endothelial cells and culture media that contained basic fibroblast growth factor and fetal bovine serum. We performed the following analysis utilizing these cultured HCECs. The older the donor was, the more frequently large senescent cells appeared in the passaged HCECs. The telomeres of HCECs were measured as terminal restriction fragments (TRF) by Southern blotting. HCECs, in vivo from donors in their seventies had a long TRFs of over 12 kilobases. Passaging shortened the TRFs but there was no difference in TRFs among donors of various ages. These results indicated that shortening of telomere length is not related to senescence of HCECs. We investigated the role of advanced glycation end products (AGEs) in the senescence of in vivo HCECs. The results indicated that AGE-protein in the aqueous humor is endocytosed into HCECs via AGE receptors expressed on the surface of HCECs and damages HCECs by producing reactive oxygen species and inducing apoptosis, suggesting that AGEs, at least partly, cause the senescence of HECEs. HCECs were cultured using adult human serum instead of bovine serum to get rid of bovine material that can be infected with prions. Primary and passage

  20. PPAR Gamma and Angiogenesis: Endothelial Cells Perspective

    PubMed Central

    2016-01-01

    We summarize the current knowledge concerning PPARγ function in angiogenesis. We discuss the mechanisms of action for PPARγ and its role in vasculature development and homeostasis, focusing on endothelial cells, endothelial progenitor cells, and bone marrow-derived proangiogenic cells. PMID:28053991

  1. Ischemia-induced endothelial cell dysfunction.

    PubMed

    Keep, R F; Andjelkovic, A V; Stamatovic, S M; Shakui, P; Ennis, S R

    2005-01-01

    Hemorrhagic transformation upon reperfusion therapy has focused attention on ischemia-induced endothelial dysfunction. This study examined whether hyperglycemia may induce hemorrhagic transformation by enhancing endothelial mitochondrial damage during ischemia and whether preconditioning (PC) stimuli may limit ischemia-induced endothelial damage. In vivo, rats received 2.8 M D-glucose or arabinose (1 ml/100 g; i.p.) prior to undergoing two hours of middle cerebral artery occlusion and transcardiac fixation for electron microscopy. In vitro, brain endothelial cells were exposed to a PC impulse (short-term oxygen glucose deprivation; OGD) prior to an injurious event (5 hours OGD). Endothelial injury was assessed by measuring lactate dehydrogenase release. Hyperglycemia during cerebral ischemia resulted in marked changes in endothelial morphology and mitochondrial swelling. Thus, in the ischemic hemisphere, there was no evidence of endothelial mitochondrial swelling in normoglycemic rats (mean profile width 0.22 +/- 0.04 vs. 0.17 +/- 0.01 microm in contralateral hemisphere) but there was marked swelling in hyperglycemic rats (0.44 +/- 0.02 microm). In vitro, cells preconditioned with one hour of OGD one day prior to 5 hours of OGD, showed reduced lactate dehydrogenase release (p < 0.05). In conclusion, hyperglycemia may have specific adverse effects on endothelial cell mitochondria during ischemia. Preventing those effects may help to ameliorate blood-brain barrier disruption on reperfusion. Insights into how to prevent endothelial injury may come from determining the mechanisms involved in endothelial preconditioning.

  2. Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

    SciTech Connect

    Vigl, Benjamin; Zgraggen, Claudia; Rehman, Nadia; Banziger-Tobler, Nadia E.; Detmar, Michael; Halin, Cornelia

    2009-01-15

    Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.

  3. Fli-1 transcription factor affects glomerulonephritis development by regulating expression of monocyte chemoattractant protein-1 in endothelial cells in the kidney.

    PubMed

    Suzuki, Eiji; Karam, Eva; Williams, Sarah; Watson, Dennis K; Gilkeson, Gary; Zhang, Xian K

    2012-12-01

    Expression of transcription factor Fli-1 is implicated in the development of glomerulonephritis. Fli-1 heterozygous knockout (Fli1(+/-)) NZM2410 mice, a murine model of lupus, had significantly improved survival and reduced glomerulonephritis. In this study, we found that infiltrated inflammatory cells were significantly decreased in the kidneys from Fli-1(+/-) NZM2410 mice. The expression of monocyte chemoattractant protein-1 (MCP-1) was significantly decreased in kidneys from Fli-1(+/-) NZM2410 mice. The primary endothelial cells isolated from the kidneys of Fli-1(+/-) NZM2410 mice produced significantly less MCP-1. In endothelial cells transfected with specific Fli-1 siRNA the production of MCP-1 was significantly reduced compared to cells transfected with negative control siRNA. By Chromatin Immunoprecipitation (ChIP) assay, we further demonstrated that Fli-1 directly binds to the promoter of the MCP-1 gene. Our data indicate that Fli-1 impacts glomerulonephritis development by regulating expression of inflammatory chemokine MCP-1 and inflammatory cell infiltration in the kidneys in the NZM2410 mice.

  4. Apicobasal polarity of brain endothelial cells

    PubMed Central

    Worzfeld, Thomas

    2015-01-01

    Normal brain homeostasis depends on the integrity of the blood–brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood–brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. PMID:26661193

  5. 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.

  6. Blood cells and endothelial barrier function

    PubMed Central

    Rodrigues, Stephen F; Granger, D Neil

    2015-01-01

    Abstract The barrier properties of endothelial cells are critical for the maintenance of water and protein balance between the intravascular and extravascular compartments. An impairment of endothelial barrier function has been implicated in the genesis and/or progression of a variety of pathological conditions, including pulmonary edema, ischemic stroke, neurodegenerative disorders, angioedema, sepsis and cancer. The altered barrier function in these conditions is often linked to the release of soluble mediators from resident cells (e.g., mast cells, macrophages) and/or recruited blood cells. The interaction of the mediators with receptors expressed on the surface of endothelial cells diminishes barrier function either by altering the expression of adhesive proteins in the inter-endothelial junctions, by altering the organization of the cytoskeleton, or both. Reactive oxygen species (ROS), proteolytic enzymes (e.g., matrix metalloproteinase, elastase), oncostatin M, and VEGF are part of a long list of mediators that have been implicated in endothelial barrier failure. In this review, we address the role of blood borne cells, including, neutrophils, lymphocytes, monocytes, and platelets, in the regulation of endothelial barrier function in health and disease. Attention is also devoted to new targets for therapeutic intervention in disease states with morbidity and mortality related to endothelial barrier dysfunction. PMID:25838983

  7. HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE

    PubMed Central

    Gimbrone, Michael A.; Cotran, Ramzi S.; Folkman, Judah

    1974-01-01

    Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration. PMID:4363161

  8. Endothelial progenitor cells in cardiovascular diseases

    PubMed Central

    Lee, Poay Sian Sabrina; Poh, Kian Keong

    2014-01-01

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells (EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vasculogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk factors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardiovascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evaluate the challenges facing EPC research and how these may be overcome. PMID:25126384

  9. Glioma-associated endothelial cells show evidence of replicative senescence

    SciTech Connect

    Charalambous, Christiana; Virrey, Jenilyn; Kardosh, Adel; Jabbour, Mark N.; Qazi-Abdullah, Lubna; Pen, Ligaya; Zidovetzki, Raphael; Schoenthal, Axel H.; Chen, Thomas C.; Hofman, Florence M. . E-mail: hofman@usc.edu

    2007-04-01

    The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated {beta}-galactosidase (SA-{beta}-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-{beta}-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.

  10. Connexin 43 expressed in endothelial cells modulates monocyte‑endothelial adhesion by regulating cell adhesion proteins.

    PubMed

    Yuan, Dongdong; Sun, Guoliang; Zhang, Rui; Luo, Chenfang; Ge, Mian; Luo, Gangjian; Hei, Ziqing

    2015-11-01

    Adhesion between circulating monocytes and vascular endothelial cells is a key initiator of atherosclerosis. In our previous studies, it was demonstrated that the expression of connexin (Cx)43 in monocytes modulates cell adhesion, however, the effects of the expression of Cx43 in endothelial cells remains to be elucidated. Therefore, the present study investigated the role of the expression of Cx43 in endothelial cells in the process of cell adhesion. A total of four different methods with distinct mechanisms were used to change the function and expression of Cx43 channels in human umbilical vein endothelial cells: Cx43 channel inhibitor (oleamide), enhancer (retinoic acid), overexpression of Cx43 by transfection with pcDNA‑Cx43 and knock‑down of the expression of Cx43 by small interfering RNA against Cx43. The results indicated that the upregulation of the expression of Cx43 enhanced monocyte‑endothelial adhesion and this was markedly decreased by downregulation of Cx43. This mechanism was associated with Cx43‑induced expression of vascular cell adhesion molecule‑1 and intercellular cell adhesion molecule‑1. The effects of Cx43 in endothelial cells was independent of Cx37 or Cx40. These experiments suggested that local regulation of endothelial Cx43 expression within the vasculature regulates monocyte‑endothelial adhesion, a critical event in the development of atherosclerosis and other inflammatory pathologies, with baseline adhesion set by the expression of Cx43. This balance may be crucial in controlling leukocyte involvement in inflammatory cascades.

  11. Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens

    PubMed Central

    1987-01-01

    The intracellular events regulating endothelial cell proliferation and organization into formalized capillaries are not known. We report that the protein kinase C activator beta-phorbol 12,13-dibutyrate (PDBu) suppresses bovine capillary endothelial (BCE) cell proliferation (K50 = 6 +/- 4 nM) and DNA synthesis in response to human hepatoma-derived growth factor, an angiogenic endothelial mitogen. In contrast, PDBu has no effect on the proliferation of bovine aortic endothelial cells and is mitogenic for bovine aortic smooth muscle and BALB/c 3T3 cells. Several observations indicate that the inhibition of human hepatoma- derived growth factor-stimulated BCE cell growth by PDBu is mediated through protein kinase C. Different phorbol compounds inhibit BCE cell growth according to their potencies as protein kinase C activators (12- O-tetradecanoylphorbol 13-acetate greater than PDBu much greater than beta-phorbol 12,13-diacetate much much greater than beta-phorbol; alpha- phorbol 12,13-dibutyrate; alpha-phorbol 12,13-didecanoate). PDBu binds to a single class of specific, saturable sites on the BCE cell with an apparent Kd of 8 nM, in agreement with reported affinities of PDBu for protein kinase C in other systems. Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol, a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2- dibutyrylglycerol, does not affect PDBu binding. A cytosolic extract from BCE cells contains a calcium/phosphatidylserine-dependent protein kinase that is activated by sn-1,2-dioctanoylglycerol and PDBu, but not by beta-phorbol. These findings indicate that protein kinase C activation can cause capillary endothelial cells to become desensitized to angiogenic endothelial mitogens. This intracellular regulatory mechanism might be invoked during certain phases of angiogenesis, for example when proliferating endothelial cells become

  12. Glassy Dynamics, Cell Mechanics and Endothelial Permeability

    PubMed Central

    Hardin, Corey; Rajendran, Kavitha; Manomohan, Greeshma; Tambe, Dhananjay T.; Butler, James P.; Fredberg, Jeffrey J.; Martinelli, Roberta; Carman, Christopher V.; Krishnan, Ramaswamy

    2013-01-01

    A key feature of all inflammatory processes is disruption of the vascular endothelial barrier. Such disruption is initiated in part through active contraction of the cytoskeleton of the endothelial cell (EC). Because contractile forces are propagated from cell to cell across a great many cell-cell junctions, this contractile process is strongly cooperative and highly nonlocal. We show here that the characteristic length scale of propagation is modulated by agonists and antagonists that impact permeability of the endothelial barrier. In the presence of agonists including thrombin, histamine, and H202, force correlation length increases, whereas in the presence of antagonists including sphingosine-1-phosphate, hepatocyte growth factor, and the rho kinase inhibitor, Y27632, force correlation length decreases. Intercellular force chains and force clusters are also evident, both of which are reminiscent of soft glassy materials approaching a glass transition. PMID:23638866

  13. Endothelial cell dynamics in vascular remodelling.

    PubMed

    Barbacena, Pedro; Carvalho, Joana R; Franco, Claudio A

    2016-01-01

    In this ESCHM 2016 conference talk report, we summarise two recently published original articles Franco et al. PLoS Biology 2015 and Franco et al. eLIFE 2016. The vascular network undergoes extensive vessel remodelling to become fully functional. Is it well established that blood flow is a main driver for vascular remodelling. It has also been proposed that vessel pruning is a central process within physiological vessel remodelling. However, despite its central function, the cellular and molecular mechanisms regulating vessel regression, and their interaction with blood flow patterns, remain largely unexplained. We investigated the cellular process governing developmental vascular remodelling in mouse and zebrafish. We established that polarised reorganization of endothelial cells is at the core of vessel regression, representing vessel anastomosis in reverse. Moreover, we established for the first time an axial polarity map for all endothelial cells together with an in silico method for the computation of the haemodynamic forces in the murine retinal vasculature. Using network-level analysis and microfluidics, we showed that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/11 renders endothelial cells more sensitive to shear, resulting in axial polarisation at lower shear stress levels. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.

  14. Rapid flow-induced responses in endothelial cells

    NASA Technical Reports Server (NTRS)

    Stamatas, G. N.; McIntire, L. V.

    2001-01-01

    Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

  15. Endothelial cells regulate neural crest and second heart field morphogenesis

    PubMed Central

    Milgrom-Hoffman, Michal; Michailovici, Inbal; Ferrara, Napoleone; Zelzer, Elazar; Tzahor, Eldad

    2014-01-01

    ABSTRACT Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio–craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio–craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio–craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1. PMID:24996922

  16. Endothelial cells regulate neural crest and second heart field morphogenesis.

    PubMed

    Milgrom-Hoffman, Michal; Michailovici, Inbal; Ferrara, Napoleone; Zelzer, Elazar; Tzahor, Eldad

    2014-07-04

    Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio-craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio-craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio-craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

  17. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  18. Mutual interaction between endothelial cells and mural cells enhances BMP9 signaling in endothelial cells.

    PubMed

    Tachida, Yuki; Izumi, Nanae; Sakurai, Toyo; Kobayashi, Hideki

    2017-03-15

    Hereditary hemorrhagic telangiectasia is characterized by the formation of abnormal vascular networks and caused by the mutation of genes involved in BMP9 signaling. It is also known that the interaction between endothelial cells (ECs) and mural cells (MCs) is critical to maintain vessel integrity. However, it has not yet fully been uncovered whether the EC-MC interaction affects BMP9 signaling or not. To elucidate this point, we analyzed BMP9 signaling in a co-culture of several types of human primary culture ECs and MCs. The co-culture activated the Notch pathway in both types of cells in a co-culture- and BMP9-dependent manner. In HUVECs, the genes induced by BMP9 were significantly and synergistically induced in the presence of pericytes, fibroblasts or mesenchymal stem cells. The synergistic induction was greatly reduced in a non-contact condition. In fibroblasts, PDGFRB expression was potently induced in the presence of HUVECs, and BMP9 additively increased this response. Taken together, these results suggest that the EC-MC interaction potentiates BMP9 signaling both in ECs and MCs and plays a critical role in the maintenance of proper vessel functions.

  19. Mutual interaction between endothelial cells and mural cells enhances BMP9 signaling in endothelial cells

    PubMed Central

    Tachida, Yuki; Izumi, Nanae; Sakurai, Toyo

    2017-01-01

    ABSTRACT Hereditary hemorrhagic telangiectasia is characterized by the formation of abnormal vascular networks and caused by the mutation of genes involved in BMP9 signaling. It is also known that the interaction between endothelial cells (ECs) and mural cells (MCs) is critical to maintain vessel integrity. However, it has not yet fully been uncovered whether the EC–MC interaction affects BMP9 signaling or not. To elucidate this point, we analyzed BMP9 signaling in a co-culture of several types of human primary culture ECs and MCs. The co-culture activated the Notch pathway in both types of cells in a co-culture- and BMP9-dependent manner. In HUVECs, the genes induced by BMP9 were significantly and synergistically induced in the presence of pericytes, fibroblasts or mesenchymal stem cells. The synergistic induction was greatly reduced in a non-contact condition. In fibroblasts, PDGFRB expression was potently induced in the presence of HUVECs, and BMP9 additively increased this response. Taken together, these results suggest that the EC–MC interaction potentiates BMP9 signaling both in ECs and MCs and plays a critical role in the maintenance of proper vessel functions. PMID:28298363

  20. Endothelial cells and the IGF system.

    PubMed

    Bach, Leon A

    2015-02-01

    Endothelial cells line blood vessels and modulate vascular tone, thrombosis, inflammatory responses and new vessel formation. They are implicated in many disease processes including atherosclerosis and cancer. IGFs play a significant role in the physiology of endothelial cells by promoting migration, tube formation and production of the vasodilator nitric oxide. These actions are mediated by the IGF1 and IGF2/mannose 6-phosphate receptors and are modulated by a family of high-affinity IGF binding proteins. IGFs also increase the number and function of endothelial progenitor cells, which may contribute to protection from atherosclerosis. IGFs promote angiogenesis, and dysregulation of the IGF system may contribute to this process in cancer and eye diseases including retinopathy of prematurity and diabetic retinopathy. In some situations, IGF deficiency appears to contribute to endothelial dysfunction, whereas IGF may be deleterious in others. These differences may be due to tissue-specific endothelial cell phenotypes or IGFs having distinct roles in different phases of vascular disease. Further studies are therefore required to delineate the therapeutic potential of IGF system modulation in pathogenic processes.

  1. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes.

  2. Proliferation status defines functional properties of endothelial cells.

    PubMed

    Lipps, Christoph; Badar, Muhammad; Butueva, Milada; Dubich, Tatyana; Singh, Vivek Vikram; Rau, Sophie; Weber, Axel; Kracht, Michael; Köster, Mario; May, Tobias; Schulz, Thomas F; Hauser, Hansjörg; Wirth, Dagmar

    2017-04-01

    Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

  3. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema.

    PubMed

    Doyle, Margaret F; Tracy, Russell P; Parikh, Megha A; Hoffman, Eric A; Shimbo, Daichi; Austin, John H M; Smith, Benjamin M; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R Graham

    2017-01-01

    Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50-79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema.

  4. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

    PubMed Central

    Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

    2017-01-01

    Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

  5. Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

    NASA Astrophysics Data System (ADS)

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

    2013-11-01

    Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

  6. Serglycin in Quiescent and Proliferating Primary Endothelial Cells

    PubMed Central

    Reine, Trine M.; Vuong, Tram T.; Rutkovskiy, Arkady; Meen, Astri J.; Vaage, Jarle; Jenssen, Trond G.; Kolset, Svein O.

    2015-01-01

    Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1β induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1β activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development. PMID:26694746

  7. Secreted Endothelial Cell Factors Immobilized on Collagen Scaffolds Enhance the Recipient Endothelial Cell Environment

    PubMed Central

    Hamilton, Charlotte; Callanan, Anthony

    2016-01-01

    Abstract Strategies to design novel vascular scaffolds are a continuing aim in tissue engineering and often such designs encompass the use of recombinant factors to enhance the performance of the scaffold. The established use of cell secretion utilized in feeder systems and conditioned media offer a source of paracrine factors, which has potential to be used in tissue-engineered (TE) scaffolds. Here we utilize this principle from endothelial cells (ECs), to create a novel TE scaffold by harnessing secreted factors and immobilizing these to collagen scaffolds. This research revealed increased cellular attachment and positive angiogenic gene upregulation responses in recipient ECs grown on these conditioned scaffolds. Also, the conditioning method did not affect the mechanical structural integrity of the scaffolds. These results may advocate the potential use of this system to improve vascular scaffolds' in vivo performance. In addition, this process may be a future method utilized to improve other tissue engineering scaffold therapies. PMID:27057474

  8. Pathogenic hantaviruses direct the adherence of quiescent platelets to infected endothelial cells.

    PubMed

    Gavrilovskaya, Irina N; Gorbunova, Elena E; Mackow, Erich R

    2010-05-01

    Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of beta(3) integrins in hemostasis and the inactivation of beta(3) integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell beta(3) integrins and neutralized by the addition of the anti-beta(3) Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and

  9. Acetaminophen protects brain endothelial cells against oxidative stress.

    PubMed

    Tripathy, Debjani; Grammas, Paula

    2009-05-01

    Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. Drugs that affect oxidant and inflammatory stress in the brain are of interest because both processes are thought to contribute to the pathogenesis of neurodegenerative disease. The objective of this study is to determine whether acetaminophen affects the response of brain endothelial cells to oxidative stress. Cultured brain endothelial cells are pre-treated with acetaminophen and then exposed to the superoxide-generating compound menadione (25 microM). Cell survival, inflammatory protein expression, and anti-oxidant enzyme activity are measured. Menadione causes a significant (p<0.001) increase in endothelial cell death as well as an increase in RNA and protein levels of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES. Menadione also evokes a significant (p<0.001) increase in the activity of the anti-oxidant enzyme superoxide dismutase (SOD). Pre-treatment of endothelial cell cultures with acetaminophen (25-100 microM) increases endothelial cell survival and inhibits menadione-induced expression of inflammatory proteins and SOD activity. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2. Suppressing Bcl2 with siRNA blocks the pro-survival effect of acetaminophen. These data show that acetaminophen has anti-oxidant and anti-inflammatory effects on the cerebrovasculature and suggest a heretofore unappreciated therapeutic potential for this drug in neurodegenerative diseases such as Alzheimer's disease that are characterized by oxidant and inflammatory stress.

  10. Regulation of endothelial cell differentiation and specification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The circulatory system is the first organ system to develop in the vertebrate embryo and is critical throughout gestation for the delivery of oxygen and nutrients to, as well as removal of metabolic waste products from, growing tissues. Endothelial cells, which constitute the luminal layer of all bl...

  11. Acidic fibroblast growth factor modulates Staphylococcus aureus adherence to human endothelial cells.

    PubMed Central

    Blumberg, E A; Hatcher, V B; Lowy, F D

    1988-01-01

    Alteration of human endothelial cells may increase their susceptibility to staphylococcal invasion and thus may contribute to the development of intravascular staphylococcal disease. Acidic fibroblast growth factor, a potent regulator of endothelial cell function, had a significant effect on Staphylococcus aureus infection of cultured human endothelial cells. Three of four S. aureus strains had diminished adherence to endothelial cells when the latter were grown in the presence of acidic fibroblast growth factor (P less than 0.05). The diminished adherence was time dependent, maximal at 72 h, and independent of the initial bacterial inoculum. A twofold enhancement of S. aureus adherence was observed when endothelial cells were pretreated with heparitinase. Adherence was unaffected by endothelial cell activation by interleukin-1 or endotoxin. Thus, acidic fibroblast growth factor exerted a protective effect, deterring S. aureus adherence to cultured endothelial cells. Endothelial cell heparan sulfate was also directly involved in the adherence process. Subtle modulations of endothelial cells can significantly affect the ability of S. aureus to adhere to and then infect these cells. Similar alterations may contribute to the ability of S. aureus to infect endovascular tissue in vivo. PMID:3259546

  12. Endothelial cells synthesize and process apolipoprotein B.

    PubMed

    Sivaram, P; Vanni-Reyes, T; Goldberg, I J

    1996-06-21

    We reported previously that a 116-kDa lipoprotein lipase (LPL)-binding protein from endothelial cells has sequence homology to the amino-terminal region of apolipoprotein (apo) B. We now tested whether endothelial cells synthesize apoB mRNA and protein. Primers were designed to the human apoB cDNA sequence and reverse transcription polymerase chain reaction was performed using total RNA isolated from bovine and human endothelial cells. With primers to the 5' region of the apoB mRNA (amino-terminal region of apoB protein) expected size PCR products were generated from both bovine and human endothelial cells as well as from mouse liver RNA, which was used as a control. Primers designed to the 3' region of apoB mRNA generated PCR products from human endothelial cells and HepG2 cells but not from bovine or mouse cells. These data suggest that endothelial cells contain full-length apoB mRNA and that the 5' or the amino-terminal region of apoB is highly conserved from mouse to human. This was confirmed by direct sequencing of the mouse and bovine PCR products. To test whether apoB protein was produced, bovine endothelial cell proteins were metabolically labeled with [35S]methionine/cysteine or [3H]leucine and immunoprecipitated with anti-human apoB antibodies. Using extracts from cells labeled for 1 h, monoclonal antibody 47, directed to the low density lipoprotein receptor binding region of apoB, precipitated a protein of approximate molecular mass 550,000, the size of full-length apoB. Immunoprecipitation of the 550-kDa protein was abolished in the presence of added unlabeled low density lipoprotein. From cells labeled for 16 h, a 116-kDa protein was immunoprecipitated by polyclonal anti-apoB antibodies. This protein was partly released from cells by heparin treatment. Pulse-chase analysis showed that the 116-kDa fragment appeared at the same time as the full-length apoB began disappearing. The immunoprecipitated 116-kDa fragment also bound labeled LPL on ligand blot

  13. Endothelial cell promotion of early liver and pancreas development.

    PubMed

    Freedman, Deborah A; Kashima, Yasushige; Zaret, Kenneth S

    2007-01-01

    Different steps of embryonic pancreas and liver development require inductive signals from endothelial cells. During liver development, interactions between newly specified hepatic endoderm cells and nascent endothelial cells are crucial for the endoderm's subsequent growth and morphogenesis into a liver bud. Reconstitution of endothelial cell stimulation of hepatic cell growth with embryonic tissue explants demonstrated that endothelial signalling occurs independent of the blood supply. During pancreas development, midgut endoderm interactions with aortic endothelial cells induce Ptf1a, a crucial pancreatic determinant. Endothelial cells also have a later effect on pancreas development, by promoting survival of the dorsal mesenchyme, which in turn produces factors supporting pancreatic endoderm. A major goal of our laboratory is to determine the endothelial-derived molecules involved in these inductive events. Our data show that cultured endothelial cells induce Ptf1a in dorsal endoderm explants lacking an endogenous vasculature. We are purifying endothelial cell line product(s) responsible for this effect. We are also identifying endothelial-responsive regulatory elements in genes such as Ptf1a by genetic mapping and chromatin-based assays. These latter approaches will allow us to track endothelial-responsive signal pathways from DNA targets within progenitor cells. The diversity of organogenic steps dependent upon endothelial cell signalling suggests that cross-regulation of tissue development with its vasculature is a general phenomenon.

  14. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  15. Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization.

    PubMed

    Seda, Robinson; Li, David S; Fowlkes, J Brian; Bull, Joseph L

    2015-12-01

    Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.

  16. Characterization of bioeffects on endothelial cells under acoustic droplet vaporization

    PubMed Central

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bul, Joseph L.

    2015-01-01

    Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization (ADV), which result in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events leading to cell injury and cell death. An idealized monolayer of endothelial cells was exposed to ADV using a 3.5 MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional), and pulse lengths that varied from 4 to 16 input cycles. The generated bubble cloud was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application. PMID:26403698

  17. CTC-Endothelial Cell Interactions during Metastasis

    DTIC Science & Technology

    2014-06-01

    study of CTC-Endothelial interactions, as it introduced cell aggregation in the chamber, likely because of the presence of contaminating RBCs in PBMC...interactions, as it introduced cell aggregation in the chamber, likely because of the presence of contaminating RBCs in PBMC preparations, which disturbed the...microvascular endothelium via E- selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the

  18. Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

    PubMed Central

    Mantovani, A.; Introna, M.; Dejana, E.

    1995-01-01

    Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

  19. Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells

    PubMed Central

    Dushpanova, Anar; Agostini, Silvia; Ciofini, Enrica; Cabiati, Manuela; Casieri, Valentina; Matteucci, Marco; Del Ry, Silvia; Clerico, Aldo; Berti, Sergio; Lionetti, Vincenzo

    2016-01-01

    Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction. PMID:27443965

  20. Androgen receptor in human endothelial cells

    PubMed Central

    Torres-Estay, Verónica; Carreño, Daniela V; San Francisco, Ignacio F; Sotomayor, Paula; Godoy, Alejandro S; Smith, Gary J

    2015-01-01

    Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. AR expression was identified in vascular cells nearly 20 years ago, and recent research has shown that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell (HUVEC) homeostatic and pathogenic processes. Although a role for AR in the modulation of HUVEC biology is evident, the molecular mechanisms by which AR regulates HUVEC homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the processes of pathogenesis of diseases ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality. PMID:25563353

  1. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

    PubMed

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

  2. Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.

    PubMed

    Lu, Zhengchun; Sarkar, Sanjay; Zhang, Jianqiang; Balasuriya, Udeni B R

    2016-04-01

    Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation.

  3. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    PubMed

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

  4. The actin cytoskeleton in endothelial cell phenotypes

    PubMed Central

    Prasain, Nutan; Stevens, Troy

    2009-01-01

    Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell’s interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but interrelated structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes. PMID:19028505

  5. Measurements of endothelial cell-to-cell and cell-to-substrate gaps and micromechanical properties of endothelial cells during monocyte adhesion

    PubMed Central

    Kataoka, Noriyuki; Iwaki, Kanso; Hashimoto, Ken; Mochizuki, Seiichi; Ogasawara, Yasuo; Sato, Masaaki; Tsujioka, Katsuhiko; Kajiya, Fumihiko

    2002-01-01

    The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1β-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage. PMID:12434019

  6. Collective cell motion in endothelial monolayers

    PubMed Central

    Szabó, A.; Ünnep, R.; Méhes, E.; Twal, W. O.; Argraves, S. W.; Cao, Y.; Czirók, A.

    2011-01-01

    Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations, and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups, and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior. PMID:21076204

  7. Adhesion of endothelial cells and endothelial progenitor cells on peptide-linked polymers in shear flow.

    PubMed

    Wang, Xin; Cooper, Stuart

    2013-05-01

    The initial adhesion of human umbilical vein endothelial cells (HUVECs), cord blood endothelial colony-forming cells (ECFCs), and human blood outgrowth endothelial cells (HBOECs) was studied under radial flow conditions. The surface of a variable shear-rate device was either coated with polymer films or covered by synthetic fibers. Spin-coating was applied to produce smooth polymer films, while fibrous scaffolds were generated by electrospinning. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate (PEGMA), and CGRGDS peptide. The peptide was incorporated into the polymer system by coupling to an acrylate-PEG-N-hydroxysuccinimide comonomer. A shear-rate-dependent increase of the attached cells with time was observed with all cell types. The adhesion of ECs increased on RGD-linked polymer surfaces compared to polymers without adhesive peptides. The number of attached ECFCs and HBOECs are significantly higher than that of HUVECs within the entire shear-rate range and surfaces examined, especially on RGD-linked polymers at low shear rates. Their superior adhesion ability of endothelial progenitor cells under flow conditions suggests they are a promising source for in vivo seeding of vascular grafts and shows the potential to be used for self-endothelialized implants.

  8. Factors associated with graft survival and endothelial cell density after Descemet's stripping automated endothelial keratoplasty.

    PubMed

    Ishii, Nobuhito; Yamaguchi, Takefumi; Yazu, Hiroyuki; Satake, Yoshiyuki; Yoshida, Akitoshi; Shimazaki, Jun

    2016-04-28

    Postoperative endothelial cell loss leads to graft failure after corneal transplantation, and is one of the important issues for long-term prognosis. The objective of this study was to identify clinical factors affecting graft survival and postoperative endothelial cell density (ECD) after Descemet's stripping automated endothelial keratoplasty (DSAEK). A total of 198 consecutive Japanese patients (225 eyes) who underwent DSAEK were analysed using Cox proportional hazard regression and multiple linear regression models. The candidate factors included recipient age; gender; diagnosis; pre-existing iris damage state, scored based on its severity; the number of previous intraocular surgeries; graft ECD; graft diameter; simultaneous cataract surgery; surgeons experience; intraoperative iris damage; postoperative rebubbling; and graft rejection. Eyes with higher pre-existing iris damage score and more number of previous intraocular surgery had a significantly higher risk of graft failure (HR = 8.53; P < 0.0001, and HR = 2.66; P = 0.026, respectively). Higher pre-existing iris damage score, lower graft ECD, and smaller graft diameter were identified as significant predisposing factors for lower postoperative ECD. The results show that iris damage status before DSAEK may be clinically useful in predicting the postoperative course. Avoiding intraoperative iris damage, especially in eyes with low ECD can change the prognosis of future DSAEK.

  9. Endothelial cell metabolism: parallels and divergences with cancer cell metabolism

    PubMed Central

    2014-01-01

    The stromal vasculature in tumors is a vital conduit of nutrients and oxygen for cancer cells. To date, the vast majority of studies have focused on unraveling the genetic basis of vessel sprouting (also termed angiogenesis). In contrast to the widely studied changes in cancer cell metabolism, insight in the metabolic regulation of angiogenesis is only just emerging. These studies show that metabolic pathways in endothelial cells (ECs) importantly regulate angiogenesis in conjunction with genetic signals. In this review, we will highlight these emerging insights in EC metabolism and discuss them in perspective of cancer cell metabolism. While it is generally assumed that cancer cells have unique metabolic adaptations, not shared by healthy non-transformed cells, we will discuss parallels and highlight differences between endothelial and cancer cell metabolism and consider possible novel therapeutic opportunities arising from targeting both cancer and endothelial cells. PMID:25250177

  10. Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

    PubMed

    Han, Jingyan; Shuvaev, Vladimir V; Muzykantov, Vladimir R

    2011-07-01

    Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of

  11. Circulating endothelial cells: a new biomarker of endothelial dysfunction in hematological diseases.

    PubMed

    Gendron, Nicolas; Smadja, David M

    2016-08-01

    The endothelium and its integrity are in the center of numerous cardiovascular, pulmonary and tumoral diseases. Several studies identified different circulating cellular sub-populations, which allow a noninvasive exploration of endothelial dysfunction. Furthermore, angiogenesis plays a major role in the biology of benign and malignant hematologic diseases. Among these biomarkers, circulating endothelial cells could be considered as a marker of endothelial injury and/or endothelial activation as well as vascular remodeling, whereas circulating endothelial progenitor cells would be only involved in the vascular regeneration. In the future, the quantification of circulating endothelial cells in many diseases could be a noninvasive biomarker used in diagnosis, prognostic and therapeutic follow-up of lung vasculopathy and/or residual disease of hematological malignancies.

  12. Role of endothelial cells in bovine mammary gland health and disease.

    PubMed

    Ryman, Valerie E; Packiriswamy, Nandakumar; Sordillo, Lorraine M

    2015-12-01

    The bovine mammary gland is a dynamic and complex organ composed of various cell types that work together for the purpose of milk synthesis and secretion. A layer of endothelial cells establishes the blood-milk barrier, which exists to facilitate the exchange of solutes and macromolecules necessary for optimal milk production. During bacterial challenge, however, endothelial cells divert some of their lactation function to protect the underlying tissue from damage by initiating inflammation. At the onset of inflammation, endothelial cells tightly regulate the movement of plasma components and leukocytes into affected tissue. Unfortunately, endothelial dysfunction as a result of exacerbated or sustained inflammation can negatively affect both barrier integrity and the health of surrounding extravascular tissue. The objective of this review is to highlight the role of endothelial cells in supporting milk production and regulating optimal inflammatory responses. The consequences of endothelial dysfunction and sustained inflammation on milk synthesis and secretion are discussed. Given the important role of endothelial cells in orchestrating the inflammatory response, a better understanding of endothelial function during mastitis may support development of targeted therapies to protect bovine mammary tissue and mammary endothelium.

  13. Membrane Cholesterol Modulates LOX-1 Shedding in Endothelial Cells.

    PubMed

    Gioia, Magda; Vindigni, Giulia; Testa, Barbara; Raniolo, Sofia; Fasciglione, Giovanni Francesco; Coletta, Massimiliano; Biocca, Silvia

    2015-01-01

    The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor responsible for ox-LDL recognition, binding and internalization, which is up-regulated during atherogenesis. Its activation triggers endothelium dysfunction and induces inflammation. A soluble form of LOX-1 has been identified in the human blood and its presence considered a biomarker of cardiovascular diseases. We recently showed that cholesterol-lowering drugs inhibit ox-LDL binding and internalization, rescuing the ox-LDL induced apoptotic phenotype in primary endothelial cells. Here we have investigated the molecular bases of human LOX-1 shedding by metalloproteinases and the role of cell membrane cholesterol on the regulation of this event by modulating its level with MβCD and statins. We report that membrane cholesterol affects the release of different forms of LOX-1 in cells transiently and stably expressing human LOX-1 and in a human endothelial cell line (EA.hy926). In particular, our data show that i) cholesterol depletion triggers the release of LOX-1 in exosomes as a full-length transmembrane isoform and as a truncated ectodomain soluble fragment (sLOX-1); ii) endothelial cells secrete a soluble metalloproteinase which induces LOX-1 ectodomain shedding and iii) long term statins treatment enhances sLOX-1 proteolytic shedding.

  14. Endothelial cell permeability to water and antipyrine

    SciTech Connect

    Garrick, R.A.

    1986-03-05

    The endothelium provides a structural barrier between plasma constituents and the tissues. The permeability characteristics of the the endothelial cells regulate the transcellular movement of materials across this barrier while other movement is paracellular. In this study the permeability of the endothelial cells to tritiated water (/sup 3/HHO) and /sup 14/C-labeled antipyrine (AP) was investigated. The cells were isolated non-enzymatically from calf pulmonary artery and were maintained in culture and used between the seventh and fifteenth passage. The cells were removed from the T-flasks with a rubber policeman, titurated with a 22g needle and centrifuged. The cells were mixed with an extracellular marker, drawn into polyethylene tubing and packed by centrifugation for use in the linear diffusion technique. All measurements were made at 37 C. The diffusion coefficients for /sup 3/HHO through the packed cells (D), the intracellular material (D/sub 2/), and the extracellular material (D/sub 1/) were 0.682, 0.932 and 2.45 x 10/sup -5/ cm/sup 2/ s/sup -1/ and for AP were 0.273, 0.355 and 1.13 x 10/sup -5/ cm/sup 2/ s/sup -1/ respectively. The permeability coefficient calculated by the series-parallel pathway model for /sup 3/HHO was higher than that for AP and for both /sup 3/HHO and AP were lower than those calculated for isolated lung cells and erythrocytes.

  15. Endothelial progenitor cells: Exploring the pleiotropic effects of statins

    PubMed Central

    Sandhu, Kully; Mamas, Mamas; Butler, Robert

    2017-01-01

    Statins have become a cornerstone of risk modification for ischaemic heart disease patients. A number of studies have shown that they are effective and safe. However studies have observed an early benefit in terms of a reduction in recurrent infarct and or death after a myocardial infarction, prior to any significant change in lipid profile. Therefore, pleiotropic mechanisms, other than lowering lipid profile alone, must account for this effect. One such proposed pleiotropic mechanism is the ability of statins to augment both number and function of endothelial progenitor cells. The ability to augment repair and maintenance of a functioning endothelium may have profound beneficial effect on vascular repair and potentially a positive impact on clinical outcomes in patients with cardiovascular disease. The following literature review will discuss issues surrounding endothelial progenitor cell (EPC) identification, role in vascular repair, factors affecting EPC numbers, the role of statins in current medical practice and their effects on EPC number. PMID:28163831

  16. KRIT1 protein depletion modifies endothelial cell behavior via increased vascular endothelial growth factor (VEGF) signaling.

    PubMed

    DiStefano, Peter V; Kuebel, Julia M; Sarelius, Ingrid H; Glading, Angela J

    2014-11-21

    Disruption of endothelial cell-cell contact is a key event in many cardiovascular diseases and a characteristic of pathologically activated vascular endothelium. The CCM (cerebral cavernous malformation) family of proteins (KRIT1 (Krev-interaction trapped 1), PDCD10, and CCM2) are critical regulators of endothelial cell-cell contact and vascular homeostasis. Here we show novel regulation of vascular endothelial growth factor (VEGF) signaling in KRIT1-depleted endothelial cells. Loss of KRIT1 and PDCD10, but not CCM2, increases nuclear β-catenin signaling and up-regulates VEGF-A protein expression. In KRIT1-depleted cells, increased VEGF-A levels led to increased VEGF receptor 2 (VEGFR2) activation and subsequent alteration of cytoskeletal organization, migration, and barrier function and to in vivo endothelial permeability in KRIT1-deficient animals. VEGFR2 activation also increases β-catenin phosphorylation but is only partially responsible for KRIT1 depletion-dependent disruption of cell-cell contacts. Thus, VEGF signaling contributes to modifying endothelial function in KRIT1-deficient cells and microvessel permeability in Krit1(+/-) mice; however, VEGF signaling is likely not the only contributor to disrupted endothelial cell-cell contacts in the absence of KRIT1.

  17. Cell trafficking of endothelial progenitor cells in tumor progression.

    PubMed

    de la Puente, Pilar; Muz, Barbara; Azab, Feda; Azab, Abdel Kareem

    2013-07-01

    Blood vessel formation plays an essential role in many physiologic and pathologic processes, including normal tissue growth and healing, as well as tumor progression. Endothelial progenitor cells (EPC) are a subtype of stem cells with high proliferative potential that are capable of differentiating into mature endothelial cells, thus contributing to neovascularization in tumors. In response to tumor-secreted cytokines, EPCs mobilize from the bone marrow to the peripheral blood, home to the tumor site, and differentiate to mature endothelial cells and secrete proangiogenic factors to facilitate vascularization of tumors. In this review, we summarize the expression of surface markers, cytokines, receptors, adhesion molecules, proteases, and cell signaling mechanisms involved in the different steps (mobilization, homing, and differentiation) of EPC trafficking from the bone marrow to the tumor site. Understanding the biologic mechanisms of EPC cell trafficking opens a window for new therapeutic targets in cancer.

  18. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    SciTech Connect

    Vallon, Mario; Rohde, Franziska; Janssen, Klaus-Peter; Essler, Markus

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  19. Tumor-Derived Factors and Reduced p53 Promote Endothelial Cell Centrosome Over-Duplication

    PubMed Central

    Yu, Zhixian; Mouillesseaux, Kevin P.; Kushner, Erich J.; Bautch, Victoria L.

    2016-01-01

    Approximately 30% of tumor endothelial cells have over-duplicated (>2) centrosomes, which may contribute to abnormal vessel function and drug resistance. Elevated levels of vascular endothelial growth factor A induce excess centrosomes in endothelial cells, but how other features of the tumor environment affect centrosome over-duplication is not known. To test this, we treated endothelial cells with tumor-derived factors, hypoxia, or reduced p53, and assessed centrosome numbers. We found that hypoxia and elevated levels of bone morphogenetic protein 2, 6 and 7 induced excess centrosomes in endothelial cells through BMPR1A and likely via SMAD signaling. In contrast, inflammatory mediators IL-8 and lipopolysaccharide did not induce excess centrosomes. Finally, down-regulation in endothelial cells of p53, a critical regulator of DNA damage and proliferation, caused centrosome over-duplication. Our findings suggest that some tumor-derived factors and genetic changes in endothelial cells contribute to excess centrosomes in tumor endothelial cells. PMID:27977771

  20. Loss of endothelial programmed cell death 10 activates glioblastoma cells and promotes tumor growth

    PubMed Central

    Zhu, Yuan; Zhao, Kai; Prinz, Anja; Keyvani, Kathy; Lambertz, Nicole; Kreitschmann-Andermahr, Ilonka; Lei, Ting; Sure, Ulrich

    2016-01-01

    Background Neo-angiogenesis is a hallmark of glioblastoma (GBM) and is sustained by autocrine and paracrine interactions between neoplastic and nonneoplastic cells. Programmed cell death 10 (PDCD10) is ubiquitously expressed in nearly all tissues and plays crucial roles in regulating angiogenesis and apoptosis. We recently discovered the absence of PDCD10 expression in the tumor vessels of GBM patients. This raised the hypothesis that loss of endothelial PDCD10 affected GBM cell phenotyping and tumor progression. Methods Endothelial PDCD10 was silenced by siRNA and lentiviral shRNA. The tumor cell phenotype was studied in direct and indirect co-culture of endothelial cells (ECs) with U87 or LN229. Angiogenic protein array was performed in the media of PDCD10-silenced ECs. Tumor angiogenesis and tumor growth were investigated in a human GBM xenograft mouse model. Results Endothelial silence of PDCD10 significantly stimulated tumor cell proliferation, migration, adhesion, and invasion and inhibited apoptosis in co-cultures. Stable knockdown of endothelial PDCD10 increased microvessel density and the formation of a functional vascular network, leading to a 4-fold larger tumor mass in mice. Intriguingly, endothelial deletion of PDCD10 increased (≥2-fold) the release of 20 of 55 tested proangiogenic factors including VEGF, which in turn activated Erk1/2 and Akt in GBM cells. Conclusions For the first time, we provide evidence that loss of endothelial PDCD10 activates GBM cells and promotes tumor growth, most likely via a paracrine mechanism. PDCD10 shows a tumor-suppressor-like function in the cross talk between ECs and tumor cells and is potentially implicated in GBM progression. PMID:26254477

  1. Effects of ultrasound upon endothelial cell ultrastructure

    NASA Astrophysics Data System (ADS)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    A number of new brain applications for therapeutic ultrasound are emerging including drug delivery through BBB opening, enhancement of angiogenesis, sonothrombolysis and neuromodulation. Safety remains important as alterations in the cytoskeleton and tight junctions of endothelial cells have been described. In this study we characterize the in vitro effects of ultrasound on cell morphology using a new human brain cell line (hCMEC/D3). Changes in ultrastructure were analyzed with antibodies against tubulin, actin and catenin. Transport was analyzed by measuring transferrin uptake. No significant changes were seen after continuous wave ultrasound treatment of hCMEC/D3 cells grown in Opticell{trade mark, serif} chambers. We could not observe disassembled actin stress fibers or variations in the microtubule network. However, severe damage occurred in cells cultured in petri dishes.

  2. Production of soluble Neprilysin by endothelial cells

    SciTech Connect

    Kuruppu, Sanjaya; Rajapakse, Niwanthi W.; Minond, Dmitriy; Smith, A. Ian

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  3. Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells

    PubMed Central

    da Silva, Cleide Gonçalves; Specht, Anke; Wegiel, Barbara; Ferran, Christiane; Kaczmarek, Elzbieta

    2009-01-01

    Background Decreased endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production are critical contributors to endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation, however the mechanism of this observation is not fully clarified. Methods and Results To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells (EC) were treated with several nucleotides including, ATP, UTP, and ADP in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2 and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation, and demonstrated that this process was adenosine-independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase C (PKC) delta inhibitor) and PKC delta siRNA. In contrast, blockade of AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent kinase (CaMK) II, CaMK kinase (CaMKK), serine/threonine protein kinase B (Akt), protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK) and p38 mitogen-activated protein kinase (MAPK) did not affect nucleotide-mediated eNOS phosphorylation. Conclusions The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and PKC delta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction. PMID:19188511

  4. Imbalance of mitochondrial-nuclear cross talk in isocyanate mediated pulmonary endothelial cell dysfunction.

    PubMed

    Panwar, Hariom; Jain, Deepika; Khan, Saba; Pathak, Neelam; Raghuram, Gorantla V; Bhargava, Arpit; Banerjee, Smita; Mishra, Pradyumna K

    2013-01-01

    Mechanistic investigations coupled with epidemiology, case-control, cohort and observational studies have increasingly linked isocyanate exposure (both chronic and acute) with pulmonary morbidity and mortality. Though ascribed for impairment in endothelial cell function, molecular mechanisms of these significant adverse pulmonary outcomes remains poorly understood. As preliminary studies conducted in past have failed to demonstrate a cause-effect relationship between isocyanate toxicity and compromised pulmonary endothelial cell function, we hypothesized that direct exposure to isocyanate may disrupt endothelial structural lining, resulting in cellular damage. Based on this premise, we comprehensively evaluated the molecular repercussions of methyl isocyanate (MIC) exposure on human pulmonary arterial endothelial cells (HPAE-26). We examined MIC-induced mitochondrial oxidative stress, pro-inflammatory cytokine response, oxidative DNA damage response and apoptotic index. Our results demonstrate that exposure to MIC, augment mitochondrial reactive oxygen species production, depletion in antioxidant defense enzymes, elevated pro-inflammatory cytokine response and induced endothelial cell apoptosis via affecting the balance of mitochondrial-nuclear cross talk. We herein delineate the first and direct molecular cascade of isocyanate-induced pulmonary endothelial cell dysfunction. The results of our study might portray a connective link between associated respiratory morbidities with isocyanate exposure, and indeed facilitate to discern the exposure-phenotype relationship in observed deficits of pulmonary endothelial cell function. Further, understanding of inter- and intra-cellular signaling pathways involved in isocyanate-induced endothelial damage would not only aid in biomarker identification but also provide potential new avenues to target specific therapeutic interventions.

  5. Endothelial Cell Dynamics during Anastomosis in vitro

    PubMed Central

    Diaz-Santana, Anthony; Shan, Mengrou; Stroock, Abraham D.

    2015-01-01

    Vascular anastomosis –the fusion of vessels from two distinct branches of the vascular system – represents a critical step in vascular growth under both healthy and pathological conditions, in vivo, and presents an important target for engineering of vascularized tissues, in vitro. Recent works in animal models have advanced our understanding of the molecular and cellular players in vascular anastomosis, but questions remain related to cellular dynamics and control of this process, in vitro. In this study, we exploited a three-dimensional (3-D) culture platform to examine the dynamics of endothelial cell (EC) during and after vascular anastomosis by allowing angiogenesis and vasculogenesis to proceed in parallel. We show that anastomosis occurs between sprouts formed by angiogenesis from an endothelium and tubes formed by vasculogenesis in the bulk of a 3-D matrix. This fusion leads to highly connected vessels that span from the surface of the matrix into the bulk in a manner that depends on cell density and identity. Further, we observe and analyze intermixing of endothelial cells of distinct origin (surface versus bulk) within the vessels structures that are formed; we provide evidence that the cells migrate along pre-existing vessels segments as part of this intermixing process. We conclude that anastomosis can occur between vessels emerging by angiogenesis and vasculogenesis and that this process may play an important role in contexts such as wound healing. PMID:25790315

  6. RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

    SciTech Connect

    Rousseau, Matthieu; Gaugler, Marie-Helene; Rodallec, Audrey; Bonnaud, Stephanie; Paris, Francois; Corre, Isabelle

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We explore the role of RhoA in endothelial cell response to ionizing radiation. Black-Right-Pointing-Pointer RhoA is rapidly activated by single high-dose of radiation. Black-Right-Pointing-Pointer Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. Black-Right-Pointing-Pointer Radiation-induced apoptosis does not require the RhoA/ROCK pathway. Black-Right-Pointing-Pointer Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial

  7. Angiogenesis in Platelet Endothelial Cell Adhesion Molecule-1-Null Mice

    PubMed Central

    Cao, Gaoyuan; Fehrenbach, Melane L.; Williams, James T.; Finklestein, Jeffrey M.; Zhu, Jing-Xu; DeLisser, Horace M.

    2009-01-01

    Platelet endothelial cell adhesion molecule (PECAM)-1 has been previously implicated in endothelial cell migration; additionally, anti-PECAM-1 antibodies have been shown to inhibit in vivo angiogenesis. Studies were therefore performed with PECAM-1-null mice to further define the involvement of PECAM-1 in blood vessel formation. Vascularization of subcutaneous Matrigel implants as well as tumor angiogenesis were both inhibited in PECAM-1-null mice. Reciprocal bone marrow transplants that involved both wild-type and PECAM-1-deficient mice revealed that the impaired angiogenic response resulted from a loss of endothelial, but not leukocyte, PECAM-1. In vitro wound migration and single-cell motility by PECAM-1-null endothelial cells were also compromised. In addition, filopodia formation, a feature of motile cells, was inhibited in PECAM-1-null endothelial cells as well as in human endothelial cells treated with either anti-PECAM-1 antibody or PECAM-1 siRNA. Furthermore, the expression of PECAM-1 promoted filopodia formation and increased the protein expression levels of Cdc42, a Rho GTPase that is known to promote the formation of filopodia. In the developing retinal vasculature, numerous, long filamentous filopodia, emanating from endothelial cells at the tips of angiogenic sprouts, were observed in wild-type animals, but to a lesser extent in the PECAM-1-null mice. Together, these data further establish the involvement of endothelial PECAM-1 in angiogenesis and suggest that, in vivo, PECAM-1 may stimulate endothelial cell motility by promoting the formation of filopodia. PMID:19574426

  8. Sarcomere mechanics in capillary endothelial cells.

    PubMed

    Russell, Robert J; Xia, Shen-Ling; Dickinson, Richard B; Lele, Tanmay P

    2009-09-16

    Tension generation in endothelial cells of the aorta, spleen, and eye occurs in actin stress fibers, and is necessary for normal cell function. Sarcomeres are the tension-generating units of actin stress fibers in endothelial cells. How sarcomeres generate and maintain tension in stress fibers is not well understood. Using femtosecond laser ablation, we severed living stress fibers and measured sarcomere contraction under zero tension. The length of the sarcomere decreased in two phases: an instantaneous initial response, followed by a slower change in length attributed to myosin activity. The latter phase ceased abruptly after a minimum sarcomere length was reached, suggesting a rigid resistance that prevents further contraction. Furthermore, severed, contracted stress fibers did not relax when treated with myosin inhibitors, indicating that contracted stress fibers do not store elastic potential energy. These novel measurements combined with modeling suggest that myosin-generated forces in adjacent sarcomeres are directly in balance, and argue against sarcomere models with springlike elements in parallel with myosin contractile elements. We propose a new model for tension generation in the sarcomere, which provides a mechanistic interpretation for our observations and previous observations of inhomogeneous sarcomere contraction and apparent stress fiber viscoelastic behavior.

  9. Fibrinogen Induces Alterations of Endothelial Cell Tight Junction Proteins

    PubMed Central

    PATIBANDLA, PHANI K.; TYAGI, NEETU; DEAN, WILLIAM L.; TYAGI, SURESH C.; ROBERTS, ANDREW M.; LOMINADZE, DAVID

    2009-01-01

    We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. PMID:19507189

  10. Differentiation state determines neural effects on microvascular endothelial cells

    SciTech Connect

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  11. Adult cutaneous hemangiomas are composed of nonreplicating endothelial cells.

    PubMed

    Tuder, R M; Young, R; Karasek, M; Bensch, K

    1987-12-01

    Thirty-four human "cherry" dermal hemangiomas were studied by electron microscopy, immunohistochemistry, and cell culture to assess the neoplastic nature of these lesions. Electron microscopy of nine hemangiomas revealed a pronounced thickening of the basement membrane (0.6 to 14 micron) in 93% of the total 158 vascular structures examined within the lesions. This increase was caused mainly by multiple layers of basal lamina, which were irregular in outline and frequently associated with pericytes. Basement membrane changes were present both in the periphery of the hemangiomas, as well as in the center of the lesions. Immature vessels could not be identified and mitoses were absent in all endothelial cells. Using an immunohistochemical marker (Ki67) specific for proliferating cells in G2 and S phases, positive staining was not found in the endothelial cells lining the hemangiomatous vessels, whereas basal epidermal keratinocytes in the same preparations and cultured microvascular endothelial cells expressed the antigen. Endothelial cells of nine hemangiomas did not stain with an activation-related antibody (E12) specific for endothelial cells. When endothelial cells from 14 hemangiomas were isolated and cultured under conditions that support the growth of normal human skin microvascular endothelial cells, the cells of hemangiomatous origin failed to grow. We conclude that the adult hemangiomas may not be true neoplasms, but a tissue overgrowth composed of mature vessels resembling dermal venules, lined by endothelial cells with virtually no turnover.

  12. The in vitro interaction of Sporothrix schenckii with human endothelial cells is modulated by cytokines and involves endothelial surface molecules.

    PubMed

    Figueiredo, Camila Castro; De Lima, Osana Cunha; De Carvalho, Laís; Lopes-Bezerra, Leila Maria; Morandi, Verônica

    2004-04-01

    Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis that can evolve to systemic complications in immunocompromised patients. Interactions with endothelium are thought to be essential for systemic infections. In the present work, we studied the interaction between S. schenckii and human umbilical vein endothelial cells (HUVECs). S. schenckii interacts with HUVECs in a time-dependent manner. Morphological analysis showed that yeasts locate to interendothelial junctions. Ultrastructural studies showed that internalized yeasts were found inside endocytic vacuoles as early as 2 h, without causing any detectable damage to HUVECs after 24 h of infection. The viability of infected HUVECs was confirmed by the MTT assay. When HUVECs were treated with different concentrations of Interleukin-1beta or transforming growth factor-beta, a significant dose-dependent increase in cell-associated yeasts was observed. The preliminary analysis of the endothelial surface ligands for S. schenckii cells revealed two major molecules, with Mr of approximately 90 and 135 kDa. The interaction of endothelial cell surface molecules with S. schenckii yeast cells was modulated by divalent cations. This is the first demonstration that S. schenckii is able to adhere and invade endothelial cells without significantly affect cellular integrity. Our results suggest the contribution of cytokine-modulated calcium-dependent molecules to this process.

  13. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    PubMed

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  14. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  15. Method for in vitro differentiation of bone marrow mesenchymal stem cells into endothelial progenitor cells and vascular endothelial cells

    PubMed Central

    Wang, Qihong; Zhang, Weifeng; He, Guifen; Sha, Huifang; Quan, Zhe

    2016-01-01

    Vascular development is a regulated process and is dependent on the participation and differentiation of many cell types including the proliferation and migration of vascular endothelial cells and differentiation of endothelial progenitor cells (EPCs) to mesodermal precursor cells. Thus, reconstitution of this process in vitro necessitates providing ambient conditions for generating and culturing EPCs in vitro and differentiating them to vascular endothelial cells. In the present study, we developed methods to differentiate bone marrow mesenchymal stem cells (MSC) into EPCs and to vascular endothelial cells. Bone marrow MSC from canines and human sources were differentiated in vitro in to EPCs. These EPCs were able to express a variety of endothelial markers following 7 days in culture. Further culturing led to the appearance of an increased number and proportion of endothelial cells. These cells were stable even after 30 generations in culture. There was a gradual loss of CD31 and increased expression of factor VIII, VEGFR and CD133. VEGF being highly angiogenic, helps in the vascular development. These results provide the basis for the possible development of vasculature in vitro conditions for biomedical applications and in vivo for organ/tissue reconstruction therapies. PMID:27878275

  16. Purification of endothelial cells from rodent brain by immunopanning.

    PubMed

    Zhou, Lu; Sohet, Fabien; Daneman, Richard

    2014-01-01

    This protocol describes the use of immunopanning to purify endothelial cells from the rodent brain. Immunopanning permits the prospective isolation of endothelial cells from nervous tissue by relying on the binding of the endothelial cells to an anti-CD31 antibody adhered to a Petri dish. The cells are viable at the end of this gentle procedure, and they can be analyzed acutely for gene expression or cultured alone or in coculture with other central nervous system (CNS) cell types, including CNS pericytes and CNS astrocytes. This procedure can be used to isolate endothelial cells from either rat or mouse. We have suggested specific antibodies that work for each species. Note that endothelial cells from rats and mice have different morphologies; in general, rat CNS endothelial cells are longer and thinner than mouse CNS endothelial cells. This procedure can also be used to purify endothelial cells from different regions of the CNS, including brain and optic nerve. Dissociation procedures must be optimized for each tissue.

  17. Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells

    PubMed Central

    Suschek, Christoph; Kolb, Hubert; Kolb-Bachofen, Victoria

    1997-01-01

    Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.In each of the different endothelial cells Mg-Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor NG-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT–PCR. PMID:9421302

  18. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells

    PubMed Central

    Yang, Guanghua; Kramer, M. Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P.; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-01-01

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties. PMID:26612671

  19. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    PubMed

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  20. Effects of surface viscoelasticity on cellular responses of endothelial cells

    PubMed Central

    Hosseini, Motahare-Sadat; Katbab, Ali Asghar

    2014-01-01

    Background: One area of nanoscience deals with nanoscopic interactions between nanostructured materials and biological systems. To elucidate the effects of the substrate surface morphology and viscoelasticity on cell proliferation, fractal analysis was performed on endothelial cells cultured on nanocomposite samples based on silicone rubber (SR) and various concentrations of organomodified nanoclay (OC). Methods: The nanoclay/SR ratio was tailored to enhance cell behavior via changes in sample substrate surface roughness and viscoelasticity. Results: Surface roughness of the cured SR filled with negatively-charged nanosilicate layers had a greater effect than elasticity on cell growth. The surface roughness of SR nanocomposite samples increased with increasing the OC content, leading to enhanced cell growth and extracellular matrix (ECM) remodeling. This was consistent with the decrease in SR segmental motions and damping factor as the primary viscoelastic parameters by the nanosilicate layers with increasing clay concentrations. Conclusions: The inclusion of clay nanolayers affected the growth and behavior of endothelial cells on microtextured SR. PMID:26989733

  1. Hypotonic shock stimulates ascorbate release from coronary artery endothelial cells by a Ca2+ -independent pathway.

    PubMed

    Davis, Kim A; Samson, Sue E; Wilson, John X; Grover, Ashok K

    2006-10-24

    In endothelial cells, anion channels open upon osmotic swelling during shear stress and hypotonic shock. Therefore, we examined the effects of hypotonic shock on release of the antioxidant anion ascorbate from pig coronary artery endothelial cells. Hypotonic shock potentiated ascorbate release from freshly isolated or cultured pig coronary artery endothelial cells; subsequently cultured endothelial cells were used. The hypotonic shock-induced increase in Asc release was rapid, depended on the degree of hypotonic shock, and not due to membrane leakiness. Stimulating P2Y2 like receptors in endothelial cells with ATP causes ascorbate release via a Ca2+ -mediated pathway. Hypotonic shock-induced release differed from the Ca2+-mediated Asc release because: (a) the increase in release with hypotonic shock was additive to that with ATP or A23187 (Ca2+ -ionophore), (b) apyrase, suramin or removing extracellular Ca2+ did not affect the hypotonic shock-stimulated release, (c) anion channel blockers inhibited the release by the two pathways differently, and (d) hypotonic shock increased the ascorbate release from endothelial cells and cultured smooth muscle cells whereas the Ca2+ -mediated ascorbate release occurred only in endothelial cells. Accumulation of ascorbate by endothelial cells was examined at extracellular ascorbate concentrations of 10 (Na+ -ascorbate symporter not saturated) and 5000 microM (Na+ -ascorbate symporter saturated). Hypotonic shock and A23187 decreased ascorbate accumulation at 10 microM ascorbate but increased it at 5000 microM. The effects of the two treatments were additive and also differed from each other with substitution of gluconate for extracellular chloride. Thus, ascorbate release from endothelial cells can be potentiated by two distinct pathways - hypotonic shock mediated and ATP/Ca2+ stimulated.

  2. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes.

    PubMed

    Maezawa, Yoshiro; Takemoto, Minoru; Yokote, Koutaro

    2015-01-01

    Diabetic nephropathy is the major cause of end-stage renal failure throughout the world in both developed and developing countries. Diabetes affects all cell types of the kidney, including endothelial cells, tubulointerstitial cells, podocytes and mesangial cells. During the past decade, the importance of podocyte injury in the formation and progression of diabetic nephropathy has been established and emphasized. However, recent findings provide additional perspectives on pathogenesis of diabetic nephropathy. Glomerular endothelial damage is already present in the normoalbuminuric stage of the disease when podocyte injury starts. Genetic targeting of mice that cause endothelial injury leads to accelerated diabetic nephropathy. Tubulointerstitial damage, previously considered to be a secondary effect of glomerular protein leakage, was shown to have a primary significance in the progression of diabetic nephropathy. Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment is a composite, dynamic entity where any injury of one cell type spreads to other cell types, and leads to the dysfunction of the whole apparatus. Accumulation of novel knowledge would provide a better understanding of the pathogenesis of diabetic nephropathy, and might lead to a development of a new therapeutic strategy for the disease.

  3. Cell biology of diabetic nephropathy: Roles of endothelial cells, tubulointerstitial cells and podocytes

    PubMed Central

    Maezawa, Yoshiro; Takemoto, Minoru; Yokote, Koutaro

    2015-01-01

    Diabetic nephropathy is the major cause of end-stage renal failure throughout the world in both developed and developing countries. Diabetes affects all cell types of the kidney, including endothelial cells, tubulointerstitial cells, podocytes and mesangial cells. During the past decade, the importance of podocyte injury in the formation and progression of diabetic nephropathy has been established and emphasized. However, recent findings provide additional perspectives on pathogenesis of diabetic nephropathy. Glomerular endothelial damage is already present in the normoalbuminuric stage of the disease when podocyte injury starts. Genetic targeting of mice that cause endothelial injury leads to accelerated diabetic nephropathy. Tubulointerstitial damage, previously considered to be a secondary effect of glomerular protein leakage, was shown to have a primary significance in the progression of diabetic nephropathy. Emerging evidence suggests that the glomerular filtration barrier and tubulointerstitial compartment is a composite, dynamic entity where any injury of one cell type spreads to other cell types, and leads to the dysfunction of the whole apparatus. Accumulation of novel knowledge would provide a better understanding of the pathogenesis of diabetic nephropathy, and might lead to a development of a new therapeutic strategy for the disease. PMID:25621126

  4. The coffee diterpene kahweol inhibits tumor necrosis factor-{alpha}-induced expression of cell adhesion molecules in human endothelial cells

    SciTech Connect

    Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang . E-mail: hgjeong@chosun.ac.kr

    2006-12-15

    Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNF{alpha}-induced monocytes to endothelial cells and suppressed the TNF{alpha}-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNF{alpha}-induced JAK2-PI3K/Akt-NF-{kappa}B activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells.

  5. Effects of Escherichia coli hemolysin on endothelial cell function.

    PubMed Central

    Suttorp, N; Flöer, B; Schnittler, H; Seeger, W; Bhakdi, S

    1990-01-01

    Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin. Images PMID:2121650

  6. Anti-endothelial cell antibodies in patients with Wegener's granulomatosis and micropolyarteritis.

    PubMed Central

    Ferraro, G; Meroni, P L; Tincani, A; Sinico, A; Barcellini, W; Radice, A; Gregorini, G; Froldi, M; Borghi, M O; Balestrieri, G

    1990-01-01

    Anti-endothelial cell antibodies (AECA) have been detected by cell surface radioimmunoassay in nine out of 15 patients with micropolyarteritis (MPA) and in two out of five patients with Wegener's granulomatosis. AECA mostly belonged to the IgG isotype and were present in the active phase of the diseases. These antibodies were not detectable in 10 sera from patients with essential mixed cryoglobulinaemia, suggesting that they were not a mere epiphenomenon consequent to the inflammatory vascular injury. The binding activity was not related to ABH antigens or to HLA class I antigens displayed by resting human endothelial cells in culture and was not influenced by removing immune complexes. Absorption of the anti-neutrophil cytoplasmic antibodies (ANCA), present in MPA and Wegener's granulomatosis sera, did not affect the endothelial binding. AECA-positive sera did not display lytic activity against endothelial cells, neither alone nor after addition of fresh complement or normal human peripheral blood mononuclear cells. Although AECA are not cytolytic for endothelial cell monolayers in vitro, the reactivity against intact endothelial cells suggests their possible involvement in in vivo pathological processes affecting vascular structures in small vessel primary vasculitides. PMID:2302834

  7. Lipopolysaccharide induces a fibrotic-like phenotype in endothelial cells.

    PubMed

    Echeverría, César; Montorfano, Ignacio; Sarmiento, Daniela; Becerra, Alvaro; Nuñez-Villena, Felipe; Figueroa, Xavier F; Cabello-Verrugio, Claudio; Elorza, Alvaro A; Riedel, Claudia; Simon, Felipe

    2013-06-01

    Endothelial dysfunction is crucial in endotoxaemia-derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS-resistant ECs exhibit a fibroblast-like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor-like kinase 5 (ALK5) activity-dependent mechanism. LPS-challenged ECs showed an up-regulation of both fibroblast-specific protein expression and extracellular matrix proteins secretion, as well as a down-regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5-dependent mechanism. It is noteworthy that LPS-induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia-derived sepsis syndrome and other inflammatory diseases.

  8. Transcriptional targeting of tumor endothelial cells for gene therapy

    PubMed Central

    Dong, Zhihong; Nör, Jacques E.

    2009-01-01

    It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy. PMID:19393703

  9. Ionizing Radiation-Induced Endothelial Cell Senescence and Cardiovascular Diseases

    PubMed Central

    Wang, Yingying; Boerma, Marjan; Zhou, Daohong

    2016-01-01

    Exposure to ionizing radiation induces not only apoptosis but also senescence. While the role of endothelial cell apoptosis in mediating radiation-induced acute tissue injury has been extensively studied, little is known about the role of endothelial cell senescence in the pathogenesis of radiation-induced late effects. Senescent endothelial cells exhibit decreased production of nitric oxide and expression of thrombomodulin, increased expression of adhesion molecules, elevated production of reactive oxygen species and inflammatory cytokines and an inability to proliferate and form capillary-like structures in vitro. These findings suggest that endothelial cell senescence can lead to endothelial dysfunction by dysregulation of vasodilation and hemostasis, induction of oxidative stress and inflammation and inhibition of angiogenesis, which can potentially contribute to radiation-induced late effects such as cardiovascular diseases (CVDs). In this article, we discuss the mechanisms by which radiation induces endothelial cell senescence, the roles of endothelial cell senescence in radiation-induced CVDs and potential strategies to prevent, mitigate and treat radiation-induced CVDs by targeting senescent endothelial cells. PMID:27387862

  10. Unidirectional transfer of prostaglandin endoperoxides between platelets and endothelial cells.

    PubMed Central

    Schafer, A I; Crawford, D D; Gimbrone, M A

    1984-01-01

    An important determinant of platelet-vessel wall interactions is the local balance of production of endothelial prostacyclin (PGI2) and platelet thromboxane (TX) A2, labile eicosanoids with opposing effects on hemostasis. Disputed evidence suggests that platelet-derived prostaglandin endoperoxide intermediates may be utilized as substrates for vascular PGI2 synthesis. Using several different approaches, we have found that platelets can transfer endoperoxides to cultured endothelial cells for efficient conversion to PGI2, but a reciprocal transfer of endothelial endoperoxides for utilization by platelet thromboxane synthetase does not occur under the same experimental conditions. However, platelets can utilize arachidonic acid released by endothelial cells for lipoxygenase metabolism. We have directly demonstrated the production of [3H]6-keto-PGF1 alpha (the breakdown product of [3H]PGI2) by aspirin-treated endothelial cells in the presence of platelets stimulated with [3H]arachidonic acid. In coincubation experiments using either arachidonate or ionophore A23187 as a stimulus, radioimmunoassay of the net production of arachidonic acid metabolites showed that 6-keto-PGF1 alpha generation by aspirin-treated endothelial cells in the presence of platelets may actually exceed its generation by uninhibited endothelial cells alone. In functional assays, platelet aggregation was inhibited in the presence of aspirin-treated endothelial cells after stimulation with either arachidonate or ionophore A23187. In contrast, the inverse experiments, using aspirin-treated platelets and uninhibited endothelial cells, failed to demonstrate platelet utilization of endothelial endoperoxides for TXA2 production by any of the above methods. These studies thus provide evidence that efficient unidirectional transfer and utilization of platelet-derived endoperoxides for endothelial PGI2 production can occur. This process may serve to amplify PGI2 generation adjacent to areas of vascular

  11. Actin Filament Stress Fibers in Vascular Endothelial Cells in vivo

    NASA Astrophysics Data System (ADS)

    Wong, Albert J.; Pollard, Thomas D.; Herman, Ira M.

    1983-02-01

    Fluorescence microscopy with 7-nitrobenz-2-oxa-3-diazole phallacidin was used to survey vertebrate tissues for actin filament bundles comparable to the stress fibers of cultured cells. Such bundles were found only in vascular endothelial cells. Like the stress fibers of cultured cells, these actin filament bundles were stained in a punctate pattern by fluorescent antibodies to both alpha-actinin and myosin. The stress fibers were oriented parallel to the direction of blood flow and were prominent in endothelial cells from regions exposed to high-velocity flow, such as the left ventricle, aortic valve, and aorta. Actin bundles may help the endothelial cell to withstand hemodynamic stress.

  12. Endothelial Cell Density to Predict Endothelial Graft Failure After Penetrating Keratoplasty

    PubMed Central

    Lass, Jonathan H.; Sugar, Alan; Benetz, Beth Ann; Beck, Roy W.; Dontchev, Mariya; Gal, Robin L.; Kollman, Craig; Gross, Robert; Heck, Ellen; Holland, Edward J.; Mannis, Mark J.; Raber, Irving; Stark, Walter; Stulting, R. Doyle

    2010-01-01

    Objective To determine whether preoperative and/or postoperative central endothelial cell density (ECD) and its rate of decline postoperatively are predictive of graft failure caused by endothelial decompensation following penetrating keratoplasty to treat a moderate-risk condition, principally, Fuchs dystrophy or pseudophakic corneal edema. Methods In a subset of Cornea Donor Study participants, a central reading center determined preoperative and postoperative ECD from available specular images for 17 grafts that failed because of endothelial decompensation and 483 grafts that did not fail. Results Preoperative ECD was not predictive of graft failure caused by endothelial decompensation (P = .91). However, the 6-month ECD was predictive of subsequent failure (P < .001). Among those that had not failed within the first 6 months, the 5-year cumulative incidence (±95% confidence interval) of failure was 13% (±12%) for the 33 participants with a 6-month ECD of less than 1700 cells/mm2 vs 2%(±3%) for the 137 participants with a 6-monthECDof 2500 cells/mm2 or higher. After 5 years’ follow-up, 40 of 277 participants (14%) with a clear graft had an ECD below 500 cells/mm2. Conclusions Preoperative ECD is unrelated to graft failure from endothelial decompensation, whereas there is a strong correlation of ECD at 6 months with graft failure from endothelial decompensation. A graft can remain clear after 5 years even when the ECD is below 500 cells/mm2. PMID:20065219

  13. Isolation and Characterization of Circulating Lymphatic Endothelial Colony Forming Cells

    PubMed Central

    DiMaio, Terri A.; Wentz, Breanna L.; Lagunoff, Michael

    2016-01-01

    Rationale The identification of circulating endothelial progenitor cells has led to speculation regarding their origin as well as their contribution to neovascular development. Two distinct types of endothelium make up the blood and lymphatic vessel system. However, it has yet to be determined whether there are distinct lymphatic-specific circulating endothelial progenitor cells. Objective This study aims to isolate and characterize the cellular properties and global gene expression of lymphatic-specific endothelial progenitor cells. Methods and Results We isolated circulating endothelial colony forming cells (ECFCs) from whole peripheral blood. These cells are endothelial in nature, as defined by their expression of endothelial markers and their ability to undergo capillary morphogenesis in three-dimensional culture. A subset of isolated colonies express markers of lymphatic endothelium, including VEGFR-3 and Prox-1, with low levels of VEGFR-1, a blood endothelial marker, while the bulk of the isolated cells express high VEGFR-1 levels with low VEGFR-3 and Prox-1 expression. The different isolates have differential responses to VEGF-C, a lymphatic endothelial specific cytokine, strongly suggesting that there are lymphatic specific and blood specific ECFCs. Global analysis of gene expression revealed key differences in the regulation of pathways involved in cellular differentiation between blood and lymphatic-specific ECFCs. Conclusion These data indicate that there are two distinguishable circulating ECFC types, blood and lymphatic, which are likely to have discrete functions during neovascularization. PMID:26597759

  14. Regulation of indoleamine 2,3-dioxygenase in primary human saphenous vein endothelial cells

    PubMed Central

    Mouratidis, Petros XE; George, Andrew JT

    2015-01-01

    Background Indoleamine 2,3-dioxygenase (IDO) is an enzyme associated with the regulation of immune responses. Cytokines such as IFNγ induce its expression in endothelial cells originating from immune-privileged sites. In this study, we investigate regulators of IDO in primary endothelial cells from a non-immune-privileged site and determine whether IDO expression affects immune cell behavior. Methods IDO expression was determined using real-time quantitative polymerase chain reaction and immunoblotting. IDO activity was estimated using an IDO enzyme assay. Primary cells were transfected using microporation, and T-cell migration was determined using a cell transmigration assay. Results IDO is expressed in human saphenous vein endothelial cells after stimulation with IFNγ but not after treatment with TNFα, IL-1β, IL-2, IL-4, IL-6, or IL-10. VEGFβ and heparin negatively regulate IFNγ-driven increases in IDO. Overexpression of IDO in endothelial cells does not affect transmigration of T-cells. Conclusion IDO is expressed in human saphenous vein endothelial cells after stimulation with IFNγ. Heparin and angiogenesis stimulators such as VEGFβ negatively regulate its expression. PMID:26056484

  15. Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

    PubMed Central

    Ryman, Valerie E.; Packiriswamy, Nandakumar

    2016-01-01

    Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA) oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1) metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE), can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE), is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC) to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis. PMID:27818578

  16. Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models.

    PubMed

    Koizumi, Noriko; Okumura, Naoki; Kinoshita, Shigeru

    2012-02-01

    This review describes our recent attempts to develop new therapeutic modalities for corneal endothelial disease using animal models including non-human primate model in which the proliferative ability of corneal endothelial cells is severely limited, as is the case in humans. First, we describe our attempt to develop new surgical treatments using cultivated corneal endothelial cells for advanced corneal endothelial dysfunction. It includes two different approaches; a "corneal endothelial cell sheet transplantation" with cells grown on a type-I collagen carrier, and a "cell-injection therapy" combined with the application of Rho-kinase (ROCK) inhibitor. Recently, it was reported that the selective ROCK inhibitor, Y-27632, promotes cell adhesion and proliferation and inhibits the apoptosis of primate corneal endothelial cells in culture. When cultivated corneal endothelial cells were injected into the anterior chamber of animal eyes in the presence of ROCK inhibitor, endothelial cell adhesion was promoted and the cells achieved a high cell density and a morphology similar to corneal endothelial cells in vivo. We are also trying to develop a novel medical treatment for the early phase of corneal endothelial disease by the use of ROCK inhibitor eye drops. In rabbit and monkey experiments using partial endothelial dysfunction models, corneal endothelial wound healing was accelerated by the topical application of ROCK inhibitor to the ocular surface, and resulted in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. We are now trying to advance the clinical application of these new therapies for patients with corneal endothelial dysfunction.

  17. Vascular mechanobiology: endothelial cell responses to fluid shear stress.

    PubMed

    Ando, Joji; Yamamoto, Kimiko

    2009-11-01

    Endothelial cells (ECs) lining blood vessel walls respond to shear stress, a fluid mechanical force generated by flowing blood, and the EC responses play an important role in the homeostasis of the circulatory system. Abnormal EC responses to shear stress impair various vascular functions and lead to vascular diseases, including hypertension, thrombosis, and atherosclerosis. Bioengineering approaches in which cultured ECs are subjected to shear stress in fluid-dynamically designed flow-loading devices have been widely used to analyze EC responses at the cellular and molecular levels. Remarkable progress has been made, and the results have shown that ECs alter their morphology, function, and gene expression in response to shear stress. Shear stress affects immature cells, as well as mature ECs, and promotes differentiation of bone-marrow-derived endothelial progenitor cells and embryonic stem cells into ECs. Much research has been done on shear stress sensing and signal transduction, and their molecular mechanisms are gradually coming to be understood. However, much remains uncertain, and many candidates have been proposed for shear stress sensors. More extensive studies of vascular mechanobiology should increase our understanding of the molecular basis of the blood-flow-mediated control of vascular functions.

  18. Effect of selected flavones on cancer and endothelial cells.

    PubMed

    Pilátová, Martina; Stupáková, Viktória; Varinská, Lenka; Sarisský, Marek; Mirossay, Ladislav; Mirossay, Andrej; Gál, Peter; Kraus, Vladimír; Dianisková, Katarína; Mojzis, Ján

    2010-06-01

    In our study we used quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) as the reference standard to compare antiproliferative and antiangiogenic effects of chrysin (5,7-dihydroxyflavone) and 3-hydroxyflavone. Our data indicates that chrysin and 3-hydroxyflavone showed significantly higher cytotoxic effect than reference standard quercetin. These tested agents significantly decreased cell survival with the efficacy of 65-85% at the concentration 100 micromol/l for HUVEC, lung carcinoma and leukemic cells being the most sensitive. Cell cycle analysis indicates that quercetin and 3-hydroxyflavone might affect the cell cycle of Jurkat cells by a similar or the same mechanism of action which lead to G2/M arrest as well as to an increase in sub-G0/G1 fraction. Treatment of Jurkat cells with chrysin resulted only increase in the fraction of cells with sub-G0/G1 DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was confirmed by DNA fragmentation and by staining with annexin V. All three tested flavones inhibited endothelial cell migration after 24 h of incubation at a concentration 100 micromol/l. At a lower concentration (10 micromol/l) only quercetin significantly inhibited migration of endothelial cells. Furthermore, in our experiments decreased secretion of matrix metalloproteinases (MMP-2 and MMP-9) was observed after a 72 h treatment with quercetin. No decrease in secretion of MMP-2 and MMP-9 was seen after chrysin and 3-hydroxyflavone treatment. On the other hand, our results showed that none of three flavonoids blocked microcapillary tube formation. Further studies are necessary to investigate the mechanism of action and to find out the relationship between the structure, character and position of substituents of natural substances and their biological activities.

  19. Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

    NASA Technical Reports Server (NTRS)

    Melhado, Caroline; Sanford, Gary; Harris-Hooker, Sandra

    1997-01-01

    Endothelial cell migration is important to vascular wall regeneration following injury or stress. However, the mechanism(s) governing this response is not well understood. The microgravity environment of space may complicate the response of these cells to injury. To date, there are no reports in this area. We examined how bovine aortic (BAEC) and pulmonary (BPEC) endothelial cells respond to denudation injury under hypergravity (HGrav) and simulated microgravity (MGrav), using image analysis. In 10% FBS, the migration of confluent BAEC and BPEC into the denuded area was not affected by HGrav or MGrav. However, in low FBS (0.5%), signficantly retarded migration under MGrav, and increased migration under HGrav was found. MGrav also decreased the migration of postconfluent BPEC while HGrav showed no difference. Both MGrav and HGrav strongly decreased the migration of postconfluent BAEC. Also, both cell lines showed significant morphological changes by scanning electron microscopy. These studies indicate that endothelial cell function is affected by changes in gravity.

  20. Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model.

    PubMed

    Neuhaus, Winfried; Samwer, Fabian; Kunzmann, Steffen; Muellenbach, Ralf M; Wirth, Michael; Speer, Christian P; Roewer, Norbert; Förster, Carola Y

    2012-11-01

    The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

  1. Focally regulated endothelial proliferation and cell death in human synovium.

    PubMed Central

    Walsh, D. A.; Wade, M.; Mapp, P. I.; Blake, D. R.

    1998-01-01

    Angiogenesis and vascular insufficiency each may support the chronic synovial inflammation of rheumatoid arthritis. We have shown by quantitative immunohistochemistry and terminal uridyl deoxynucleotide nick end labeling that endothelial proliferation and cell death indices were each increased in synovia from patients with rheumatoid arthritis compared with osteoarthritic and noninflamed controls, whereas endothelial fractional areas did not differ significantly among disease groups. Markers of proliferation were associated with foci immunoreactive for vascular endothelial growth factor and integrin alpha(v)beta3, whereas cell death was observed in foci in which immunoreactivities for these factors were weak or absent. No association was found with thrombospondin immunoreactivity. The balance between angiogenesis and vascular regression in rheumatoid synovitis may be determined by the focal expression of angiogenic and endothelial survival factors. Increased endothelial cell turnover may contribute to microvascular dysfunction and thereby facilitate persistent synovitis. Images Figure 1 Figure 3 Figure 4 PMID:9502411

  2. Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide

    SciTech Connect

    Madden, M.C.; Eling, T.E.; Friedman, M.

    1987-09-01

    We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H/sub 2/O/sub 2/ is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.

  3. Ozone inhibits endothelial cell cyclooxygenase activity through formation of hydrogen peroxide.

    PubMed

    Madden, M C; Eling, T E; Friedman, M

    1987-09-01

    We have previously demonstrated that a 2H exposure of cultured pulmonary endothelial cells to ozone (0.0-1.0 ppm) in-vitro resulted in a concentration-dependent reduction of endothelial prostacyclin production (90% decrease at the 1.0 ppm level). Ozone-exposed endothelial cells, incubated with 20 uM arachidonate, also demonstrated a significant inhibition of prostacyclin synthesis. To further examine the mechanisms of the inhibition of prostacyclin synthesis, bovine pulmonary endothelial cells were exposed to 1.0 ppm ozone for 2H. A significant decrease in prostacyclin synthesis was found within 5 min of exposure (77 +/- 36% of air-exposed control values, p less than 0.05). Endothelial prostacyclin synthesis returned to baseline levels by 12H after ozone exposure, a time point which was similar to the recovery time of unexposed endothelium treated with 0.5 uM acetylsalicylic acid. Incubation of endothelial cells, previously exposed to 1.0 ppm ozone for 2 hours, with 4 uM PGH2 resulted in restoration of essentially normal prostacyclin synthesis. When endothelial cells were co-incubated with catalase (5 U/ml) during ozone exposure, no inhibition of prostacyclin synthesis was observed. Co-incubation with either heat-inactivated catalase or superoxide dismutase (10 U/ml) did not affect the ozone-induced inhibition of prostacyclin synthesis. These data suggest that H2O2 is a major toxic species produced in endothelial cells during ozone exposure and responsible for the inhibition of endothelial cyclooxygenase activity.

  4. The Role of Lymphatic Endothelial Cells in Liver Injury and Tumor Development

    PubMed Central

    Lukacs-Kornek, Veronika

    2016-01-01

    Lymphatics and lymphatic endothelial cells (LECs) possess multiple immunological functions besides affecting immune cell migration, such as inhibiting T cell proliferation and antigen presentation by dendritic cells. Moreover, they control the trans-endothelial transport of multiple molecules and antigens. Emerging evidence suggest their active involvements in immunregulation, tumor, and metastases formation. In the liver, increased lymphangiogenesis, specifically at the portal area has been associated with multiple liver diseases in particular primary biliary cirrhosis, idiopathic portal hypertension, and liver malignancies. Nevertheless, the exact role and contribution of LECs to liver diseases are poorly understood. The review summarizes the current understanding of LECs in liver diseases. PMID:27965673

  5. Effects of Gravitational Mechanical Unloading in Endothelial Cells: Association between Caveolins, Inflammation and Adhesion Molecules

    PubMed Central

    Grenon, S. Marlene; Jeanne, Marion; Aguado-Zuniga, Jesus; Conte, Michael S.; Hughes-Fulford, Millie

    2013-01-01

    Mechanical forces including gravity affect endothelial cell (ECs) function, and have been implicated in vascular disease as well as physiologic changes associated with low gravity environments. The goal of this study was to investigate the impact of gravitational mechanical unloading on ECs phenotype as determined by patterns of gene expression. Human umbilical vascular endothelial cells were exposed to 1-gravity environment or mechanical unloading (MU) for 24 hours, with or without periods of mechanical loading (ML). MU led to a significant decrease in gene expression of several adhesion molecules and pro-inflammatory cytokines. On the contrary, eNOS, Caveolin-1 and -2 expression were significantly increased with MU. There was a decrease in the length and width of the cells with MU. Addition of ML during the MU period was sufficient to reverse the changes triggered by MU. Our results suggest that gravitational loading could dramatically affect vascular endothelial cell function. PMID:23511048

  6. Endothelial cells direct mesenchymal stem cells toward a smooth muscle cell fate.

    PubMed

    Lin, Cho-Hao; Lilly, Brenda

    2014-11-01

    Under defined conditions, mesenchymal stem cells can differentiate into unique cell types, making them attractive candidates for cell-based disease therapies. Ischemic diseases would greatly benefit from treatments that include the formation of new blood vessels from mesenchymal stem cells. However, blood vessels are complex structures composed of endothelial cells and smooth muscle cells, and their assembly and function in a diseased environment is reliant upon joining with the pre-existing vasculature. Although endothelial cell/smooth muscle cell interactions are well known, how endothelial cells may influence mesenchymal stem cells and facilitate their differentiation has not been defined. Therefore, we sought to explore how endothelial cells might drive mesenchymal stem cells toward a smooth muscle fate. Our data show that cocultured endothelial cells induce smooth muscle cell differentiation in mesenchymal stem cells. Endothelial cells can promote a contractile phenotype, reduce proliferation, and enhance collagen synthesis and secretion. Our data show that Notch signaling is essential for endothelial cell-dependent differentiation, and this differentiation pathway is largely independent of growth factor signaling mechanisms.

  7. Activated Brain Endothelial Cells Cross-Present Malaria Antigen.

    PubMed

    Howland, Shanshan W; Poh, Chek Meng; Rénia, Laurent

    2015-06-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.

  8. Aldosterone does not modify gene expression in human endothelial cells.

    PubMed

    Verhovez, A; Williams, T A; Morello, F; Monticone, S; Brizzi, M F; Dentelli, P; Fallo, F; Fabris, B; Amenta, F; Gomez-Sanchez, C; Veglio, F; Mulatero, P

    2012-03-01

    The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

  9. Growth of Cowdria ruminantium in tissue culture endothelial cell lines from wild African mammals.

    PubMed

    Smith, G E; Anderson, E C; Burridge, M J; Peter, T F; Mahan, S M

    1998-04-01

    Endothelial cell cultures were established from several wild African mammalian species. Long-term cultures were established from three ruminants, stable antelope (Hippotragus niger), buffalo (Syncerus caffer), and eland (Tragelaphus oryx), and from an omnivore, the bushpig (Potamochoerus porcus). Cowdria ruminanntium was isolated from plasma of clinically affected animals in these four cell lines and in bovine endothelial cells used routinely for C. ruminantium propagation. Nineteen different strains of C. ruminantium from Africa and the Caribbean region were grown and maintained in these cell lines and their growth was comparable with growth in the bovine endothelial cells. The role of sable antelope, eland, and bushpigs in the epidemiology of heartwater is unknown. However, these results extend the number of cell lines that can be used to isolate and grow C. ruminantium.

  10. Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells.

    PubMed

    Huang, Lan; Nakayama, Hironao; Klagsbrun, Michael; Mulliken, John B; Bischoff, Joyce

    2015-01-01

    Endothelial glucose transporter 1 (GLUT1) is a definitive and diagnostic marker for infantile hemangioma (IH), a vascular tumor of infancy. To date, GLUT1-positive endothelial cells in IH have not been quantified nor directly isolated and studied. We isolated GLUT1-positive and GLUT1-negative endothelial cells from IH specimens and characterized their proliferation, differentiation, and response to propranolol, a first-line therapy for IH, and to rapamycin, an mTOR pathway inhibitor used to treat an increasingly wide array of proliferative disorders. Although freshly isolated GLUT1-positive cells, selected using anti-GLUT1 magnetic beads, expressed endothelial markers CD31, VE-Cadherin, and vascular endothelial growth factor receptor 2, they converted to a mesenchymal phenotype after 3 weeks in culture. In contrast, GLUT1-negative endothelial cells exhibited a stable endothelial phenotype in vitro. GLUT1-selected cells were clonogenic when plated as single cells and could be induced to redifferentiate into endothelial cells, or into pericytes/smooth muscle cells or into adipocytes, indicating a stem cell-like phenotype. These data demonstrate that, although they appear and function in the tumor as bona fide endothelial cells, the GLUT1-positive endothelial cells display properties of facultative stem cells. Pretreatment with rapamycin for 4 days significantly slowed proliferation of GLUT1-selected cells, whereas propranolol pretreatment had no effect. These results reveal for the first time the facultative nature of GLUT1-positive endothelial cells in IH.

  11. Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions

    PubMed Central

    1996-01-01

    Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE- cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE- cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen- reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE

  12. The impact of microgravity and hypergravity on endothelial cells.

    PubMed

    Maier, Jeanette A M; Cialdai, Francesca; Monici, Monica; Morbidelli, Lucia

    2015-01-01

    The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

  13. The Impact of Microgravity and Hypergravity on Endothelial Cells

    PubMed Central

    Maier, Jeanette A. M.

    2015-01-01

    The endothelial cells (ECs), which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature. PMID:25654101

  14. Classification of signals for blocking apoptosis in vascular endothelial cells.

    PubMed

    Hase, M; Araki, S; Kaji, K; Hayashi, H

    1994-10-01

    The survival and death of human umbilical vascular endothelial cells in culture are affected by several factors, such as fibroblast growth factor (FGF), serum, phorbol ester (TPA), and vanadate. In order to identify common aspects of the various signal-transduction processes during the course of apoptotic or programmed cell death, we designed experiments to distinguish between these factors in terms of the pathway that is responsible for the processing of each stimulus. We found, for example, that the effect of removal of FGF was specifically overcome by the addition of the phorbol ester. Our results indicated that two distinct pathways were operative, one specific for signal transduction initiated by FGF and phorbol ester and another specific for signal transduction initiated by serum and vanadate. These two pathways merged down-stream of the individual signal-processing pathways.

  15. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells

    PubMed Central

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R.; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells. PMID:26977592

  16. Activation of Endothelial Nitric Oxide (eNOS) Occurs through Different Membrane Domains in Endothelial Cells.

    PubMed

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  17. Chemosensitizing AML cells by targeting bone marrow endothelial cells.

    PubMed

    Bosse, Raphael C; Wasserstrom, Briana; Meacham, Amy; Wise, Elizabeth; Drusbosky, Leylah; Walter, Glenn A; Chaplin, David J; Siemann, Dietmar W; Purich, Daniel L; Cogle, Christopher R

    2016-05-01

    Refractory disease is the greatest challenge in treating patients with acute myeloid leukemia (AML). Blood vessels may serve as sanctuary sites for AML. When AML cells were co-cultured with bone marrow endothelial cells (BMECs), a greater proportion of leukemia cells were in G0/G1. This led us to a strategy of targeting BMECs with tubulin-binding combretastatins, causing BMECs to lose their flat phenotype, degrade their cytoskeleton, cease growth, and impair migration despite unchanged BMEC viability and metabolism. Combretastatins also caused downregulation of BMEC adhesion molecules known to tether AML cells, including vascular cell adhesion molecule (VCAM)-1 and vascular endothelial (VE)-cadherin. When AML-BMEC co-cultures were treated with combretastatins, a significantly greater proportion of AML cells dislodged from BMECs and entered the G2/M cell cycle, suggesting enhanced susceptibility to cell cycle agents. Indeed, the combination of combretastatins and cytotoxic chemotherapy enhanced additive AML cell death. In vivo mice xenograft studies confirmed this finding by revealing complete AML regression after treatment with combretastatins and cytotoxic chemotherapy. Beyond highlighting the pathologic role of BMECs in the leukemia microenvironment as a protective reservoir of disease, these results support a new strategy for using vascular-targeting combretastatins in combination with cytotoxic chemotherapy to treat AML.

  18. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    SciTech Connect

    Askari, Ara A.; Thomson, Scott; Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C.; Bishop-Bailey, David

    2014-04-04

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.

  19. Endothelial cell tumor growth is Ape/ref-1 dependent

    PubMed Central

    Biswas, Ayan; Khanna, Savita; Roy, Sashwati; Pan, Xueliang; Sen, Chandan K.

    2015-01-01

    Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation. PMID:26108661

  20. Expression of the beta 7 integrin by human endothelial cells.

    PubMed Central

    Brezinschek, R. I.; Brezinschek, H. P.; Lazarovits, A. I.; Lipsky, P. E.; Oppenheimer-Marks, N.

    1996-01-01

    Integrin adhesion receptors mediate fundamental intercellular interactions of many cell types as well as cellular interactions with specific extracellular matrix molecules. To date, the beta 7 integrin has been shown to be expressed by leukocyte subsets and to mediate interactions of these cells with extracellular matrix molecules as well as with endothelial and epithelial cells. The data presented here indicate that human endothelial cells also express the beta 7 integrin both in vitro and in situ. Analysis of cDNA indicated that endothelial beta 7 was identical to that expressed by leukocytes. Cell surface expression of beta 7 was increased by exposure of the endothelium to the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1 beta. In leukocytes, beta 7 complexes with alpha 4 or alpha E integrin chains. Endothelial cells also expressed a number of alpha-integrin chains, including alpha 4, but not alpha E. The expression and utilization of beta 7, presumably complexed with alpha 4, by endothelial cells may be instrumental in the maintenance of the function or phenotype of endothelial cells. Images Figure 2 Figure 4 Figure 6 Figure 7 PMID:8909254

  1. The natural antioxidants, pomegranate extract and soy isoflavones, favourably modulate canine endothelial cell function.

    PubMed

    Baumgartner-Parzer, Sabina M; Waldenberger, Ferdinand Rudolf; Freudenthaler, Angelika; Ginouvès-Guerdoux, Amandine; McGahie, David; Gatto, Hugues

    2012-01-01

    Cardiovascular disease, preceded by vascular endothelial dysfunction, is a prominent cause of death in dogs. L-carnitine and taurine, well known for their antioxidative capacity, beneficially affect cardiovascular disease as well as certain dog cardiomyopathies. It is well established that vascular endothelial dysfunction precedes cardiovascular disease and that "vasoprotective factors" (NO and antioxidants) prevent apoptosis, whereas "risk factors" such as oxidized LDL, hyperglycemia, and free fatty acids trigger it in cultured human vascular endothelial cells. Whereas human vascular cell in vitro models are widely established and used for the characterisation of potential vasoprotective substances, such models are not available for canine endothelial cells. In the present study we therefore developed an in vitro model, which allows the testing of the effects of different substances on proliferation and apoptosis in canine aortic endothelial cells. This model was used to test L-carnitine, taurine, pomegranate extract, and Soy Isoflavones in comparison to reference substances (glutathione and pioglitazone) previously shown to modulate human endothelial cell function. L-carnitine and taurine neither exhibited antiproliferative nor antiapoptotic activities in the context of this study. However extracts from pomegranate and soy isoflavones dramatically reduced proliferation and apoptosis in a dose dependent fashion, being in line with a vasoprotective activity in dogs.

  2. The Natural Antioxidants, Pomegranate Extract and Soy Isoflavones, Favourably Modulate Canine Endothelial Cell Function

    PubMed Central

    Baumgartner-Parzer, Sabina M.; Waldenberger, Ferdinand Rudolf; Freudenthaler, Angelika; Ginouvès-Guerdoux, Amandine; McGahie, David; Gatto, Hugues

    2012-01-01

    Cardiovascular disease, preceded by vascular endothelial dysfunction, is a prominent cause of death in dogs. L-carnitine and taurine, well known for their antioxidative capacity, beneficially affect cardiovascular disease as well as certain dog cardiomyopathies. It is well established that vascular endothelial dysfunction precedes cardiovascular disease and that “vasoprotective factors” (NO and antioxidants) prevent apoptosis, whereas “risk factors” such as oxidized LDL, hyperglycemia, and free fatty acids trigger it in cultured human vascular endothelial cells. Whereas human vascular cell in vitro models are widely established and used for the characterisation of potential vasoprotective substances, such models are not available for canine endothelial cells. In the present study we therefore developed an in vitro model, which allows the testing of the effects of different substances on proliferation and apoptosis in canine aortic endothelial cells. This model was used to test L-carnitine, taurine, pomegranate extract, and Soy Isoflavones in comparison to reference substances (glutathione and pioglitazone) previously shown to modulate human endothelial cell function. L-carnitine and taurine neither exhibited antiproliferative nor antiapoptotic activities in the context of this study. However extracts from pomegranate and soy isoflavones dramatically reduced proliferation and apoptosis in a dose dependent fashion, being in line with a vasoprotective activity in dogs. PMID:23762588

  3. Plasmodium falciparum Malaria: reduction of endothelial cell apoptosis in vitro.

    PubMed

    Hemmer, Christoph Josef; Lehr, Hans Anton; Westphal, Kathi; Unverricht, Marcus; Kratzius, Manja; Reisinger, Emil Christian

    2005-03-01

    Organ failure in Plasmodium falciparum malaria is associated with neutrophil activation and endothelial damage. This study investigates whether neutrophil-induced endothelial damage involves apoptosis and whether it can be prevented by neutralization of neutrophil secretory products. Endothelial cells from human umbilical veins were coincubated with neutrophils from healthy donors and with sera from eight patients with P. falciparum malaria, three patients with P. vivax malaria, and three healthy controls. Endothelial apoptosis was demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V staining. The rate of apoptosis of cells was markedly increased after incubation with patient serum compared to that with control serum. Apoptosis was most pronounced after incubation with sera from two patients with fatal cases of P. falciparum malaria, followed by sera of survivors with severe P. falciparum malaria and, finally, by sera of patients with mild P. falciparum and P. vivax malaria. Ascorbic acid, tocopherol, and ulinastatin reduced the apoptosis rate, but gabexate mesilate and pentoxifylline did not. Furthermore, in fatal P. falciparum malaria, apoptotic endothelial cells were identified in renal and pulmonary tissue by TUNEL staining. These findings show that apoptosis caused by neutrophil secretory products plays a major role in endothelial cell damage in malaria. The antioxidants ascorbic acid and tocopherol and the protease inhibitor ulinastatin can reduce malaria-associated endothelial apoptosis in vitro.

  4. Primary monocytes regulate endothelial cell survival through secretion of Angiopoietin-1 and activation of endothelial Tie2

    PubMed Central

    Schubert, Shai Y.; Benarroch, Alejandro; Monter-Solans, Juan; Edelman, Elazer R.

    2011-01-01

    Objective Monocyte recruitment and interaction with the endothelium is imperative to vascular recovery. Tie2 plays a key role in endothelial health and vascular remodeling. We studied monocyte-mediated Tie2/Angiopoietin signaling following interaction of primary monocytes with endothelial cells and its role in endothelial cell survival. Methods and results The direct interaction of primary monocytes with subconfluent endothelial cells resulted in transient secretion of Angiopoietin-1 from monocytes and the activation of endothelial Tie2. This effect was abolished by preactivation of monocytes with TNFα. While primary monocytes contained high levels of both Angiopoietin 1 and 2, endothelial cells contained primarily Angiopoietin 2. Seeding of monocytes on serum starved endothelial cells reduced caspase-3 activity by 46% ± 5.1%, and 52% ± 5.8% after TNFα treatment, and decreased detected single strand DNA levels by 41% ± 4.2% and 40± 3.5% respectively. This protective effect of monocytes on endothelial cells was reversed by Tie2 silencing with specific siRNA. The anti-apoptotic effect of monocytes was further supported by the activation of cell survival signaling pathways involving PI3K, STAT3 and AKT. Conclusions Monocytes and endothelial cells form a unique Tie2/Angiopoietin-1 signaling system which effects endothelial cell survival and may play critical a role in vascular remodeling and homeostasis. PMID:21273558

  5. Tipping off endothelial tubes: nitric oxide drives tip cells.

    PubMed

    Priya, Mani Krishna; Sahu, Giriraj; Soto-Pantoja, David R; Goldy, Naga; Sundaresan, Abaya Meenakshi; Jadhav, Vivek; Barathkumar, T R; Saran, Uttara; Jaffar Ali, B M; Roberts, David D; Bera, Amal Kanti; Chatterjee, Suvro

    2015-04-01

    Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

  6. How to utilize Ca2+ signals to rejuvenate the repairative phenotype of senescent endothelial progenitor cells in elderly patients affected by cardiovascular diseases: a useful therapeutic support of surgical approach?

    PubMed Central

    2013-01-01

    Endothelial dysfunction or loss is the early event that leads to a host of severe cardiovascular diseases, such as atherosclerosis, hypertension, brain stroke, myocardial infarction, and peripheral artery disease. Ageing is regarded among the most detrimental risk factor for vascular endothelium and predisposes the subject to atheroscleorosis and inflammatory states even in absence of traditional comorbid conditions. Standard treatment to restore blood perfusion through stenotic arteries are surgical or endovascular revascularization. Unfortunately, ageing patients are not the most amenable candidates for such interventions, due to high operative risk or unfavourable vascular involvement. It has recently been suggested that the transplantation of autologous bone marrow-derived endothelial progenitor cells (EPCs) might constitute an alternative and viable therapeutic option for these individuals. Albeit pre-clinical studies demonstrated the feasibility of EPC-based therapy to recapitulate the diseased vasculature of young and healthy animals, clinical studies provided less impressive results in old ischemic human patients. One hurdle associated to this kind of approach is the senescence of autologous EPCs, which are less abundant in peripheral blood and display a reduced pro-angiogenic activity. Conversely, umbilical cord blood (UCB)-derived EPCs are more suitable for cellular therapeutics due to their higher frequency and sensitivity to growth factors, such as vascular endothelial growth factor (VEGF). An increase in intracellular Ca2+ concentration is central to EPC activation by VEGF. We have recently demonstrated that the Ca2+ signalling machinery driving the oscillatory Ca2+ response to this important growth factor is different in UCB-derived EPCs as compared to their peripheral counterparts. In particular, we focussed on the so-called endothelial colony forming cells (ECFCs), which are the only EPC population belonging to the endothelial lineage and able to

  7. [Systemic inflammatory response syndrome (SIRS) and endothelial cell injury].

    PubMed

    Gando, Satoshi

    2004-12-01

    During recent years, evidences have been accumulated demonstrating bidirectional crosstalk between coagulation and inflammation. This review outlines the influences that coagulation and inflammation exert on each other to the endothelium and how these systems induce systemic inflammatory response syndrome (SIRS). Then we discussed the implications of leucocyte-endothelial activation to endothelial cell injury followed by multiple organ dysfunction syndrome (MODS) in patients with sustained SIRS. Last we demonstrated an important role of inflammatory circulation disturbance induced by endothelial cell injury for the pathogenesis of MODS in SIRS and sepsis.

  8. High-density lipoprotein exerts vasculoprotection via endothelial progenitor cells

    PubMed Central

    Petoumenos, Vasileios; Nickenig, Georg; Werner, Nikos

    2009-01-01

    Endothelial progenitor cells (EPC) enhance endothelial cell repair, improve endothelial dysfunction and are a predictor for cardiovascular mortality. High-density lipoprotein (HDL) cholesterol levels inversely correlate with cardiovascular events and have vasculoprotective effects. Here we postulate that HDL influences EPC biology. HDL and EPC were isolated according to standard procedures. Differentiation of mononuclear cells into DiLDL/lectin positive cells was enhanced after HDL treatment compared to vehicle. HDL was able to inhibit apoptosis (TUNEL assay, annexin V staining) while proliferation (BrdU incorporation) of early outgrowth colonies after extended cell cultivation (14 days) was increased. Flow chamber experiments revealed an improved adhesion of HDL pre-incubated EPC on human coronary artery endothelial cells (HCAEC) compared to vehicle while HDL treatment of HCAEC prevented adhesion of inflammatory cells. Flow cytometry demonstrated an up-regulation of β2- and α4-integrins on HDL pre-incubated EPC. Blocking experiments revealed a unique role of β2-integrin in EPC adhesion. Treatment of wild-type mice with recombinant HDL after endothelial denudation resulted in enhanced re-endothelialization compared to vehicle. Finally, in patients with coronary artery disease a correlation between circulating EPC and HDL concentrations was demonstrated. We provide evidence that HDL mediates important vasculoprotective action via the improvement of function of circulating EPC. PMID:18705697

  9. Alk1 controls arterial endothelial cell migration in lumenized vessels.

    PubMed

    Rochon, Elizabeth R; Menon, Prahlad G; Roman, Beth L

    2016-07-15

    Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs.

  10. Novel Identity and Functional Markers for Human Corneal Endothelial Cells

    PubMed Central

    Bartakova, Alena; Alvarez-Delfin, Karen; Weisman, Alejandra D.; Salero, Enrique; Raffa, Gabriella A.; Merkhofer, Richard M.; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2016-01-01

    Purpose Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. Methods Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. Results Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. Conclusions In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction. PMID:27196322

  11. Cellular and Molecular Biology of Aging Endothelial Cells

    PubMed Central

    Donato, Anthony J.; Morgan, R. Garrett; Walker, Ashley E.; Lesniewski, Lisa A.

    2015-01-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage have been shown to trigger cell senescence via the p53/p21 pathway that results in increased inflammatory signaling in arteries from older adults. This review will discuss the current

  12. Cellular and molecular biology of aging endothelial cells.

    PubMed

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  13. Changes of myoid and endothelial cells in the peritubular wall during contraction of the seminiferous tubule.

    PubMed

    Losinno, Antonella D; Sorrivas, Viviana; Ezquer, Marcelo; Ezquer, Fernando; López, Luis A; Morales, Alfonsina

    2016-08-01

    The wall of the seminiferous tubule in rodents consists of an inner layer of myoid cells covered by an outer layer of endothelial cells. Myoid cells are a type of smooth muscle cell containing α-actin filaments arranged in two independent layers that contract when stimulated by endothelin-1. The irregular surface relief of the tubular wall is often considered a hallmark of contraction induced by a variety of stimuli. We examine morphological changes of the rat seminiferous tubule wall during contraction by a combination of light, confocal, transmission and scanning electron microscopy. During ET-1-induced contraction, myoid cells changed from a flat to a conical shape, but their actin filaments remained in independent layers. As a consequence of myoid cell contraction, the basement membrane became wavy, orientation of collagen fibers in the extracellular matrix was altered and the endothelial cell layer became folded. To observe the basement of the myoid cell cone, the endothelial cell monolayer was removed by collagenase digestion prior to SEM study. In contracted tubules, it is possible to distinguish cell relief: myoid cells have large folds on the external surface oriented parallel to the tubular axis, whereas endothelial cells have numerous cytoplasmic projections facing the interstitium. The myoid cell cytoskeleton is unusual in that the actin filaments are arranged in two orthogonal layers, which adopt differing shapes during contraction with myoid cells becoming cone-shaped. This arrangement impacts on other components of the seminiferous tubule wall and affects the propulsion of the tubular contents to the rete testis.

  14. [Effects of radiation on capillary endothelial cells derived from Mongolian gerbil brain].

    PubMed

    Mori, S; Tanaka, R; Minakawa, T; Onda, K

    1990-09-01

    Confluent monolayers of capillary endothelial cells derived from Mongolian gerbil brain were irradiated with a single exposure of x-rays, and their radiosensitivity and sequential changes in morphology, staining intensity for factor VIII-related antigen (F VIII RAg), and capacity to produce prostacyclin (PGI2) were examined. The radiobiologic parameters that characterized the dose-response survival curve for these cells were found to be n = 1.9, Dq = 140 rad, and D0 = 190 rad. Morphologically, nuclear and cytoplasmic swelling, vacuolation of cytoplasm, and giant cell formation occurred in a dose dependent manner after 24 hours from irradiation. Decreased staining intensity for F VIII RAg was observed in morphologically affected cells. The capacity to synthesize PGI2 was significantly enhanced at 24 hours, but less significant at 72 hours after irradiation. The present data suggest that the radiosensitivity of brain capillary endothelial cells may be somewhat lower than that of endothelial cells originated from larger vessels, and that radiation induced morphological and functional changes in the brain capillary endothelial cells may be quantitatively similar to the changes in endothelial cells of larger vessels.

  15. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

    SciTech Connect

    Zhou Yijun . E-mail: zhou-yijun@hotmail.com; Wang Jiahe; Zhang Jin

    2006-06-02

    Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-{kappa}B, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-{kappa}B, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.

  16. Putative Key Role of Inositol Messengers in Endothelial Cells in Preeclampsia

    PubMed Central

    Kunjara, Sirilaksana; McLean, Patricia; Rademacher, Laurens; Rademacher, Thomas W.; Fascilla, Fabiana; Bettocchi, Stefano

    2016-01-01

    Immunological alterations, endothelial dysfunction, and insulin resistance characterize preeclampsia. Endothelial cells hold the key role in the pathogenesis of this disease. The signaling pathways mediating these biological abnormalities converge on PKB/Akt, an intracellular kinase regulating cell survival, proliferation, and metabolism. Inositol second messengers are involved in metabolic and cell signaling pathways and are highly expressed during preeclampsia. Intracellular action of these molecules is deeply affected by zinc, manganese, and calcium. To evaluate the pathophysiological significance, we present the response of the intracellular pathways of inositol phosphoglycans involved in cellular metabolism and propose a link with the disease. PMID:27738431

  17. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    SciTech Connect

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-12-01

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain.

  18. Substrates for Expansion of Corneal Endothelial Cells towards Bioengineering of Human Corneal Endothelium

    PubMed Central

    Navaratnam, Jesintha; Utheim, Tor P.; Rajasekhar, Vinagolu K.; Shahdadfar, Aboulghassem

    2015-01-01

    Corneal endothelium is a single layer of specialized cells that lines the posterior surface of cornea and maintains corneal hydration and corneal transparency essential for vision. Currently, transplantation is the only therapeutic option for diseases affecting the corneal endothelium. Transplantation of corneal endothelium, called endothelial keratoplasty, is widely used for corneal endothelial diseases. However, corneal transplantation is limited by global donor shortage. Therefore, there is a need to overcome the deficiency of sufficient donor corneal tissue. New approaches are being explored to engineer corneal tissues such that sufficient amount of corneal endothelium becomes available to offset the present shortage of functional cornea. Although human corneal endothelial cells have limited proliferative capacity in vivo, several laboratories have been successful in in vitro expansion of human corneal endothelial cells. Here we provide a comprehensive analysis of different substrates employed for in vitro cultivation of human corneal endothelial cells. Advances and emerging challenges with ex vivo cultured corneal endothelial layer for the ultimate goal of therapeutic replacement of dysfunctional corneal endothelium in humans with functional corneal endothelium are also presented. PMID:26378588

  19. Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension.

    PubMed

    Tuder, R M; Radisavljevic, Z; Shroyer, K R; Polak, J M; Voelkel, N F

    1998-12-01

    Anorexigens such as aminorex fumarate and dexfenfluramine are associated with the development of severe pulmonary hypertension (PH), which clinically and histopathologically is considered indistinguishable from idiopathic or primary pulmonary hypertension (PPH). For the current study, we asked whether anorexigen-associated PH is characterized by monoclonal pulmonary endothelial cell proliferation (such as in PPH) or, alternatively, is associated with a polyclonal endothelial cell proliferation as found in secondary PH. Analysis of clonality by the human androgen receptor assay was performed in microdissected endothelial cells of plexiform lesions of two patients with anorexigen-associated PH. The four plexiform lesions of Patient 1 and the six of Patient 2 with anorexigen-associated PH exhibited a monoclonal expansion of pulmonary endothelial cells, with a mean clonality ratio of 0.03 +/- 0.01 SE. Our results indicate that appetite suppressant-associated PH is identical to PPH not only in clinical and histopathologic features but also, at a molecular level, in terms of the monoclonal nature of the endothelial cell proliferation. The anorexigens may accelerate the growth of pulmonary endothelial cells in patients with predisposition to develop PPH.

  20. Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells.

    PubMed

    Nishinaga, M; Ozawa, T; Shimada, K

    1993-09-01

    Previous studies showed that homocysteine, a thrombo-atherogenic and atherogenic agent, inhibits an endothelial thrombomodulin-protein C anticoagulant pathway. We examined whether homocysteine might affect another endothelial anticoagulant mechanism; i.e., heparin-like glycosaminoglycan-antithrombin III interactions. Incubations of porcine aortic endothelial cell cultures with homocysteine reduced the amount of antithrombin III bound to the cell surface in a dose- and time-dependent fashion. The inhibitory effect was observed at a homocysteine concentration as low as 0.1 mM, and the maximal suppression occurred at 1 mM of homocysteine after 24 h. In contrast with a marked reduction in the maximal antithrombin III binding capacity (approximately 30% of control), the radioactivity of [35S]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative charge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reduction in antithrombin III binding capacity of heparan sulfate isolated from homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III binding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in binding activity was observed after preincubation with the same concentration of methionine, alanine, or valine. This sulfhydryl effect may be caused by generation of hydrogen peroxide, as incubation of catalase, but not superoxide dismutase, with homocysteine-treated endothelial cells prevented this reduction, whereas copper augmented the inhibitory effects of the metabolite. Thus, our data suggest that the inhibited expression of anticoagulant heparan sulfate may contribute to the thrombogenic property resulting from the

  1. Comparative Evaluation for Potential Differentiation of Endothelial Progenitor Cells and Mesenchymal Stem Cells into Endothelial-Like Cells

    PubMed Central

    Sabry, Dina; Noh, Olfat; Samir, Mai

    2016-01-01

    Understanding the mechanisms of vascular remodeling could lead to more effective treatments for ischemic conditions. We aimed to compare between the abilities of both human Wharton jelly derived mesenchymal stem cells (hMSCs) and human cord blood endothelial progenitor cells (hEPCs) and CD34+ to induce angiogenesis in vitro. hMSCs, hEPCs, and CD34+ were isolated from human umbilical cord blood using microbead (MiniMacs). The cells characterization was assessed by flow cytometry following culture and real-time PCR for vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand factor (vWF) to prove stem cells differentiation. The study revealed successful isolation of hEPCs, CD34+, and hMSCs. The hMSCs were identified by gaining CD29+ and CD44+ using FACS analysis. The hEPCs were identified by having CD133+, CD34+, and KDR. The potential ability of hEPCs and CD34+ to differentiate into endothelial-like cells was more than hMSCs. This finding was assessed morphologically in culture and by higher significant VEGFR2 and vWF genes expression (p<0.05) in differentiated hEPCs and CD34+ compared to differentiated hMSCs. hEPCs and CD34+ differentiation into endothelial-like cells were much better than that of hMSCs. PMID:27426085

  2. Traction Forces of Endothelial Cells under Slow Shear Flow

    PubMed Central

    Perrault, Cecile M.; Brugues, Agusti; Bazellieres, Elsa; Ricco, Pierre; Lacroix, Damien; Trepat, Xavier

    2015-01-01

    Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress. PMID:26488643

  3. Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis

    PubMed Central

    Gimbrone, Michael A.; García-Cardeña, Guillermo

    2016-01-01

    Dysfunction of the endothelial lining of lesion-prone areas of the arterial vasculature is an important contributor to the pathobiology of atherosclerotic cardiovascular disease. Endothelial cell dysfunction (ECD), in its broadest sense, encompasses a constellation of various non-adaptive alterations in functional phenotype, which have important implications for the regulation of hemostasis and thrombosis, local vascular tone and redox balance, and the orchestration of acute and chronic inflammatory reactions within the arterial wall. In this review, we trace the evolution of the concept of endothelial cell dysfunction, focusing on recent insights into the cellular and molecular mechanisms that underlie its pivotal roles in atherosclerotic lesion initiation and progression; explore its relationship to classic, as well as more recently defined, clinical risk factors for atherosclerotic cardiovascular disease; consider current approaches to the clinical assessment of endothelial cell dysfunction; and outline some promising new directions for its early detection and treatment. PMID:26892962

  4. Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells

    PubMed Central

    Huang, Lan; Nakayama, Hironao; Klagsbrun, Michael; Mulliken, John B.; Bischoff, Joyce

    2014-01-01

    Endothelial glucose transporter 1 (GLUT1) is a definitive and diagnostic marker for infantile hemangioma (IH), a vascular tumor of infancy. To date, GLUT1-positive endothelial cells in IH have not been quantified nor directly isolated and studied. We isolated GLUT1-positive and GLUT1-negative endothelial cells from IH specimens and characterized their proliferation, differentiation and response to propranolol, a first-line therapy for IH, and to rapamycin, an mTOR pathway inhibitor used to treat an increasingly wide array of proliferative disorders. Although freshly isolated GLUT1-positive cells, selected using anti-GLUT1 magnetic beads, expressed endothelial markers CD31, VE-Cadherin and VEGFR2, they converted to a mesenchymal phenotype after three weeks in culture. In contrast, GLUT1-negative endothelial cells exhibited a stable endothelial phenotype in vitro. GLUT1-selected cells were clonogenic when plated as single cells and could be induced to re-differentiate into endothelial cells, or into pericyte/smooth muscle cells or into adipocytes, indicating a stem cell-like phenotype. These data demonstrate that, although they appear and function in the tumor as bona fide endothelial cells, the GLUT1-positive endothelial cells display properties of facultative stem cells. Pretreatment with rapamycin for 4 days significantly slowed proliferation of GLUT1-selected cells, whereas propranolol pretreatment had no effect. These results reveal for the first time the facultative nature of GLUT1-positive endothelial cells in infantile hemangioma. PMID:25187207

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

    SciTech Connect

    Kim, Woo Ho; Lee, Sun Hee; Jung, Myung Hwan; Seo, Ji Heun; Kim, Jin; Kim, Min A; Lee, You Mie

    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 is 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.

  6. Cell biology and pathology of liver sinusoidal endothelial cells.

    PubMed

    Enomoto, Katsuhiko; Nishikawa, Yuji; Omori, Yasufumi; Tokairin, Takuo; Yoshida, Masayuki; Ohi, Naoto; Nishimura, Takuya; Yamamoto, Youhei; Li, Qinchang

    2004-12-01

    Growing evidence revealed that liver sinusoidal endothelial cells (SEC) play several important roles in physiology and pathology of the liver. It has been well understood that their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhancement of hepatic metabolic activity. In addition, SEC is now regarded as a member of the scavenger endothelial cells, which have potential to eliminate a variety of macromolecules from the blood circulation by receptor-mediated endocytosis. It is reported that molecules preferentially eliminated by SEC are denatured or modified proteins such as advanced glycation end products, extracellular matrix components including hyaluronic acid, and some lipoproteins. The nature of the scavenger receptors corresponding to these molecules remains to be clarified. Recently, it was noted that SEC has an antigen-presenting function similar to dendritic cells. Taken together, it is suggested that SEC, cooperating with Kupffer cells and hepatic dendritic cells, may partake of immunoregulatory functions in the liver. SEC also plays a pivotal role in the pathological process of ischemia-reperfusion injury following liver surgery and liver transplantation. Thus, it is of importance to elucidate the mechanisms of apoptosis and proliferation of SEC. Recent results on the regulation of growth and apoptotic signaling of SEC are discussed.

  7. Obstructive sleep apnea syndrome, vascular pathology, endothelial function and endothelial cells and circulating microparticles.

    PubMed

    Stiefel, Pablo; Sánchez-Armengol, Maria Angeles; Villar, José; Vallejo-Vaz, Antonio; Moreno-Luna, Rafael; Capote, Francisco

    2013-08-01

    Accelerated atherosclerosis and increased cardiovascular risk are frequently reported in patients with obstructive sleep apnea (OSA) syndrome. In this article the authors attempt a review of the current understanding of the relationship between vascular risk and OSA syndrome based on large cohort studies that related the disease to several cardiovascular risk factors and vascular pathologies. We also discuss the pathophysiological mechanisms that may be involved in this relationship, starting with endothelial dysfunction and its mediators. These include an increased oxidative stress and inflammation as well as several disorders of coagulation and lipid metabolism. Moreover, circulating microparticles from activated leukocytes (CD62L_MPs) are higher in patients with OSA and there is a positive correlation between circulating levels of CD62L_MPs and nocturnal hypoxemia severity. Finally, circulating level of endothelial microparticles and circulating endothelial cells seem to be increased in patients with OSA. Also, endothelial progenitor cells are reduced and plasma levels of the vascular endothelial growth factor are increased.

  8. Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors.

    PubMed

    Hah, Young-Sool; Jun, Jin-Su; Lee, Seong-Gyun; Park, Bong-Wook; Kim, Deok Ryong; Kim, Uk-Kyu; Kim, Jong-Ryoul; Byun, June-Ho

    2011-02-01

    Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF(121), VEGF(165), VEGF(189), and VEGF(206)), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF(165) elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF(165) resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.

  9. Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells

    PubMed Central

    Cuvelier, Susan L.; Paul, Smitha; Shariat, Neda; Colarusso, Pina; Patel, Kamala D.

    2005-01-01

    Leukocyte transmigration can be affected by shear stress; however, the mechanisms by which shear stress modulates transmigration are unknown. We found that adhesion of eosinophils or an eosinophilic cell line to intereukin 4–stimulated endothelial cells led to a shear-dependent increase in endothelial cell intracellular calcium and increased phosphorylation of extracellular signal-regulated kinase (ERK) 2, but not c-Jun NH2-terminal kinase or p38 mitogen-activated protein kinase. Latex beads coated with antibodies were used to characterize the role of specific endothelial cell surface molecules in initiating signaling under shear conditions. We found that ligation of either vascular cell adhesion molecule–1 or E-selectin, but not major histocompatibility complex class I, induced a shear-dependent increase in ERK2 phosphorylation in cytokine-stimulated endothelial cells. Disassembly of the actin cytoskeleton with latrunculin A prevented ERK2 phosphorylation after adhesion under flow conditions, supporting a role for the cytoskeleton in mechanosensing. Rapid phosphorylation of focal adhesion kinase and paxillin occurred under identical conditions, suggesting that focal adhesions were also involved in mechanotransduction. Finally, we found that Rho-associated protein kinase and calpain were both critical in the subsequent transendothelial migration of eosinophils under flow conditions. These data suggest that ligation of leukocyte adhesion molecules under flow conditions leads to mechanotransduction in endothelial cells, which can regulate subsequent leukocyte trafficking. PMID:16172263

  10. Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells.

    PubMed

    Cuvelier, Susan L; Paul, Smitha; Shariat, Neda; Colarusso, Pina; Patel, Kamala D

    2005-09-19

    Leukocyte transmigration can be affected by shear stress; however, the mechanisms by which shear stress modulates transmigration are unknown. We found that adhesion of eosinophils or an eosinophilic cell line to intereukin 4-stimulated endothelial cells led to a shear-dependent increase in endothelial cell intracellular calcium and increased phosphorylation of extracellular signal-regulated kinase (ERK) 2, but not c-Jun NH2-terminal kinase or p38 mitogen-activated protein kinase. Latex beads coated with antibodies were used to characterize the role of specific endothelial cell surface molecules in initiating signaling under shear conditions. We found that ligation of either vascular cell adhesion molecule-1 or E-selectin, but not major histocompatibility complex class I, induced a shear-dependent increase in ERK2 phosphorylation in cytokine-stimulated endothelial cells. Disassembly of the actin cytoskeleton with latrunculin A prevented ERK2 phosphorylation after adhesion under flow conditions, supporting a role for the cytoskeleton in mechano-sensing. Rapid phosphorylation of focal adhesion kinase and paxillin occurred under identical conditions, suggesting that focal adhesions were also involved in mechanotransduction. Finally, we found that Rho-associated protein kinase and calpain were both critical in the subsequent transendothelial migration of eosinophils under flow conditions. These data suggest that ligation of leukocyte adhesion molecules under flow conditions leads to mechanotransduction in endothelial cells, which can regulate subsequent leukocyte trafficking.

  11. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells

    PubMed Central

    Kusuma, Sravanti; Zhao, Stephen; Gerecht, Sharon

    2012-01-01

    Extracellular matrix (ECM) production is critical to preserve the function and integrity of mature blood vessels. Toward the engineering of blood vessels, studies have centered on ECM production by supporting cells, whereas few studies implicate endothelial cells (ECs) with ECM synthesis. Here, we elucidate variations between cultured human arterial, venous, and progenitor ECs with respect to ECM deposition assembly, composition, and response to biomolecular and physiological factors. Our studies reveal that progenitor ECs, endothelial colony-forming cells (ECFCs), deposit collagen IV, fibronectin, and laminin that assemble to an organized weblike structure, as confirmed by decellularized cultures. Mature ECs only express these ECM proteins intracellularly. ECFC-derived ECM is abrogated in response to TGFβ signaling inhibition and actin cytoskeleton disruption. Hypoxic (1%) and physiological (5%) O2 tension stimulate ECM deposition from mature ECs. Interestingly, deposition of collagen I is observed only under 5% O2 tension. ECM production from all ECs is found to be regulated by hypoxia-inducible factors 1α and 2α but differentially in the different cell lines. Collectively, we suggest that ECM deposition and assembly by ECs is dependent on maturation stage and oxygen supply and that these findings can be harnessed to advance engineered vascular therapeutics.—Kusuma, S., Zhao, S., Gerecht, S. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells. PMID:22919069

  12. The effects of glucocorticoids on cultured human endothelial cells.

    PubMed

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man.

  13. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

    PubMed Central

    Yu, Cai-Guo; Zhang, Ning; Yuan, Sha-Sha; Ma, Yan; Yang, Long-Yan; Feng, Ying-Mei; Zhao, Dong

    2016-01-01

    Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs) in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF) treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on “VEGF uncoupling with nitric oxide” and “competitive angiopoietin 1/angiopoietin 2” mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics. PMID:27313624

  14. Stereological Analysis of Neuron, Glial and Endothelial Cell Numbers in the Human Amygdaloid Complex

    PubMed Central

    García-Amado, María; Prensa, Lucía

    2012-01-01

    Cell number alterations in the amygdaloid complex (AC) might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL), corticomedial and central groups), 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm3 and mean cell numbers (x106) were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals’ age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions. PMID:22719923

  15. Stereological analysis of neuron, glial and endothelial cell numbers in the human amygdaloid complex.

    PubMed

    García-Amado, María; Prensa, Lucía

    2012-01-01

    Cell number alterations in the amygdaloid complex (AC) might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL), corticomedial and central groups), 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm(3) and mean cell numbers (x10(6)) were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals' age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions.

  16. Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte-endothelial cell coculture model.

    PubMed

    Liu, Xin; Sun, Jiao

    2014-01-01

    Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte-endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease.

  17. Effects of simulated microgravity on cell cycle in human endothelial cells

    NASA Astrophysics Data System (ADS)

    Sokolovskaya, Alisa A.; Ignashkova, Tatiana I.; Bochenkova, Anna V.; Moskovtsev, Aleksey A.; Baranov, Victor M.; Kubatiev, Aslan A.

    2014-06-01

    The aim of the current study is to investigate effects of simulated microgravity on the cell cycle of endothelial cells. We analyze changes in the cell cycle after exposure of endothelial-like EA.hy 926 cells to simulated microgravity using a Desktop random positioning machine (RPM). Cell cycle profiles determined by flow cytometry show, that the percentage of the cells in the G0/G1 phase after 24 and 96 h of RPM-simulated microgravity is significantly increased as compared to the control group. However, no significant difference is observed after 120 h of RPM-simulated microgravity. In regard to S phase, the percentage of cells is significantly decreased after 24 and 96 h of RPM, respectively; whereas 120 h later, the number of S-phase cells is comparable to the control group. Thus, we show that simulated microgravity inhibits cell cycle progression of human EA.hy 926 cells from the G0/G1 phase to the S phase. We observe an effect of a hibernation-like state, when the growth of the cells in the RPM group slows down, but does not stop. Our results further show that simulated microgravity can affect adhesion of endothelial cells, and alpha-tubulin expression, as most cells begin to detach from the surface of OptiCell unit after 24 h, form aggregates after 48 h, and exhibit accumulation of alpha-tubulin around the nucleus after 48 h of exposure to simulated microgravity conditions. Our results demonstrate a chance in the cell cycle in a low gravitational field.

  18. Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation

    PubMed Central

    Zeng, Gefei; Taylor, Sarah M.; McColm, Janet R.; Kappas, Nicholas C.; Kearney, Joseph B.; Williams, Lucy H.; Hartnett, Mary E.; Bautch, Victoria L.

    2007-01-01

    New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell–derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gain-of-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis. PMID:17068148

  19. Genetic manipulation of sinusoidal endothelial cells.

    PubMed

    Takei, Yoshiyuki; Maruyama, Atsushi; Ikejima, Kenichi; Enomoto, Nobuyuki; Yamashina, Shunhei; Lemasters, John J; Sato, Nobuhiro

    2007-06-01

    Altered gene expression in liver sinusoidal endothelial cells (SEC) is associated with a variety of aspects of liver pathophysiology. It is, therefore, possible to envision a new therapeutic strategy for treatment of intractable liver diseases and achievement of graft-specific immunotolerance through modulation of SEC functions by genetic engineering. The SEC possesses unique hyaluronan receptors that recognize and internalize hyaluronic acid (HA). This characteristic was used in the development of a system for targeting foreign DNA to SEC. A gene carrier system was prepared by coupling HA oligomers to poly L-lysine (PLL) in a 1:1 weight ratio by reductive amination reaction. The resulting copolymer (PLL-g-HA) was mixed with various amounts of DNA in 154 mM NaCl. Inter-polyelectrolyte complex formation between PLL-g-HA and DNA exhibited minimal self-aggregation, explaining the highly soluble nature of the complex. Complex formation between PLL-g-HA and DNA was further assessed with a gel retardation assay. The titration point representing the minimum proportion of PLL-g-HA required to retard the DNA completely occurred at a 1:1 copolymer (based on PLL) to DNA charge ratio. Following intravenous injection of (32)P-labeled pSV beta-Gal plasmid complexed to PLL-g-HA in Wistar rats, >90% of the injected counts were shown to be taken up by the liver. Further, it was shown that the PLL-g-HA/DNA complex was distributed exclusively in the SEC. At 72 h after injection of 90 mug of pSV beta-Gal in a PLL-g-HA-complexed form, a large number of SEC expressing beta-galactosidase were detected. So, the PLL-g-HA/DNA system permits targeted delivery of exogenous nucleotide agents selectively to the liver SEC, providing a novel strategy for manipulation of SEC functions.

  20. Methamphetamine is not Toxic but Disrupts the Cell Cycle of Blood-Brain Barrier Endothelial Cells.

    PubMed

    Fisher, D; Gamieldien, K; Mafunda, P S

    2015-07-01

    The cytotoxic effects of methamphetamine (MA) are well established to be caused via induced oxidative stress which in turn compromises the core function of the blood-brain barrier (BBB) by reducing its ability to regulate the homeostatic environment of the brain. While most studies were conducted over a period of 24-48 h, this study investigated the mechanisms by which chronic exposure of MA adversely affect the endothelial cells of BBB over an extended period of 96 h. MA induced significant depression of cell numbers at 96 h. This result was supported by flow cytometric data on the cell cycle which showed that brain endothelial cells (bEnd5) at 96 h were significantly suppressed in the S-phase of the cell cycle. In contrast, at 24-72 h control cell numbers for G1, S and G2-M phases were similar to MA-exposed cells. MA (0-1,000 µM) did not, however, statistically affect the viability and cytotoxicity of the bEnd5 cells, and the profile of ATP production and DNA synthesis (BrdU) across 96 h did not provide a rationale for the suppression of cell division. Our study reports for the first time that chronic exposure to MA results in long-term disruption of the cell cycle phases which eventuates in the attenuation of brain capillary endothelial cell growth after 96 h, compounding and contributing to the already well-known adverse short-term permeability effects of MA exposure on the BBB.

  1. Endothelial Progenitor Cell Dysfunction in Polycystic Ovary Syndrome: Implications for The Genesis of Cardiovascular Diseases

    PubMed Central

    Kao, Yu-Hsun; Chiu, Wan-Chun; Hsu, Ming-I; Chen, Yi-Jen

    2013-01-01

    Polycystic ovary syndrome (PCOS), the most common endocrine disorder affecting women of reproductive age, is characterized by hyperandrogenism and insulin resistance. Women with PCOS have a higher risk for cardiovascular diseases (CVDs) and endothelial dysfunction. The mechanisms underlying these risks are unclear. Human peripheral blood contains circulating endothelial progenitor cells (EPCs) derived from bone marrow that have the ability to proliferate and differentiate into mature endothelial cells, which may contribute to vessel homeostasis and repair. PCOS is associated with insulin resistance, hyperinsulinemia, and dyslipidemia, which may result in EPC dysfunction. In this review, we summarize the potential mechanisms of EPC dysfunction in PCOS, which possibly result in a higher genesis of CVDs in PCOS-affected subjects. PMID:24520442

  2. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    PubMed

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  3. Characterization of iron uptake from transferrin by murine endothelial cells.

    PubMed

    Hallmann, R; Savigni, D L; Morgan, E H; Baker, E

    2000-01-01

    Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

  4. Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor.

    PubMed

    Schwafertz, Carolin; Schinner, Sven; Kühn, Markus C; Haase, Matthias; Asmus, Amelie; Mülders-Opgenoorth, Birgit; Ansurudeen, Ishrath; Hornsby, Peter J; Morawietz, Henning; Oetjen, Elke; Schott, Matthias; Willenberg, Holger S

    2017-02-05

    Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of β-catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect β-catenin activity in adrenocortical NCIH295R cells. Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of β-catenin and an increase in β-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway. Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced β-catenin-dependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM. Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate β-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.

  5. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    EPA Pesticide Factsheets

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  6. Oxidized low-density lipoprotein alters endothelial progenitor cell populations.

    PubMed

    Cui, Yuqi; Narasimhulu, Chandrakala A; Liu, Lingjuan; Li, Xin; Xiao, Yuan; Zhang, Jia; Xie, Xiaoyun; Hao, Hong; Liu, Jason Z; He, Guanglong; Cowan, Peter J; Cui, Lianqun; Zhu, Hua; Parthasarathy, Sampath; Liu, Zhenguo

    2015-06-01

    Oxidized low-density lipoprotein (ox-LDL) is critical to atherosclerosis in hyperlipidemia. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are important to preventing atherosclerosis, and significantly decreased in hyperlipidemia. This study was to demonstrate ox-LDL and hyperlipidemia could exhibit similar effect on EPC population and the role of reactive oxygen species (ROS). ROS production in BM and blood was significantly increased in male C57BL/6 mice with intravenous ox-LDL treatment, and in hyperlipidemic LDL receptor knockout mice with 4-month high-fat diet. ROS formation was effectively blocked with overexpression of antioxidant enzymes or N-acetylcysteine treatment. In hyperlipidemic and ox-LDL-treated mice, c-Kit(+)/CD31(+) cell number in BM and blood, and Sca-1(+)/Flk-1(+) cell number in blood, not in BM, were significantly decreased, which were not affected by inhibiting ROS production, while blood CD34(+)/Flk-1(+) cell number was significantly increased that was prevented with reduced ROS formation. However, blood CD34(+)/CD133(+) cell number increased in ox-LDL-treated mice, while decreased in hyperlipidemic mice. These data suggested that ox-LDL produced significant changes in BM and blood EPC populations similar (but not identical) to chronic hyperlipidemia with predominantly ROS-independent mechanism(s).

  7. Effect of Hypergravity on Endothelial Cell Function and Gene Expression

    NASA Astrophysics Data System (ADS)

    Morbidelli, Lucia; Marziliano, Nicola; Basile, Venere; Pezzatini, Silvia; Romano, Giovanni; Conti, Antonio; Monici, Monica

    2009-01-01

    It is well known that endothelial cells (ECs), which play a major role in cardiovascular system functioning, are very sensitive to mechanical stimuli. It has been demonstrated that changes in inertial conditions (i.e. microgravity and hypergravity) can affect both phenotypic and genotypic expression in ECs. In this report we describe the effects of hypergravity on ECs isolated from bovine aorta (BAECs). ECs were repeatedly exposed to discontinuous hypergravity conditions (5 × 10 min at 10× g with 10 min at 1× g between sets), simulated in a hyperfuge. Then, cell morphology and metabolism were analyzed by autofluorescence techniques. The phenotypic expression of cytoskeleton constituents ( β-actin, vimentin, tubulin), adhesion and survival signals (integrins), mediators of inflammation and angiogenesis was evaluated by immunocytofluorescence. Quantitative PCR (Q-PCR) with Low Density Arrays (LDAs) was used to evaluate modifications in gene expression. After hypergravity exposure, no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substratum, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. On the whole, the results documented that hypergravity exposure maintained EC survival and function by activation of adaptive mechanisms.

  8. Facile endothelial cell micropatterning induced by reactive oxygen species on polydimethylsiloxane substrates.

    PubMed

    Choi, Jong Seob; Kim, Do Hyun; Seo, Tae Seok

    2016-04-01

    Sophisticated cell pattern provides unique cellular assay platform for studying cell to cell interaction, cellular differentiation and signaling, high-throughput cell response to chemicals. In this study, we demonstrated reactive oxygen species (ROS) mediated endothelial cell micropatterning on a polydimethylsiloxane (PDMS) substrate. The exposure of UV/O radiation led to the formation of ROS on the surface of PDMS, which could selectively prevent adhesion of endothelial cells. The degree of ROS amount was monitored according to the UV/O irradiation time, and at least 36 μM of ROS resulted in the precise cellular micropattern on the PDMS. The presence of ROS affected not only cellular detachment from the substrate, but also endothelial cell morphology such as cell spreading area, confluence, nuclear area and nuclear inverse aspect ratio. In addition, we could observe that the actin cytoskeleton of cells was also constricted due to ROS, thereby minimizing the focal adhesion area of vinculin. Compared with previously reported methods which use chemical treatment or nano/microstructure on the substrate, the proposed methodology is quite simple, accurate, and harmless to the patterned endothelial cells.

  9. Descending vasa recta endothelial cells and pericytes form mural syncytia

    PubMed Central

    Zhang, Zhong; Lin, Hai; Cao, Chunhua; Payne, Kristie

    2013-01-01

    Using patch clamp, we induced depolarization of descending vasa recta (DVR) pericytes or endothelia and tested whether it was conducted to distant cells. Membrane potential was measured with the fluorescent voltage dye di-8-ANEPPS or with a second patch-clamp electrode. Depolarization of an endothelial cell induced responses in other endothelia within a millisecond and was slowed by gap junction blockade with heptanol. Endothelial response to pericyte depolarization was poor, implying high-resistance myo-endothelial coupling. In contrast, dual patch clamp of neighboring pericytes revealed syncytial coupling. At high sampling rate, the spread of depolarization between pericytes and endothelia occurred in 9 ± 2 or 12 ± 2 μs, respectively. Heptanol (2 mM) increased the overall input resistance of the pericyte layer to current flow and prevented transmission of depolarization between neighboring cells. The fluorescent tracer Lucifer yellow (LY), when introduced through ruptured patches, spread between neighboring endothelia in 1 to 7 s, depending on location of the flanking cell. LY diffused to endothelial cells on the ipsilateral but not contralateral side of the DVR wall and minimally between pericytes. We conclude that both DVR pericytes and endothelia are part of individual syncytia. The rate of conduction of membrane potential exceeds that for diffusion of hydrophilic molecules by orders of magnitude. Gap junction coupling of adjacent endothelial cells may be spatially oriented to favor longitudinal transmission along the DVR axis. PMID:24381184

  10. Factors associated with graft survival and endothelial cell density after Descemet’s stripping automated endothelial keratoplasty

    PubMed Central

    Ishii, Nobuhito; Yamaguchi, Takefumi; Yazu, Hiroyuki; Satake, Yoshiyuki; Yoshida, Akitoshi; Shimazaki, Jun

    2016-01-01

    Postoperative endothelial cell loss leads to graft failure after corneal transplantation, and is one of the important issues for long-term prognosis. The objective of this study was to identify clinical factors affecting graft survival and postoperative endothelial cell density (ECD) after Descemet’s stripping automated endothelial keratoplasty (DSAEK). A total of 198 consecutive Japanese patients (225 eyes) who underwent DSAEK were analysed using Cox proportional hazard regression and multiple linear regression models. The candidate factors included recipient age; gender; diagnosis; pre-existing iris damage state, scored based on its severity; the number of previous intraocular surgeries; graft ECD; graft diameter; simultaneous cataract surgery; surgeons experience; intraoperative iris damage; postoperative rebubbling; and graft rejection. Eyes with higher pre-existing iris damage score and more number of previous intraocular surgery had a significantly higher risk of graft failure (HR = 8.53; P < 0.0001, and HR = 2.66; P = 0.026, respectively). Higher pre-existing iris damage score, lower graft ECD, and smaller graft diameter were identified as significant predisposing factors for lower postoperative ECD. The results show that iris damage status before DSAEK may be clinically useful in predicting the postoperative course. Avoiding intraoperative iris damage, especially in eyes with low ECD can change the prognosis of future DSAEK. PMID:27121659

  11. Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

    PubMed

    Schumacher, V L; Martel, A; Pasmans, F; Van Immerseel, F; Posthaus, H

    2013-07-01

    Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

  12. Vascular endothelial growth factor signalling in endothelial cell survival: A role for NF{kappa}B

    SciTech Connect

    Grosjean, Jennifer . E-mail: Jennifer.grosjean@imperial.ac.uk; Kiriakidis, Serafim; Reilly, Kerri; Feldmann, Marc; Paleolog, Ewa

    2006-02-17

    Angiogenesis is the development of blood capillaries from pre-existing vessels. Vascular endothelial growth factor (VEGF) is a key regulator of vessel growth and regression, and acts as an endothelial survival factor by protecting endothelial cells from apoptosis. Many genes involved in cell proliferation and apoptosis are regulated by the nuclear factor kappa B (NF{kappa}B) transcription factor family. This study aimed to address the hypothesis that VEGF-mediated survival effects on endothelium involve NF{kappa}B. Using an NF{kappa}B-luciferase reporter adenovirus, we observed activation of NF{kappa}B following VEGF treatment of human umbilical vein endothelial cells. This was confirmed using electrophoretic mobility shift assay and found to involve nuclear translocation of NF{kappa}B sub-unit p65. However, NF{kappa}B activation occurred without degradation of inhibitory I{kappa}B proteins (I{kappa}B{alpha}, I{kappa}B{beta}, and I{kappa}B{epsilon}). Instead, tyrosine phosphorylation of I{kappa}B{alpha} was observed following VEGF treatment, suggesting NF{kappa}B activation was mediated by degradation-independent dissociation of I{kappa}B{alpha} from NF{kappa}B. Adenovirus-mediated over-expression of either native I{kappa}B{alpha}, or of I{kappa}B{alpha} in which tyrosine residue 42 was mutated to phenylalanine, inhibited induction of NF{kappa}B-dependent luciferase activity in response to VEGF. Furthermore, VEGF-induced upregulation of mRNA for the anti-apoptotic protein Bcl-2 and cell survival following serum withdrawal was reduced following I{kappa}B{alpha} over-expression. This study highlights that different molecular mechanisms of NF{kappa}B activation may be involved downstream of stimuli which activate the endothelial lining of blood vessels.

  13. Shaping of Peripheral T Cell Responses by Lymphatic Endothelial Cells

    PubMed Central

    Humbert, Marion; Hugues, Stéphanie; Dubrot, Juan

    2017-01-01

    Lymph node stromal cells (LNSCs) have newly been promoted to the rank of new modulators of T cell responses. The different non-hematopoietic cell subsets in lymph node (LN) were considered for years as a simple scaffold, forming routes and proper environment for antigen (Ag)-lymphocyte encountering. Deeper characterization of those cells has recently clearly shown their impact on both dendritic cell and T cell functions. In particular, lymphatic endothelial cells (LECs) control lymphocyte trafficking and homeostasis in LNs and limit adaptive immune responses. Therefore, the new role of LECs in shaping immune responses has drawn the attention of immunologists. Striking is the discovery that LECs, among other LNSCs, ectopically express a large range of peripheral tissue-restricted Ags (PTAs), and further present PTA-derived peptides through major histocompatibility class I molecules to induce self-reactive CD8+ T cell deletional tolerance. In addition, both steady-state and tumor-associated LECs were described to be capable of exogenous Ag cross-presentation. Whether LECs can similarly impact CD4+ T cell responses through major histocompatibility class II restricted Ag presentation is still a matter of debate. Here, we review and discuss our current knowledge on the contribution of Ag-presenting LECs as regulators of peripheral T cell responses in different immunological contexts, including autoimmunity and cancer. PMID:28127298

  14. Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier

    PubMed Central

    Bonakdar, Mohammad; Wasson, Elisa M.; Lee, Yong W.; Davalos, Rafael V.

    2016-01-01

    The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway. PMID:26789772

  15. Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier.

    PubMed

    Bonakdar, Mohammad; Wasson, Elisa M; Lee, Yong W; Davalos, Rafael V

    2016-01-19

    The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway.

  16. Generation and Characterization of Telomerase-Transfected Human Lymphatic Endothelial Cells with an Extended Life Span

    PubMed Central

    Nisato, Riccardo E.; Harrison, Jillian A.; Buser, Raphaele; Orci, Lelio; Rinsch, Chris; Montesano, Roberto; Dupraz, Philippe; Pepper, Michael S.

    2004-01-01

    The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin α9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro. PMID:15215158

  17. Extra- and intracellular innate immune recognition in endothelial cells.

    PubMed

    Opitz, Bastian; Hippenstiel, Stefan; Eitel, Julia; Suttorp, Norbert

    2007-08-01

    The innate immune system represents the principal sensor of infections in multicellular organisms and might also mediate responses to some endogenous molecules. In this context, endothelial cells are among the first cells coming into contact with microbial or endogenous (danger-associated) molecules or whole pathogens entering the bloodstream. Since many bacteria and viruses invade the endothelium, endothelial cells are equipped with both extracellular and cytosolic surveillance systems capable of sensing microbial components, and endogenous danger-associated molecules. The receptor molecules, called pattern recognition receptors (PRRs), are classified as transmembrane or cytosolic molecules. While the transmembrane PRRs recognize extracellular and membrane-enclosed foreign organisms, the cytosolic PRRs appear to sense intracellular infections. Here we focus on both PRR classes in general, and outline the current knowledge of extra- and intracellular pattern recognition in endothelial cells and its potential role in vascular diseases and sepsis.

  18. Human cultured endothelial cells do secrete endothelin-1

    SciTech Connect

    Clozel, M.; Fischli, W. )

    1989-01-01

    Endothelin-1 (ET-1) has been identified in the conditioned medium of porcine endothelial cells. Human endothelin (ET-1) cloned from a placenta cDNA library is similar to porcine, but it is not known whether endothelin itself is secreted by human endothelial cells. To answer this question, a conditioned medium taken every 48 h from confluent cultures of umbilical vein endothelial cells was analyzed by HPLC and all fractions were tested for their ability to inhibit ({sup 125}I)ET-1 binding on human placenta membranes. Only one fraction did inhibit ({sup 125}I)ET-1 binding. When the conditioned medium was spiked with ET-1, the same single fraction inhibited ({sup 125}I)ET-1 binding showing that ET-1, itself, is present in the conditioned medium of human endothelial cells. ET-1 accumulates with time, reaching a plateau at 48 h. ET-1 secretion is not increased by a 24-h incubation of endothelial cells with phorbol myristate acetate, interleukin-1, tumor necrosis factor, thrombin or neuropeptide Y.

  19. Fibroblast nemosis induces angiogenic responses of endothelial cells

    SciTech Connect

    Enzerink, Anna; Rantanen, Ville; Vaheri, Antti

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  20. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    PubMed Central

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  1. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

    PubMed

    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering.

  2. Principles of targeting endothelial cell metabolism to treat angiogenesis and endothelial cell dysfunction in disease

    PubMed Central

    Goveia, Jermaine; Stapor, Peter; Carmeliet, Peter

    2014-01-01

    The endothelium is the orchestral conductor of blood vessel function. Pathological blood vessel formation (a process termed pathological angiogenesis) or the inability of endothelial cells (ECs) to perform their physiological function (a condition known as EC dysfunction) are defining features of various diseases. Therapeutic intervention to inhibit aberrant angiogenesis or ameliorate EC dysfunction could be beneficial in diseases such as cancer and cardiovascular disease, respectively, but current strategies have limited efficacy. Based on recent findings that pathological angiogenesis and EC dysfunction are accompanied by EC-specific metabolic alterations, targeting EC metabolism is emerging as a novel therapeutic strategy. Here, we review recent progress in our understanding of how EC metabolism is altered in disease and discuss potential metabolic targets and strategies to reverse EC dysfunction and inhibit pathological angiogenesis. PMID:25063693

  3. Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells.

    PubMed

    Scarritt, Michelle E; Pashos, Nicholas C; Motherwell, Jessica M; Eagle, Zachary R; Burkett, Brian J; Gregory, Ashley N; Mostany, Ricardo; Weiss, Daniel J; Alvarez, Diego F; Bunnell, Bruce A

    2016-12-12

    Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that hemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells (HUVECs) were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells (PAECs) and rat pulmonary vein endothelial cells (PVECs) attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells (MVECs) deposited mostly in microvessels. Combination seeding of PAECs, PVECs, and MVECs led to positive VE-cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. This article is protected by copyright. All rights reserved.

  4. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    NASA Astrophysics Data System (ADS)

    Shen, Qin; Goderie, Susan K.; Jin, Li; Karanth, Nithin; Sun, Yu; Abramova, Natalia; Vincent, Peter; Pumiglia, Kevin; Temple, Sally

    2004-05-01

    Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

  5. Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke

    PubMed Central

    Yu, Qi-jin; Tao, Hong; Wang, Xin; Li, Ming-chang

    2015-01-01

    Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions. PMID:26807131

  6. 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells.

    PubMed

    Ladurner, Angela; Atanasov, Atanas G; Heiss, Elke H; Baumgartner, Lisa; Schwaiger, Stefan; Rollinger, Judith M; Stuppner, Hermann; Dirsch, Verena M

    2012-09-15

    Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity. DPPB (1-10 μM) treatment for 24 h induced a significant and dose-dependent increase in eNOS activity as determined by the [(14)C]L-arginine/[(14)C]L-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser(1177) and decrease in eNOS-Thr(495) phosphorylation, as well as increased AMPK phosphorylation at Thr(172), whereas Akt phosphorylation at Ser(473) was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca(2+) chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser(1177) and AMPK-Thr(172) phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca(2+) concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca(2+) levels and increases signaling through a CaMKKβ-AMPK dependent pathway.

  7. Directed Endothelial Cell Morphogenesis in Micropatterned Gelatin Methacrylate Hydrogels

    PubMed Central

    Nikkhah, Mehdi; Eshak, Nouran; Zorlutuna, Pinar; Annabi, Nasim; Castello, Marco; Kim, Keekyoung; Dolatshahi-Pirouz, Alireza; Edalat, Faramarz; Bae, Hojae; Yang, Yunzhi; Khademhosseini, Ali

    2013-01-01

    Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50–150 µm height). We demonstrated the significance dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 µm-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs. PMID:23018132

  8. Magnetizable stent-grafts enable endothelial cell capture

    NASA Astrophysics Data System (ADS)

    Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

  9. Role of the Retinal Vascular Endothelial Cell in Ocular Disease

    PubMed Central

    Bharadwaj, Arpita S.; Appukuttan, Binoy; Wilmarth, Phillip A.; Pan, Yuzhen; Stempel, Andrew J.; Chipps, Timothy J.; Benedetti, Eric E.; Zamora, David O.; Choi, Dongseok; David, Larry L.; Smith, Justine R.

    2012-01-01

    Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell. PMID:22982179

  10. Cerebral Cavernous Malformations: Somatic Mutations in Vascular Endothelial Cells

    PubMed Central

    Gault, Judith; Awad, Issam A.; Recksiek, Peter; Shenkar, Robert; Breeze, Robert; Handler, Michael; Kleinschmidt-DeMasters, Bette Kay

    2009-01-01

    OBJECTIVE Germline mutations in three genes have been found in familial cases of cerebral cavernous malformations (CCM). We previously discovered somatic and germline truncating mutations in the KRIT1 gene supporting the “two-hit” mechanism of CCM lesion formation in a single lesion. The purpose of this study was to screen for somatic, nonheritable, mutations in three more lesions from different patients and identify the cell type(s) in which somatic mutations occur. METHODS Somatic mutations were sought in DNA from three surgically excised, fresh-frozen CCM lesions by cloning and screening PCR products generated from KRIT1 or PDCD10 coding regions. Laser capture microdissection (LCM) was used to isolated endothelial and nonendothelial cells in order to determine if somatic mutations were found in endothelial cells. RESULTS A CCM lesion harbored somatic and germline KRIT1 mutations on different chromosomes and are therefore biallelic. Both mutations are predicted to truncate the protein. The KRIT1 somatic mutations (novel c.1800delG mutation and previously identified 34 nucleotide deletion) in CCMs from two different patients were only found in the vascular endothelial cells lining caverns. No obvious somatic mutations were identified in the two other lesions; however, the results were inconclusive possibly due to the technical limitations or the fact that these specimens had a small proportion of vascular endothelial cells lining pristine caverns. CONCLUSION The “two-hit” mechanism occurs in vascular endothelial cells lining CCM caverns from two patients with somatic and Hispanic-American KRIT1 germline mutations. Methods for somatic mutation detection should focus on vascular endothelial cells lining pristine caverns. PMID:19574835

  11. INTACT AND INJURED ENDOTHELIAL CELLS DIFFERENTIALLY MODULATE POSTNATAL MURINE FOREBRAIN NEURAL STEM CELLS

    PubMed Central

    Plane, Jennifer M.; Andjelkovic, Anuska V.; Keep, Richard F.; Parent, Jack M.

    2010-01-01

    Neural stem cells (NSCs) persist in the forebrain subventricular zone (SVZ) within a niche containing endothelial cells. Evidence suggests that endothelial cells stimulate NSC expansion and neurogenesis. Experimental stroke increases neurogenesis and angiogenesis, but how endothelial cells influence stroke-induced neurogenesis is unknown. We hypothesized intact or oxygen-glucose deprived (OGD) endothelial cells secrete factors that enhance neurogenesis. We co-cultured mouse SVZ neurospheres (NS) with endothelial cells, or differentiated NS in endothelial cell-conditioned medium (ECCM). NS also were expanded in ECCM from OGD-exposed (OGD-ECCM) endothelial cells to assess injury effects. ECCM significantly increased NS production. NS co-cultured with endothelial cells or ECCM generated more immature-appearing neurons and oligodendrocytes, and astrocytes with radial glial-like/reactive morphology than controls. OGD-ECCM stimulated neuroblast migration and yielded neurons with longer processes and more branching. These data indicate that intact and injured endothelial cells exert differing effects on NSCs, and suggest targets for stimulating regeneration after brain insults. PMID:19837162

  12. Tumor endothelial cells express high pentraxin 3 levels.

    PubMed

    Hida, Kyoko; Maishi, Nako; Kawamoto, Taisuke; Akiyama, Kosuke; Ohga, Noritaka; Hida, Yasuhiro; Yamada, Kenji; Hojo, Takayuki; Kikuchi, Hiroshi; Sato, Masumi; Torii, Chisaho; Shinohara, Nobuo; Shindoh, Masanobu

    2016-12-01

    It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

  13. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    NASA Astrophysics Data System (ADS)

    Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

    1988-05-01

    The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

  14. The antiangiogenic agent Neovastat (AE-941) induces endothelial cell apoptosis.

    PubMed

    Boivin, Dominique; Gendron, Sébastien; Beaulieu, Edith; Gingras, Denis; Béliveau, Richard

    2002-08-01

    Neovastat (AE-941), a naturally occurring multifunctional antiangiogenic agent, has been shown to inhibit key components of the angiogenic process, including matrix metalloproteinases and vascular endothelial growth factor-mediated signaling events. In this study, we report the presence of a proapoptotic activity within this compound. Neovastat treatment of bovine aortic endothelial cells caused cell death with characteristics of apoptosis, including chromatin condensation and DNA fragmentation. Neovastat markedly induced caspase-3, caspase-8, and caspase-9 activities, at similar levels to those measured in cells treated with tumor necrosis factor-alpha. Activation of caspases by Neovastat appears to be essential for its proapoptotic effects because all apoptotic features were blocked by zVAD-fmk, a broad-spectrum caspase inhibitor. The activation of caspases was correlated with the cleavage of the nuclear substrate poly(ADP-ribose) polymerase, and by a concomitant release of cytochrome c from mitochondria to the cytoplasm. Neovastat-induced apoptosis appears to be specific to endothelial cells because treatment of other cell types such as U-87, COS-7, NIH-3T3, and SW1353 did not result in increased caspase-3 activity. These results demonstrate that Neovastat contains a proapoptotic factor that specifically induces the activation of caspases in endothelial cells and the resulting apoptosis of these cells.

  15. Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

    PubMed Central

    Kanda, Shigeru; Kanetake, Hiroshi; Miyata, Yasuyoshi

    2007-01-01

    Abstract The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells.LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis.Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation.Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells. PMID:17521372

  16. Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation

    PubMed Central

    Wilson, Calum; Lee, Matthew D.

    2016-01-01

    Key points The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. Abstract Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction. PMID:27730645

  17. The effects of adiponectin and leptin on human endothelial cell proliferation: a live-cell study.

    PubMed

    Alvarez, Granada; Visitación Bartolomé, M; Miana, María; Jurado-López, Raquel; Martín, Ruben; Zuluaga, Pilar; Martinez-Martinez, Ernesto; Nieto, M Luisa; Alvarez-Sala, Luis A; Millán, Jesús; Lahera, Vicente; Cachofeiro, Victoria

    2012-01-01

    The effect of adiponectin and leptin on the proliferation of the human microvascular endothelial cell line (HMEC-1) was studied in the absence or presence of fetal bovine serum (FBS). The participation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) pathways in this effect were evaluated. We studied the effect of both adipokines on the motility, mitosis, proliferation and cell death processes of HMEC-1 cells using live-cell imaging techniques. Adiponectin but not leptin further increased the proliferative effect induced by FBS on HMEC-1. This effect seems to be the consequence of an increase in the mitotic index in adiponectin-treated cells when compared to untreated ones. The presence of either the mitogen-activated protein kinase (MAPK) inhibitor (PD98059), or PI-3K inhibitor (LY294002), reduced the effect of adiponectin in a dose-dependent manner. Neither adipokine was able to affect HMEC-1 proliferation in FBS-free conditions. Duration of mitosis, cell motility and the cell death process were similar in all conditions. These data suggest that adiponectin and leptin exert different effects on endothelial cell function. Adiponectin was able to potentiate proliferation of HMEC-1. This effect involves the activation of both PI3-K/Akt and ERK/MAPK pathways. However, it seems to exert minimal effects on HMEC-1 function in the case of leptin.

  18. Methamphetamine disrupts blood-brain barrier function by induction of oxidative stress in brain endothelial cells.

    PubMed

    Ramirez, Servio H; Potula, Raghava; Fan, Shongshan; Eidem, Tess; Papugani, Anil; Reichenbach, Nancy; Dykstra, Holly; Weksler, Babette B; Romero, Ignacio A; Couraud, Pierre O; Persidsky, Yuri

    2009-12-01

    Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood-brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. In this study, we provide evidence that METH alters BBB function through direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human brain microvascular endothelial cells (BMVEC) to METH diminishes the tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing the expression of cell membrane-associated tight junction (TJ) proteins. These changes were accompanied by the enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Antioxidant treatment attenuated or completely reversed all tested aspects of METH-induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.

  19. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

    NASA Astrophysics Data System (ADS)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

    1983-11-01

    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  20. Decellularized extracellular matrix of human umbilical vein endothelial cells promotes endothelial differentiation of stem cells from exfoliated deciduous teeth.

    PubMed

    Gong, Ting; Heng, Boon Chin; Xu, Jianguang; Zhu, Shaoyue; Yuan, Changyong; Lo, Edward Chin Man; Zhang, Chengfei

    2017-04-01

    Dental stem cells can serve as a potential source of functional endothelial cells for tissue engineering applications, but the endothelial-lineage differentiation efficiency is rather low even with growth factors and mechanical stimuli, which greatly limits their clinical applications. This is partly due to the deficiency of standard two-dimensional (2-D) culture systems, which is unable to recapitulate the three-dimensional (3-D) in vivo milieu that is rich in extracellular matrix. Hence, we extracted decellularized extracellular matrix from human umbilical vein endothelial cells (HUVECs-DECM) to provide a bioactive substratum conducive to the endothelial differentiation of dental stem cells. Compared to cells plated on tissue culture polystyrene (TCP), stem cells from exfoliated deciduous teeth (SHED) cultured on the HUVECs-DECM demonstrated more regular arrangement and elongated morphology. HUVECs-DECM significantly enhanced the rapid adhesion and proliferation rates of SHED, as demonstrated by WST-8 assay and immunocytochemistry indicating higher expression levels of vinculin by newly adherent SHED on HUVECs-DECM versus TCP. In addition, there was twofold to fivefold higher mRNA expression levels of endothelial-specific markers CD31 and VEGFR-2 in SHED after seven days of culture on DECM versus TCP. Functional testing with in vitro matrigel angiogenesis assay identified more capillary-like structure formation with significantly higher tubule length in SHED induced by DECM versus TCP. Hence, the results of this study provide a better understanding of the unique characteristics of cell-specific ECM and demonstrated the potential use of HUVECs-DECM as a culture substratum conducive for stimulating the endothelial differentiation of SHED for therapeutic angiogenic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1083-1093, 2017.

  1. Bidirectional Crosstalk between Lymphatic Endothelial Cell and T Cell and Its Implications in Tumor Immunity

    PubMed Central

    Yeo, Kim Pin; Angeli, Veronique

    2017-01-01

    Lymphatic vessels have been traditionally considered as passive transporters of fluid and lipids. However, it is apparent from recent literature that the function of lymphatic vessels is not only restricted to fluid balance homeostasis but also extends to regulation of immune cell trafficking, antigen presentation, tolerance, and immunity, all which may impact the progression of inflammatory responses and diseases such as cancer. The lymphatic system and the immune system are intimately connected, and there is emergent evidence for a crosstalk between T cell and lymphatic endothelial cell (LEC). This review describes how LECs in lymph nodes can affect multiple functional properties of T cells and the impact of these LEC-driven effects on adaptive immunity and, conversely, how T cells can modulate LEC growth. The significance of such crosstalk between T cells and LECs in cancer will also be discussed. PMID:28220121

  2. Effects of Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells.

    PubMed

    Dietmann, Anelia; Millonig, Alban; Combes, Valery; Couraud, Pierre-Olivier; Kachlany, Scott C; Grau, Georges E

    2013-01-01

    Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 μg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 μg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.

  3. Zinc and dexamethasone induce metallothionein accumulation by endothelial cells

    SciTech Connect

    Briske-Anderson, M.; Bobilya, D.J.; Reeves, P.G. )

    1991-03-11

    Several tissues increase their metallothionein (MT) concentration when exposed to elevated amounts of plasma Zn. Endothelial cells form the blood vessels that supply all tissues and constitute a barrier between cells of tissues and the blood. This study examined the ability of endothelial cells to synthesize MT and accumulate Zn in response to high amounts of Zn and dexamethasone. Bovine pulmonary endothelial cells were grown to confluence in Minimum Essential Medium with Earle's salts and 10% fetal calf serum. The monolayer was maintained for 2 d prior to use in medium containing EDTA-dialyzed serum. This low Zn medium was replaced with one containing 1, 6, 25, 50, 100, 150, or 200 {mu}M Zn and incubated for 24 hr before harvesting the cells. MT was quantified by the cadmium binding assay. Cellular Zn concentrations were analyzed by atomic absorption after a nitric acid digestion. The MT concentration was elevated in response to Zn concentrations of 100 {mu}M or more. Cellular Zn concentration was elevated when media Zn was 25 {mu}M or more. MT and cellular Zn concentrations were positively correlated. In another study, inclusion of 0.1 {mu}M dexamethasone in the media increased concentration at all Zn concentrations studied. However, the inclusion of 0.3 {mu}M cis-platinum had no effect. In conclusion, endothelial cells in culture respond to elevated amounts of Zn and dexamethasone in the media by accumulating Zn and MT.

  4. Contractile proteins of endothelial cells, platelets and smooth muscle.

    PubMed

    Becker, C G; Nachman, R L

    1973-04-01

    In experiments described herein it was observed, by direct and indirect immunofluorescence technics, that rabbit antisera to human platelet actomyosin (thrombosthenin) stained mature megakaryocytes, blood platelets, endothelial cells and smooth muscle cells of arteries and veins, endothelial cells of liver sinusoids and certain capillaries, uterine smooth muscle cells, myoepithelial cells, perineurial cells of peripheral nerves and "fibroblastic" cells of granulation tissue. The specificity of immunohistologic staining was confirmed by appropriate absorption and blocking studies and immunodiffusional analysis in agarose gel. It was also observed by immunodiffusional analysis in agarose gel, electrophoresis of actomyosin fragments in polyacrylamide gels, immune inhibition of actomyosin ATPase activity and immune aggregation of platelets that uterine and platelet actomyosin are partially, but not completely, identical.

  5. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    NASA Astrophysics Data System (ADS)

    Arbeitman, Claudia R.; del Grosso, Mariela F.; Behar, Moni; García Bermúdez, Gerardo

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  6. Calcium handling in porcine coronary endothelial cells by gastrin-17.

    PubMed

    Grossini, E; Molinari, C; Sigaudo, L; Biella, M; Mary, D A S G; Vacca, G

    2013-04-01

    In porcine coronary artery endothelial cells (PCAEC), gastrin-17 has recently been found to increase nitric oxide (NO) production by the endothelial NO synthase (eNOS) isoform through cholecystokinin 1/2 (CCK1/2) receptors and the involvement of protein kinase A (PKA), PKC and the β2-adrenoreceptor-related pathway. As eNOS is the Ca(2)(+)-dependent isoform of the enzyme, we aimed to examine the effects of gastrin-17 on Ca(2)(+) movements. Thus, experiments were performed in Fura-2-acetoxymethyl-ester-loaded PCAEC, where changes of cytosolic Ca(2)(+) ([Ca(2)(+)]c) caused by gastrin-17 were analysed and compared with those of CCK receptors and β2-adrenoreceptors agonists/antagonists. In addition, some experiments were performed by stimulating cells with gastrin-17 in the presence or absence of cAMP/PKA activator/inhibitor and of phospholipase C (PLC) and Ca(2)(+)-calmodulin dependent protein kinase II (CaMKII) blockers. The results have shown that gastrin-17 can promote a transient increase in [Ca(2)(+)]c mainly originating from an intracellular pool sensitive to thapsigargin and from the extracellular space. In addition, the response of cells to gastrin-17 was increased by the adenylyl cyclase activator and the β2-adrenoreceptor agonists and affected mainly by the CCK2 receptor agonists/antagonists. Moreover, the effects of gastrin-17 were prevented by β2-adrenoreceptors and CaMKII blockers and the adenylyl cyclase/PKA and PLC inhibitors. Finally, in PCAEC cultured in Na(+)-free medium or loaded with the plasma membrane Ca(2)(+) pump inhibitor, the gastrin-17-evoked Ca(2)(+) transient was long lasting. In conclusion, this study shows that gastrin-17 affected intracellular Ca(2)(+) homeostasis in PCAEC by both promoting a discharge of an intracellular pool and by interfering with the operation of store-dependent channels through mainly CCK2 receptors and PKA/PLC- and CaMKII-related signalling downstream of β2-adrenoreceptor stimulation.

  7. Matrix Gla protein regulates differentiation of endothelial cells derived from mouse embryonic stem cells.

    PubMed

    Yao, Jiayi; Guihard, Pierre J; Blazquez-Medela, Ana M; Guo, Yina; Liu, Ting; Boström, Kristina I; Yao, Yucheng

    2016-01-01

    Matrix Gla protein (MGP) is an antagonist of bone morphogenetic proteins and expressed in vascular endothelial cells. Lack of MGP causes vascular abnormalities in multiple organs in mice. The objective of this study is to define the role of MGP in early endothelial differentiation. We find that expression of endothelial markers is highly induced in Mgp null organs, which, in wild type, contain high MGP expression. Furthermore, Mgp null embryonic stem cells express higher levels of endothelial markers than wild-type controls and an abnormal temporal pattern of expression. We also find that the Mgp-deficient endothelial cells adopt characteristics of mesenchymal stem cells. We conclude that loss of MGP causes dysregulation of early endothelial differentiation.

  8. Endothelial colony forming cells ameliorate endothelial dysfunction via secreted factors following ischemia-reperfusion injury.

    PubMed

    Collett, Jason A; Mehrotra, Purvi; Crone, Allison; Shelley, W Christopher; Yoder, Mervin C; Basile, David P

    2017-02-22

    Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFCs) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFCs may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia/reperfusion (I/R). Rat pulmonary microvascular ECs (PMVEC) with high proliferative potential were compared with pulmonary artery ECs (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 hours of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of adhesion molecules such as ICAM-1 and p-selectin, and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.

  9. Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

    SciTech Connect

    Li Aihua; Cheng Guangli; Zhu Genghui; Tarnawski, Andrzej S. . E-mail: atarnawski@yahoo.com

    2007-02-09

    Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increased in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.

  10. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  11. Biomechanical changes in endothelial cells result from an inflammatory response

    NASA Astrophysics Data System (ADS)

    Vaitkus, Janina; Stroka, Kimberly; Aranda-Espinoza, Helim

    2012-02-01

    During periods of infection and disease, the immune system induces the release of TNF-α, an inflammatory cytokine, from a variety of cell types, such as macrophages. TNF-α, while circulating in the vasculature, binds to the apical surface of endothelial cells and causes a wide range of biological and mechanical changes to the endothelium. While the biological changes have been widely studied, the biomechanical aspects have been largely unexplored. Here, we investigated the biomechanical changes of the endothelium as a function of TNF-α treatment. First, we studied the traction forces applied by the endothelium, an effect that is much less studied than others. Through the use of traction force microscopy, we found that TNF-α causes an increase in traction forces applied by the endothelial cells as compared to non-treated cells. Then, we investigated cell morphology, cell mechanics, migration, and cytoskeletal dynamics. We found that in addition to increasing applied traction forces, TNF-α causes an increase in cell area and aspect ratio on average, as well as a shift in the organization of F-actin filaments within the cell. Combining these findings together, our results show that an inflammatory response heavily impacts the morphology, cell mechanics, migration, cytoskeletal dynamics, and applied traction forces of endothelial cells.

  12. Galvanotactic migration of EA.Hy926 endothelial cells in a novel designed electric field bioreactor.

    PubMed

    Long, Haiyan; Yang, Gang; Wang, Zhengrong

    2011-12-01

    Endogenous direct current electric fields (dcEFs) play a significant role in major biological processes such as embryogenesis, wound healing, and tissue regeneration. In this study, the galvanotaxis of human umbilical vein endothelial cell line EA.Hy926 was investigated by using a novel designed bioreactor. The physical features of the bioreactor were discussed and analyzed by both numerical simulation method and equivalent circuit model method. EA.Hy926 cells were cultured in the bioreactor for 10-24 h under 50-250 mV/mm dcEFs. Cell migration direction, distance, and velocity were recorded under an online time-lapse microscope. The effects of serum and growth factor on cell galvanotatic migration were investigated. To further explore the role of dcEFs in regulating endothelial cells, we analyzed the endothelial cell proliferation and secretion of nitric oxide (NO), endothelin-1 (ET-1) in response to dcEFs of physiological strength. Our results showed that EA.Hy926 cells had an obvious directional migration to the cathode, and the EF-directed migration was voltage dependent. The results also showed dcEFs did not affect cell proliferation, but affected the productions of NO and ET-1. Our study also showed the novel bioreactor, with a compact and planar style, makes it more convenient and more reasonable for EF stimulation experiments than earlier chamber designs.

  13. Superficial vimentin mediates DENV-2 infection of vascular endothelial cells.

    PubMed

    Yang, Jie; Zou, Lingyun; Yang, Yi; Yuan, Jizhen; Hu, Zhen; Liu, Hui; Peng, Huagang; Shang, Weilong; Zhang, Xiaopeng; Zhu, Junmin; Rao, Xiancai

    2016-12-02

    Damage to vascular endothelial cells (VECs) is a critical hallmark of hemorrhagic diseases caused by dengue virus (DENV). However, the precise molecular event involved in DENV binding and infection of VECs has yet to be clarified. In this study, vimentin (55 kDa) was identified to be involved in DENV-2 adsorption into VECs. This protein is located on the surface of VECs and interacts with DENV-2 envelope protein domain III (EDIII). The expression level of the superficial vimentin on VECs was not affected by viral infection or siRNA interference, indicating that the protein exists in a particular mode. Furthermore, the rod domain of the vimentin protein mainly functions in DENV-2 adsorption into VECs. Molecular docking results predicted several residues in vimentin rod and DENV EDIII; these residues may be responsible for cell-virus interactions. We propose that the superficial vimentin could be a novel molecule involved in DENV binding and infection of VECs. DENV EDIII directly interacts with the rod domain of vimentin on the VEC surface and thus mediates the infection.

  14. VUV modification promotes endothelial cell proliferation on PTFE vascular grafts

    NASA Astrophysics Data System (ADS)

    Cezeaux, J. L.; Romoser, C. E.; Benson, R. S.; Buck, C. K.; Sackman, J. E.

    1998-05-01

    Small diameter (⩽6 mm ID ) synthetic vascular grafts, used as lower-limb vessel replacements in patients without suitable autologous saphenous veins, have a failure rate of 53% after 4 yr. Graft failure is due to thrombosis and intimal hyperplasia, an increase in smooth muscle cells in the lumen of the vessel which leads to progressive closing and ultimate occlusion of the vessel. In an effort to increase patency rates of synthetic grafts, investigators have seeded vascular grafts with endothelial cells prior to implantation in an attempt to control both thrombosis and smooth muscle proliferation. This technique has been successful for the development of an endothelial monolayer in animal trials, but has met with limited success in humans. The hydrophobicity, low surface energy, and weak electrical charge of expanded polytetrafluoroethylene (ePTFE) provides conditions which are not optimal for endothelial cell attachment. The purpose of this study is to evaluate the effect of vacuum ultraviolet (VUV) modification of ePTFE on endothelial cell adhesion and proliferation. Pieces of ePTFE graft material were exposed to 10, 20 or 40 W VUV radiation for 10, 20 or 40 min using a UV excimer lamp. Prior to cell adhesion and proliferation experiments, the grafts pieces were autoclaved and cut into pledgets. Half of the pledgets were precoated with fibronectin ( 20 μg/ml). Cell adhesion was measured by seeding 3H-thymidine labeled human umbilical vein endothelial cells (HUVEC) onto the pledgets for 60 min. The pledgets were then washed and the remaining radioactivity assayed using scintillation counting. For the cell proliferation experiments, pledgets were seeded with unlabeled HUVEC which were allowed to adhere to the graft material for 18 h. The cells were then exposed to 3H-thymidine ( 1 μCi/ml) for approximately 48 h and then washed to remove any unincorporated 3H-thymidine. Incorporation of 3H-thymidine was measured using scintillation counting. Four replicate

  15. CD3 directed bispecific antibodies induce increased lymphocyte–endothelial cell interactions in vitro

    PubMed Central

    Molema, G; Tervaert, J W Cohen; Kroesen, B J; Helfrich, W; Meijer, D K F; Leij, L F M H de

    2000-01-01

    Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells. Intravenous administration of BIS-1 F(ab′)2to carcinoma patients in a phase I/II clinical trial, caused immunomodulation as demonstrated by a rapid lymphopenia prior to a rise in plasma tumour necrosis factor-α and interferon-γ levels. Yet, no lymphocyte accumulation in the tumour tissue and no anti-tumour effect could be observed. These data suggest a BsMAb-induced lymphocyte adhesion to blood vessel walls and/or generalized redistribution of the lymphocytes into tissues. In this study, we describe the effects of BIS-1 F(ab′)2binding to peripheral blood mononuclear cells (PBMC) on their capacity to interact with resting endothelial cells in vitro. Resting and pre-activated PBMC exhibited a significant increase in adhesive interaction with endothelial cells when preincubated with BIS-1 F(ab′)2, followed by an increase in transendothelial migration (tem). Binding of BIS-1 F(ab′)2to PBMC affected the expression of a number of adhesion molecules involved in lymphocyte adhesion/migration. Furthermore, PBMC preincubated with BIS-1 F(ab′)2induced the expression of endothelial cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 during adhesion/tem. These phenomena were related to the CD3 recognizing antibody fragment of the BsMAb and dependent on lymphocyte–endothelial cell contact. Possibly, in patients, the BIS-1 F(ab′)2infusion induced lymphopenia is a result of generalized activation of endothelial cells, leading to the formation of a temporary sink for lymphocytes. This process may distract the lymphocytes from homing to the tumour cells, and hence prevent the occurrence of BIS-1 F(ab′)2– CTL-mediated tumour cell lysis. © 2000 Cancer Research Campaign PMID:10646907

  16. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    PubMed

    Myojo, Masahiro; Nagata, Daisuke; Fujita, Daishi; Kiyosue, Arihiro; Takahashi, Masao; Satonaka, Hiroshi; Morishita, Yoshiyuki; Akimoto, Tetsu; Nagai, Ryozo; Komuro, Issei; Hirata, Yasunobu

    2014-01-01

    Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  17. Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

    PubMed Central

    Myojo, Masahiro; Nagata, Daisuke; Fujita, Daishi; Kiyosue, Arihiro; Takahashi, Masao; Satonaka, Hiroshi; Morishita, Yoshiyuki; Akimoto, Tetsu; Nagai, Ryozo; Komuro, Issei; Hirata, Yasunobu

    2014-01-01

    Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling. PMID:24827148

  18. Isoflurane protects against human endothelial cell apoptosis by inducing sphingosine kinase-1 via ERK MAPK.

    PubMed

    Bakar, Adnan M; Park, Sang Won; Kim, Mihwa; Lee, H Thomas

    2012-01-01

    Endothelial dysfunction is a major clinical problem affecting virtually every patient requiring critical care. Volatile anesthetics are frequently used during the perioperative period and protect the heart and kidney against ischemia and reperfusion injury. We aimed to determine whether isoflurane, the most commonly used volatile anesthetic in the USA, protects against endothelial apoptosis and necrosis and the mechanisms involved in this protection. Human endothelial EA.hy926 cells were pretreated with isoflurane or carrier gas (95% room air + 5% CO(2)) then subjected to apoptosis with tumor necrosis factor-α or to necrosis with hydrogen peroxide. DNA laddering and in situ Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick-End Labeling (TUNEL) staining determined EA.hy926 cell apoptosis and percent LDH released determined necrosis. We also determined whether isoflurane modulates the expression and activity of sphingosine kinase-1 (SK1) and induces the phosphorylation of extracellular signal regulated kinase (ERK MAPK) as both enzymes are known to protect against cell death. Isoflurane pretreatment significantly decreased apoptosis in EA.hy926 cells as evidenced by reduced TUNEL staining and DNA laddering without affecting necrosis. Mechanistically, isoflurane induces the phosphorylation of ERK MAPK and increased SK1 expression and activity in EA.hy926 cells. Finally, selective blockade of SK1 (with SKI-II) or S1P(1) receptor (with W146) abolished the anti-apoptotic effects of isoflurane. Taken together, we demonstrate that isoflurane, in addition to its potent analgesic and anesthetic properties, protects against endothelial apoptosis most likely via SK1 and ERK MAPK activation. Our findings have significant clinical implication for protection of endothelial cells during the perioperative period and patients requiring critical care.

  19. CARD14 expression in dermal endothelial cells in psoriasis.

    PubMed

    Harden, Jamie L; Lewis, Steven M; Pierson, Katherine C; Suárez-Fariñas, Mayte; Lentini, Tim; Ortenzio, Francesca S; Zaba, Lisa C; Goldbach-Mansky, Raphaela; Bowcock, Anne M; Lowes, Michelle A

    2014-01-01

    Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31(+) endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14(+)ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14(+) CD31(+) ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated CARD14 mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin.

  20. Zinc modulates PPARgamma signaling and activation of porcine endothelial cells.

    PubMed

    Meerarani, Purushothaman; Reiterer, Gudrun; Toborek, Michal; Hennig, Bernhard

    2003-10-01

    Dietary zinc has potent antioxidant and anti-inflammatory properties and is a critical component of peroxisome proliferator-activated receptor (PPAR) gene expression and regulation. To assess the protective mechanisms of PPARgamma in endothelial cell dysfunction and the role of zinc in the modulation of PPARgamma signaling, cultured porcine pulmonary artery endothelial cells were exposed to the membrane-permeable zinc chelator N,N,N'N'-tetrakis (2-pyridylmethyl)-ethylene diamine (TPEN), thiazolidinedione (TZD; PPARgamma agonist) or bisphenol A diglycidyl ether (BADGE; PPARgamma antagonist). Subsequently, endothelial cells were activated by treatment with linoleic acid (90 micro mol/L) for 6 h. Zinc chelation by TPEN increased the DNA binding activity of nuclear factor (NF)-kappaB and activator protein (AP)-1, decreased PPARgamma expression and activation as well as up-regulated interleukin (IL)-6 expression and production. These effects were fully reversed by zinc supplementation. In addition, exposure to TZD down-regulated linoleic acid-induced DNA binding activity of NF-kappaB and AP-1, whereas BADGE further induced activation of these oxidative stress-sensitive transcription factors. Most importantly, the TZD-mediated down-regulation of NF-kappaB and AP-1 and reduced inflammatory response were impaired during zinc chelation. These data suggest that zinc plays a critical role in PPARgamma signaling in linoleic acid-induced endothelial cell activation and indicate that PPARgamma signaling is impaired during zinc deficiency.

  1. METABOLIC CAPACITY REGULATES IRON HOMEOSTATIS IN ENDOTHELIAL CELLS

    EPA Science Inventory

    The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuat...

  2. NAP reduces murine microvascular endothelial cells proliferation induced by hyperglycemia.

    PubMed

    D'Amico, Agata Grazia; Scuderi, Soraya; Maugeri, Grazia; Cavallaro, Sebastiano; Drago, Filippo; D'Agata, Velia

    2014-11-01

    Hyperglycemia has been identified as a risk factor responsible for micro- and macrovascular complications in diabetes. NAP (Davunetide) is a peptide whose neuroprotective actions are widely demonstrated, although its biological role on endothelial dysfunctions induced by hyperglycemia remains uninvestigated. In the present study we hypothesized that NAP could play a protective role on hyperglycemia-induced endothelial cell proliferation. To this end we investigated the effects of NAP on an in vitro model of murine microvascular endothelial cells grown in high glucose for 7 days. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cyclin D1 protein expression analysis revealed that NAP treatment significantly reduces viability and proliferation of the cells. Hyperglycemia induced the activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphatidylinositol-3 kinase/Akt pathways in a time-dependent manner. NAP treatment reduced the phosphorylation levels of ERK and AKT in cells grown in high glucose. These evidences suggest that NAP might be effective in the regulation of endothelial dysfunction induced by hyperglycemia.

  3. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    PubMed Central

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  4. Benidipine, a dihydropyridine-Ca2+ channel blocker, increases the endothelial differentiation of endothelial progenitor cells in vitro.

    PubMed

    Ando, Hiroshi; Nakanishi, Kosuke; Shibata, Mami; Hasegawa, Kazuhide; Yao, Kozo; Miyaji, Hiromasa

    2006-12-01

    Benidipine is a dihydropyridine-Ca2+ channel blocker used in the treatment of hypertension and angina pectoris. In the present study, we examined the effects of benidipine on the endothelial differentiation of circulating endothelial progenitor cells (EPCs) using an in vitro culture method. Peripheral blood derived mononuclear cells (PBMCs) containing EPCs were isolated from C57BL/6 mice, and then the cells were cultured on vitronectin/gelatin-coated slide glasses. After 7 days of culture, endothelial cells differentiated from EPCs were identified as adherent cells with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-labeled acetylated low density lipoprotein (Dil-Ac-LDL) uptake and lectin binding under a fluorescent microscope. Incubation of PBMCs for 7 days with benidipine (0.01-1 micromol/l) significantly increased the number of Dil-Ac-LDL+/fluorescein isothiocyanate-lectin (FITC-Lectin)+ cells. Wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor, selectively attenuated the effect of benidipine on the endothelial differentiation. In addition, benidipine treatment augmented the phosphorylation of Akt, indicating that the PI3K/Akt pathway contributed, at least in part, to the endothelial differentiation induced by benidipine. These results suggest that the treatment with benidipine may increase the endothelial differentiation of circulating EPCs and contribute to endothelial protection, prevention of cardiovascular disease, and/or an improvement of the prognosis after ischemic damage.

  5. [Effects of crocetin on VCAM-1 expression in human umbilical vein endothelial cells and monocyte-endothelial cell adhesion].

    PubMed

    Zheng, Shu-guo; Zhao, Meng-qiu; Ren, You-nan; Yang, Jie-ren; Qian, Zhi-yu

    2015-01-01

    Crocetin, a naturally occurring carotenoid, possesses antioxidant and antiatherosclerotic properties, of which the underlying mechanism remains unclear. In the present study, we examined the effects of crocetin (0.1, 1, 10 μmol·L(-1)) on angiotensin II (Ang II, 0.1 μmol·L(-1)) induced expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and monocyte-endothelial cell adhesion. The effects of crocetin on the activation of nuclear factor kappa B (NF-κB) and intracellular reactive oxygen species (ROS) were also observed. The results demonstrated that crocetin notably suppressed Ang II induced NF-κB activation (P<0.01) and VCAM-1 expression (P<0.05, P<0.01) in HUVECs, accompanied by a markedly reduced monocyte-endothelial cell adhesion (P<0.05, P<0.01). In addition, preincubation with crocetin resulted in a significant enhancement of cellular antioxidant capacity (P<0.05, P<0.01), while Ang II induced intracellular ROS decreased markedly (P<0.05, P<0.01). These results indicated that crocetin was capable of suppressing Ang II induced VCAM-1 expression and monocyte-endothelial cell adhesion by suppression of NF-κB activation, which might be derived from the enhancement of antioxidant capacity and subsequent reduction of intracellular ROS.

  6. Vascular endothelial cells and dysfunctions: role of melatonin.

    PubMed

    Rodella, Luigi Fabrizio; Favero, Gaia; Foglio, Eleonora; Rossini, Claudia; Castrezzati, Stefania; Lonati, Claudio; Rezzani, Rita

    2013-01-01

    Several pathological conditions, including hypertension, atherosclerosis, diabetes, ischemia/reperfusion injury and nicotine-induced vasculopathy, are associated with vascular endothelial dysfunction characterized by altered secretory output of endothelial cells. Therefore there is a search for molecules and interventions that could restore endothelial function, in particular augmenting NO production, reducing the generation of free radicals and vasoconstrictors and preventing undesired inflammation. The pineal hormone melatonin exhibits several endothelium protective properties: it scavenges free radicals, activates antioxidant defence enzymes, normalizes lipid and blood pressure profile and increases NO bioavailability. Melatonin improved vascular function in experimental hypertension, reducing intimal infiltration and restoring NO production. Melatonin improved the NO pathway also in animal models for the study of diabetes and prevented NO down-regulation and adhesive molecules up-regulation in nicotine-induced vasculopathy. The protection against endothelial damage, vasoconstriction, platelet aggregation and leukocyte infiltration might contribute to the beneficial effects against ischemia-reperfusion injury by melatonin. Therefore, melatonin administration has endothelium-protective potential in several pathological conditions. Nevertheless, it still needs to be established, whether melatonin is able to revert already established endothelial dysfunction in these conditions.

  7. Selective human endothelial cell activation by chemokines as a guide to cell homing.

    PubMed

    Crola Da Silva, Claire; Lamerant-Fayel, Nathalie; Paprocka, Maria; Mitterrand, Michèle; Gosset, David; Dus, Danuta; Kieda, Claudine

    2009-03-01

    An original model of organo-specific, immortalized and stabilized endothelial cell lines was used to delineate the part played by some chemokines (CCL21, CX3CL1, CCL5 and CXCL12) and their receptors in endothelium organo-specificity. Chemokine receptor expression and chemokine presentation were investigated on organo-specific human endothelial cell lines. Although the chemokines showed distinct binding patterns for the various endothelial cell lines, these were not correlated with the expression of the corresponding receptors (CX3CR1, CXCR4, CCR5 and CCR7). Experiments with CCL21 on peripheral lymph node endothelial cells demonstrated that the chemokine did not co-localize with its receptor but was associated with extracellular matrix components. The specific activity of chemokines was clearly shown to be related to the endothelial cell origin. Indeed, CX3CL1 and CCL21 promoted lymphocyte recruitment by endothelial cells from the appendix and peripheral lymph nodes, respectively, while CX3CL1 pro-angiogenic activity was restricted to endothelial cells from the appendix and skin. The high specificity of the chemokine/endothelium interaction allowed the design of a direct in vitro endothelial cell targeting assay. This unique cellular model demonstrated a fundamental role for chemokines in conferring on the endothelium its organo-specificity and its potential for tissue targeting through the selective binding, presentation and activation properties of chemokines.

  8. Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells.

    PubMed

    Tancharoen, Waleephan; Aungsuchawan, Sirinda; Pothacharoen, Peraphan; Markmee, Runchana; Narakornsak, Suteera; Kieodee, Junjira; Boonma, Nonglak; Tasuya, Witoon

    2017-03-01

    Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering.

  9. Vascular endothelial growth factor directly stimulates tumour cell proliferation in non-small cell lung cancer.

    PubMed

    Devery, Aoife M; Wadekar, Rekha; Bokobza, Sivan M; Weber, Anika M; Jiang, Yanyan; Ryan, Anderson J

    2015-09-01

    Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is found predominantly on endothelial cells. The purpose of this study was to determine the role of VEGFR2 expression in NSCLC cells. NSCLC cells and tissue sections were stained for VEGFR2 expression by immunohistochemistry (IHC). Immunoblotting and ELISA were used to determine the activation and inhibition of VEGFR2 and its downstream signalling pathways. Five-day proliferation assays were carried out in the presence or absence of VEGF. IHC analysis of NSCLC demonstrated tumour cell VEGFR2 expression in 20% of samples. Immunoblot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation was apparent in NSCLC cells, and was associated with increased tumor cell proliferation. Cediranib treatment or siRNA against VEGFR2 inhibited VEGF-dependent increases in cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Our data suggest that a subset of NSCLC tumour cells express functional VEGFR2 which can act to promote VEGF-dependent tumour cell growth. In this tumour subset, therapies targeting VEGFR2 signalling, such as cediranib, have the potential to inhibit both tumour cell proliferation and angiogenesis.

  10. Serum factors involved in human microvascular endothelial cell morphogenesis.

    PubMed

    Harvey, Kevin; Siddiqui, Rafat A; Sliva, Daniel; Garcia, Joe G N; English, Denis

    2002-09-01

    Our previous studies have demonstrated that lipid and protein angiogenic factors operate in tandem to induce optimal angiogenic responses in vivo. This study was undertaken to clarify the nature of the substances in human serum that are responsible for its remarkable ability to promote capillary morphogenesis in vitro. The ability of dilute (2%) human serum to promote the morphogenic differentiation of human dermal microvascular endothelial cells on Matrigel supports was depleted by more than 50% by treatment of the serum with activated charcoal, a procedure that effectively removes biologically active lipid growth factors. The remainder of the activity within serum was lost on heating to 60 degrees C for 60 minutes, indicating the involvement of a protein in the response. The ability of charcoal-treated serum to promote capillary morphogenesis was completely restored by the addition of sphingosine 1-phosphate (SPP, 500 nmol/L), but other lipids thought to be released into serum during clotting were ineffective. In addition, basic fibroblast growth factor (bFGF) effectively restored the ability of heat-treated serum to promote endothelial cell morphogenesis, but other protein growth factors, including vascular endothelial growth factor and platelet-derived growth factor, were ineffective. Together, SPP and bFGF were as effective as whole serum in promoting capillary morphogenesis. Responses to purified SPP were entirely sensitive to the effects of preexposure of the cells to pertussis toxin, whereas responses to bFGF were entirely pertussis toxin-resistant. Consistent with our hypothesis that two distinct factors in serum play a role in promoting capillary morphogenesis, responses induced by serum were inhibited approximately 50% by preexposure of endothelial cells to pertussis toxin. We conclude that platelet-released SPP acts in conjunction with circulating bFGF to promote capillary formation by microvascular endothelial cells. Lipid and protein growth factors

  11. Pancreatic tumor cell secreted CCN1/Cyr61 promotes endothelial cell migration and aberrant neovascularization.

    PubMed

    Maity, Gargi; Mehta, Smita; Haque, Inamul; Dhar, Kakali; Sarkar, Sandipto; Banerjee, Sushanta K; Banerjee, Snigdha

    2014-05-16

    The complex signaling networks between cancer cells and adjacent endothelial cells make it challenging to unravel how cancer cells send extracellular messages to promote aberrant vascularization or tumor angiogenesis. Here, in vitro and in vivo models show that pancreatic cancer cell generated unique microenvironments can underlie endothelial cell migration and tumor angiogenesis. Mechanistically, we find that pancreatic cancer cell secreted CCN1/Cyr61 matricellular protein rewires the microenvironment to promote endothelial cell migration and tumor angiogenesis. This event can be overcome by Sonic Hedgehog (SHh) antibody treatment. Collectively, these studies identify a novel CCN1 signaling program in pancreatic cancer cells which activates SHh through autocrine-paracrine circuits to promote endothelial cell migration and tumor angiogenesis and suggests that CCN1 signaling of pancreatic cancer cells is vital for the regulation of tumor angiogenesis. Thus CCN1 signaling could be an ideal target for tumor vascular disruption in pancreatic cancer.

  12. Endothelial cells and human cerebral small vessel disease.

    PubMed

    Hainsworth, Atticus H; Oommen, Asho T; Bridges, Leslie R

    2015-01-01

    Brain endothelial cells have unique properties in terms of barrier function, local molecular signaling, regulation of local cerebral blood flow (CBF) and interactions with other members of the neurovascular unit. In cerebral small vessel disease (arteriolosclerosis; SVD), the endothelial cells in small arteries survive, even when mural pathology is advanced and myocytes are severely depleted. Here, we review aspects of altered endothelial functions that have been implicated in SVD: local CBF dysregulation, endothelial activation and blood-brain barrier (BBB) dysfunction. Reduced CBF is reported in the diffuse white matter lesions that are a neuroradiological signature of SVD. This may reflect an underlying deficit in local CBF regulation (possibly via the nitric oxide/cGMP signaling pathway). While many laboratories have observed an association of symptomatic SVD with serum markers of endothelial activation, it is apparent that the origin of these circulating markers need not be brain endothelium. Our own neuropathology studies did not confirm local endothelial activation in small vessels exhibiting SVD. Local BBB failure has been proposed as a cause of SVD and associated parenchymal lesions. Some groups find that computational analyses of magnetic resonance imaging (MRI) scans, following systemic injection of a gadolinium-based contrast agent, suggest that extravasation into brain parenchyma is heightened in people with SVD. Our recent histochemical studies of donated brain tissue, using immunolabeling for large plasma proteins [fibrinogen, immunoglobulin G (IgG)], do not support an association of SVD with recent plasma protein extravasation. It is possible that a trigger leakage episode, or a size-selective loosening of the BBB, participates in SVD pathology.

  13. Endothelial progenitor cells and asymmetric dimethylarginine after renal transplantation.

    PubMed

    Teplan, Vladimír; Mahrová, Andrea; Králová-Lesná, Ivana; Racek, Jaroslav; Valkovský, Ivo; Štollová, Milena

    2015-03-01

    Levels of the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) are elevated and endothelial progenitor cells (EPCs) decreased in patients undergoing renal transplantation (Tx) and may contribute to cardiovascular complications. In this study, we tested the hypothesis that elevated ADMA and decreased EPC can be positively influenced with regular physical exercise early after Tx. Blood samples for analysis of ADMA and EPC were obtained from randomly selected 64 patients after Tx who agreed to participate in a supervised aerobic exercise program for 6 months (group I). Samples were collected before the training began, 1 month after surgery (with stabilized renal function), and at 6 months after initiation. Sixty-two age, sex, human leukocyte antigens (HLA) typing, duration of previous dialysis, history of cardiovascular disease, and immunosupression regimen-matched transplant patients who did not exercise regularly were examined as controls (group II). There were no differences in ADMA levels and EPC count between both groups before the training program began. After 6 months of exercise, ADMA concentration in the group I decreased (3.50 ± 0.45 vs. 2.11 ± 0.35 μmol/L; P < .01) and was also lower comparing with group II (2.11 ± 0.23 vs. 3.25 ± 0.35 μmol/L; P < .01). In the same period, EPC cells increased from 2.085 ± 650 cells/mL versus 3.991 ± 560 cells/mL, P < .01 in group I; but in group II, changes were nonsignificant (P = .11). Blood lipids, HbA1c, insulin, and systolic blood pressure were also affected by the training program. Elevated ADMA level and decreased EPC count were significantly influenced by early regular exercise in patients after Tx.

  14. Effect of Excessive Potassium Iodide on Rat Aorta Endothelial Cells.

    PubMed

    Zhang, Man; Zou, Xiaoyan; Lin, Xinying; Bian, Jianchao; Meng, Huicui; Liu, Dan

    2015-08-01

    The aim of the current study was to investigate the effect of excess iodine on rat aorta endothelial cells and the potential underlying mechanisms. Rat aorta endothelial cells were cultured with iodide ion (3506, 4076, 4647, 5218, 5789, 6360, 6931, and 7512 mg/L) for 48 h. Morphological changes of cells were observed with microscope after Wright-Giemsa staining and acridine orange staining. Cell proliferation was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell apoptosis was assessed with flow cytometry. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and concentrations of malondialdehyde (MDA), glutathione (GSH), and protein carbonyl in culture medium were determined with colorimetric method. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by enzyme linked immunosorbent assay. The results showed that excess iodine induced abnormal morphologic changes of cells, inhibited cell proliferation, and increased apoptosis rate. Iodine also reduced the activity of SOD, GSH-Px, and concentrations of GSH and increased the concentrations of MDA and protein carbonyl in a dose-dependent manner. Moreover, excess iodine decreased the activity of eNOS and increased the activity of iNOS and the expression of ICAM-1 and VCAM-1 in culture medium. Our results suggested that excess iodine exposure increased oxidative stress, caused damage of vascular endothelial cells, and altered the expression of adhesion factors and the activity of NOS. These changes may explain the mechanisms underlying excess iodine-induced vascular injury.

  15. Killing Prostate Cancer Cells and Endothelial Cells with a VEGF-Triggered Cell Death Receptor

    DTIC Science & Technology

    2005-06-01

    The goal of this project was to test a novel chimeric cell death receptor (termed R2Fas) that is triggered by vascular endothelial growth factor...cells that overexpress VEGF activates apoptotic signaling and induces cell death ; (iii) we demonstrated that adenoviral-mediated expression of R2Fas in

  16. Arteries are formed by vein-derived endothelial tip cells.

    PubMed

    Xu, Cong; Hasan, Sana S; Schmidt, Inga; Rocha, Susana F; Pitulescu, Mara E; Bussmann, Jeroen; Meyen, Dana; Raz, Erez; Adams, Ralf H; Siekmann, Arndt F

    2014-12-15

    Tissue vascularization entails the formation of a blood vessel plexus, which remodels into arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endothelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to developing arteries when in association with wild-type cells, suggesting collective migration of endothelial cells. Together, our findings reveal specific cell migratory behaviours in the developing blood vessel plexus and uncover a conserved mode of artery formation.

  17. Functional CB1 cannabinoid receptors in human vascular endothelial cells.

    PubMed Central

    Liu, J; Gao, B; Mirshahi, F; Sanyal, A J; Khanolkar, A D; Makriyannis, A; Kunos, G

    2000-01-01

    Cannabinoid CB1 receptor mRNA was detected using reverse transcription-polymerase chain reaction (RT-PCR) in endothelial cells from human aorta and hepatic artery and in the ECV304 cell line derived from human umbilical vein endothelial cells. CB1 receptor-binding sites were detected by the high-affinity antagonist radioligand [(125)I]AM-251. In ECV304 cells, both the highly potent synthetic cannabinoid agonist HU-210 and the endogenous ligand anandamide induce activation of mitogen-activated protein (MAP) kinase, and the effect of HU-210 was completely blocked, whereas the effect of anandamide was partially inhibited by SR141716A, a selective CB1 receptor antagonist. Transfection of ECV304 cells with CB1 receptor antisense, but not sense, oligonucleotides caused the same pattern of inhibition as SR141716A. This provides more definitive evidence for the involvement of CB1 receptors in MAP kinase activation and suggests that anandamide may also activate MAP kinase via an additional, CB1 receptor-independent, SR141716A-resistant mechanism. The MAP kinase activation by anandamide in ECV304 cells requires genistein-sensitive tyrosine kinases and protein kinase C (PKC), and anandamide also activates p38 kinase and c-Jun kinase. These findings indicate that CB1 receptors located in human vascular endothelium are functionally coupled to the MAP kinase cascade. Activation of protein kinase cascades by anandamide may be involved in the modulation of endothelial cell growth and proliferation. PMID:10698714

  18. Arteries are formed by vein-derived endothelial tip cells

    PubMed Central

    Xu, Cong; Hasan, Sana S.; Schmidt, Inga; Rocha, Susana F.; Pitulescu, Mara E.; Bussmann, Jeroen; Meyen, Dana; Raz, Erez; Adams, Ralf H.; Siekmann, Arndt F.

    2014-01-01

    Tissue vascularization entails the formation of a blood vessel plexus, which remodels into arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endothelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to developing arteries when in association with wild-type cells, suggesting collective migration of endothelial cells. Together, our findings reveal specific cell migratory behaviours in the developing blood vessel plexus and uncover a conserved mode of artery formation. PMID:25502622

  19. Isolation, characterization, and biologic features of bone marrow endothelial cells.

    PubMed

    Almeida-Porada, G; Ascensão, J L

    1996-10-01

    Bone marrow endothelial cells (BMECs) are an integral part of the bone marrow microenvironment and are likely to play an important role in the regulation of hematopoiesis, either by producing growth factors or inhibitory cytokines or by displaying adhesion molecules that can interact with hematopoietic progenitors. In the present study we demonstrate the isolation, propagation, and characterization of BMECs with regard to morphology, growth characteristics, phenotype, and production of cytokines. Furthermore, we report the creation of a cell line with "BMEC-like" characteristics and compare the characteristics of primary BMEC cultures to those of the immortalized cell line. In addition, we demonstrate that BMECs are susceptible to infection by a laboratory strain of human cytomegalovirus (CMV), suggesting that CMV infection of endothelial cells in vivo could potentially play a role in the hematologic abnormalities observed during CMV infection.

  20. Klotho Prevents NFκB Translocation and Protects Endothelial Cell From Senescence Induced by Uremia.

    PubMed

    Buendía, Paula; Carracedo, Julia; Soriano, Sagrario; Madueño, Juan Antonio; Ortiz, Alberto; Martín-Malo, Alejandro; Aljama, Pedro; Ramírez, Rafael

    2015-10-01

    In patients with renal disease, uremia raises oxidative stress and senescence in endothelial cells, which can lead to endothelial dysfunction and cardiovascular disease. Klotho protein is a β-glucuronidase capable of hydrolyzing steroid β-glucuronides. This protein is recognized as an antiaging gene, that modulate both stress-induced senescence and functional response. The aim of the study was to investigate how senescence and oxidative stress induced by uremia in endothelial cells affects Klotho expression and whether intra or extracellular Klotho has effects on the response of these cells. Senescence and oxidative stress was obtained by exposure to uremic serum. Telomere length, the enzyme β-galactosidase, and oxidative stress were studied by flow cytometry. Nuclear factor kappa B activity was determined by electrophoretic mobility shift assay. The expression of Klotho decreased with the uremia and preceded the manifestations of cell aging. Levels of intracellular Klotho decreases associated to endothelial senescence, and exogenous Klotho prevents cellular senescence by inhibiting the increase in oxidative stress induced by uremia and diminished the nuclear factor kappa B-DNA binding ability.

  1. Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

    PubMed

    Baldo, C; Lopes, D S; Faquim-Mauro, E L; Jacysyn, J F; Niland, S; Eble, J A; Clissa, P B; Moura-da-Silva, A M

    2015-12-15

    Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms.

  2. Obesity and aging: determinants of endothelial cell dysfunction and atherosclerosis.

    PubMed

    Barton, Matthias

    2010-10-01

    Endothelial cells are both the source and target of factors contributing to atherosclerosis. After the discovery of the endothelium-derived relaxing factor (EDRF) by Robert F. Furchgott in 1980 it soon became clear that endothelial cells also release vasoactive factors distinct from nitric oxide (NO) namely, endothelium-derived contracting factors (EDCF) as well as hyperpolarizing factors (EDHF). Vasoactive factors derived from endothelial cells include NO/EDRF, reactive oxygen species, endothelins and angiotensins which have either EDRF or EDCF functions, cyclooxygenase-derived EDCFs and EDRFs, and EDHFs. Endothelial factors are formed by enzymes such as NO synthase, cyclooxygenase, converting enyzmes, NADPH oxidases, and epoxigenases, among others, and participate in the regulation of vascular homeostasis under physiological conditions; however, their abnormal regulation due to endothelial cell dysfunction contributes to disease processes such as atherosclerosis, arterial hypertension, and renal disease. Because of recent changes in world demographics and the declining health status of the world's population, both aging and obesity as independent risk factors for atherosclerosis-related diseases such as coronary artery disease and stroke, will continue to increase in the years to come. Obesity and associated conditions such as arterial hypertension and diabetes are now also some of the primary health concerns among children and adolescents. The similarities of pathomechanisms activated in obesity and aging suggest that obesity--at least in the vasculature--can be considered to have effects consistent with accelerated, "premature" aging. Pathomechanisms as well as the clinical issues of obesity- and aging-associated vascular changes important for atherosclerosis development and prevention are discussed.

  3. Superficial vimentin mediates DENV-2 infection of vascular endothelial cells

    PubMed Central

    Yang, Jie; Zou, Lingyun; Yang, Yi; Yuan, Jizhen; Hu, Zhen; Liu, Hui; Peng, Huagang; Shang, Weilong; Zhang, Xiaopeng; Zhu, Junmin; Rao, Xiancai

    2016-01-01

    Damage to vascular endothelial cells (VECs) is a critical hallmark of hemorrhagic diseases caused by dengue virus (DENV). However, the precise molecular event involved in DENV binding and infection of VECs has yet to be clarified. In this study, vimentin (55 kDa) was identified to be involved in DENV-2 adsorption into VECs. This protein is located on the surface of VECs and interacts with DENV-2 envelope protein domain III (EDIII). The expression level of the superficial vimentin on VECs was not affected by viral infection or siRNA interference, indicating that the protein exists in a particular mode. Furthermore, the rod domain of the vimentin protein mainly functions in DENV-2 adsorption into VECs. Molecular docking results predicted several residues in vimentin rod and DENV EDIII; these residues may be responsible for cell–virus interactions. We propose that the superficial vimentin could be a novel molecule involved in DENV binding and infection of VECs. DENV EDIII directly interacts with the rod domain of vimentin on the VEC surface and thus mediates the infection. PMID:27910934

  4. Hyperoxia, Endothelial Progenitor Cell Mobilization, and Diabetic Wound Healing

    PubMed Central

    Liu, Zhao-Jun

    2008-01-01

    Abstract Diabetic foot disease is a major health problem, which affects 15% of the 200 million patients with diabetes worldwide. Diminished peripheral blood flow and decreased local neovascularization are critical factors that contribute to the delayed or nonhealing wounds in these patients. The correction of impaired local angiogenesis may be a key component in developing therapeutic protocols for treating chronic wounds of the lower extremity and diabetic foot ulcers. Endothelial progenitor cells (EPCs) are the key cellular effectors of postnatal neovascularization and play a central role in wound healing, but their circulating and wound-level numbers are decreased in diabetes, implicating an abnormality in EPC mobilization and homing mechanisms. The deficiency in EPC mobilization is presumably due to impairment of eNOS-NO cascade in bone marrow (BM). Hyperoxia, induced by a clinically relevant hyperbaric oxygen therapy (HBO) protocol, can significantly enhance the mobilization of EPCs from the BM into peripheral blood. However, increased circulating EPCs failed to reach to wound tissues. This is partly a result of downregulated production of SDF-1α in local wound lesions with diabetes. Administration of exogenous SDF-1α into wounds reversed the EPC homing impairment and, with hyperoxia, synergistically enhanced EPC mobilization, homing, neovascularization, and wound healing. Antioxid. Redox Signal. 10, 1869–1882. PMID:18627349

  5. Nylon-3 polymers that enable selective culture of endothelial cells.

    PubMed

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  6. Endothelial cells and cathepsins: biochemical and biomechanical regulation

    PubMed Central

    Platt, Manu O.; Shockey, W. Andrew

    2015-01-01

    Cathepsins are mechanosensitive proteases that are regulated not only by biochemical factors, but are also responsive to biomechanical forces in the cardiovascular system that regulate their expression and activity to participate in cardiovascular tissue remodeling. Their elastinolytic and collagenolytic activity have been implicated in atherosclerosis, abdominal aortic aneurysms, and in heart valve disease, all of which are lined by endothelial cells that are the mechanosensitive monolayer of cells that sense and respond to fluid shear stress as the blood flows across the surfaces of the arteries and valve leaflets. Inflammatory cytokine signaling is integrated with biomechanical signaling pathways by the endothelial cells to transcribe, translate, and activate either the cysteine cathepsins to remodel the tissue or to express their inhibitors to maintain healthy cardiovascular tissue structure. Other cardiovascular diseases should now be included in the study of the cysteine cathepsin activation because of the additional biochemical cues they provide that merges with the already existing hemodynamics driving cardiovascular disease. Sickle cell disease causes a chronic inflammation including elevated TNFα and increased numbers of circulating monocytes that alter the biochemical stimulation while the more viscous red blood cells due to the sickling of hemoglobin alters the hemodynamics and is associated with accelerated elastin remodeling causing pediatric strokes. HIV-mediated cardiovascular disease also occurs earlier in than the broader population and the influence of HIV-proteins and antiretrovirals on endothelial cells must be considered to understand these accelerated mechanisms in order to identify new therapeutic targets for prevention. PMID:26458976

  7. Endothelial Progenitor Cells Physiology and Metabolic Plasticity in Brain Angiogenesis and Blood-Brain Barrier Modeling

    PubMed Central

    Malinovskaya, Natalia A.; Komleva, Yulia K.; Salmin, Vladimir V.; Morgun, Andrey V.; Shuvaev, Anton N.; Panina, Yulia A.; Boitsova, Elizaveta B.; Salmina, Alla B.

    2016-01-01

    Currently, there is a considerable interest to the assessment of blood-brain barrier (BBB) development as a part of cerebral angiogenesis developmental program. Embryonic and adult angiogenesis in the brain is governed by the coordinated activity of endothelial progenitor cells, brain microvascular endothelial cells, and non-endothelial cells contributing to the establishment of the BBB (pericytes, astrocytes, neurons). Metabolic and functional plasticity of endothelial progenitor cells controls their timely recruitment, precise homing to the brain microvessels, and efficient support of brain angiogenesis. Deciphering endothelial progenitor cells physiology would provide novel engineering approaches to establish adequate microfluidically-supported BBB models and brain microphysiological systems for translational studies. PMID:27990124

  8. Arterial identity of endothelial cells is controlled by local cues.

    PubMed

    Othman-Hassan, K; Patel, K; Papoutsi, M; Rodriguez-Niedenführ, M; Christ, B; Wilting, J

    2001-09-15

    The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

  9. XIAP reverses various functional activities of FRNK in endothelial cells

    SciTech Connect

    Ahn, Sunyoung; Kim, Hyun Jeong; Chi, Sung-Gil; Park, Heonyong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  10. Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue

    PubMed Central

    Moss, Stephanie C.; Bates, Michael; Parrino, Patrick E.; Woods, T. Cooper

    2007-01-01

    Analyses of vascular smooth muscle cell and endothelial cell function through tissue culture techniques are often employed to investigate the underlying mechanisms regulating cardiovascular disease. As diseases such as diabetes mellitus and chronic kidney disease increase a patient's risk of cardiovascular disease, the development of methods for examining the effects of these diseases on vascular smooth muscle cells and endothelial cells is needed. Commercial sources of endothelial cells and vascular smooth muscle cells generally provide minimal donor information and are in limited supply. This study was designed to determine if vascular smooth muscle cells and endothelial cells could be isolated from human internal mammary arteries obtained from donors undergoing coronary artery bypass graft surgery. As coronary artery bypass graft surgery is a commonly performed procedure, this method would provide a new source for these cells that when combined with the donor's medical history will greatly enhance our studies of the effects of complicating diseases on vascular biology. Internal mammary artery tissue was obtained from patients undergoing coronary artery bypass graft surgery. Through a simple method employing two separate tissue digestions, vascular smooth muscle cells and endothelial cells were isolated and characterized. The isolated vascular smooth muscle cells and endothelial cells exhibited the expected morphology and were able to be passaged for further analysis. The vascular smooth muscle cells exhibited positive staining for α-smooth muscle actin and the endothelial cells exhibited positive staining for CD31. The overall purity of the isolations was > 95%. This method allows for the isolation of endothelial cells and vascular smooth muscle cells from internal mammary arteries, providing a new tool for investigations into the interplay of vascular diseases and complicating diseases such as diabetes and kidney disease. PMID:21603530

  11. Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation.

    PubMed

    Lanier, Viola; Gillespie, Corey; Leffers, Merle; Daley-Brown, Danielle; Milner, Joy; Lipsey, Crystal; Webb, Nia; Anderson, Leonard M; Newman, Gale; Waltenberger, Johannes; Gonzalez-Perez, Ruben Rene

    2016-10-01

    Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.

  12. Suprabasin as a novel tumor endothelial cell marker

    PubMed Central

    Alam, Mohammad T; Nagao-Kitamoto, Hiroko; Ohga, Noritaka; Akiyama, Kosuke; Maishi, Nako; Kawamoto, Taisuke; Shinohara, Nobuo; Taketomi, Akinobu; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2014-01-01

    Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker. PMID:25283635

  13. Time analysis of corneal endothelial cell density after cataract extraction.

    PubMed

    Galin, M A; Lin, L L; Fetherolf, E; Obstbaum, S A; Sugar, A

    1979-07-01

    Serial endothelial photographs were taken preoperatively and postoperatively in 200 eyes; 111 eyes contained a Rayner iris clip lens, 54 eyes contained a Fyodorov Sputnik lens, and 35 eyes had no lens. Central endothelial cell density was changed in all instances, with counts in implanted eyes declining 25 to 30%, and in nonimplanted eyes 10 to 15%. In both instances, the decline essentially ceased at about three months. The cause of the greater decline in implanted eyes appeared to be mechanical and subsequent cell loss after the 90-day period was virtually equal for the two groups. Methods that may be used to alter the difference in cell density occurring with implantation are best analyzed by using the 90-day period data for comparison.

  14. Microenvironmental Regulation of the Sinusoidal Endothelial Cell Phenotype In Vitro

    PubMed Central

    March, Sandra; Hui, Elliot E.; Underhill, Gregory H.; Khetani, Salman; Bhatia, Sangeeta N.

    2010-01-01

    Liver Sinusoidal Endothelial Cells (LSEC) differ, both structurally and functionally, from endothelial cells (EC) lining blood vessels of other tissues. For example, in contrast to other EC, LSEC posses fenestrations, have low detectable levels of PECAM-1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli, namely, extracellular matrix (ECM) and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte-fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as Factor VIII activity in co-cultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease. PMID:19585615

  15. Effects of amniotic epithelial cell transplantation in endothelial injury

    PubMed Central

    Vácz, Gabriella; Cselenyák, Attila; Cserép, Zsuzsanna; Benkő, Rita; Kovács, Endre; Pankotai, Eszter; Lindenmair, Andrea; Wolbank, Susanne; Schwarz, Charlotte M.; Horváthy, Dénes B.; Kiss, Levente; Hornyák, István; Lacza, Zsombor

    2016-01-01

    Purpose Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. Results Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs’ transplanted vessels. Conclusion We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration. PMID:28180006

  16. The expression of ADAMTS13 in human microvascular endothelial cells.

    PubMed

    Wang, Anyou; Duan, Qiaohong; Wu, Jingsheng; Liu, Xin; Sun, Zimin

    2016-06-01

    ADAMTS13, as a specific von Willebrand factor (VWF)-cleaving protease, prevents microvascular thrombosis of VWF/platelet thrombi. It has been reported that human vascular endothelial cells could also synthesize and secrete ADAMTS13, and these reports were focused in human umbilical vascular endothelial cells. Considering the particularity of its huge quantity and structure of human microvascular endothelial cells (HMECs) in the body, whether ADAMTS13 is expressed in HMECs also needs to be confirmed. To investigate whether ADAMTS13 is expressed in HMECs. Real-time PCR (RT-PCR) amplification detected ADAMTS13 mRNA in HMEC-1 cell line. The expression and distribution of ADAMTS13 protein and VWF were detected by fluorescence immunoassay and western blot. We observed the expression and distribution of ADAMTS13 in HMECs. We confirmed the expression of ADAMTS13 mRNA in HMEC-1, and found that there were some partly common distributions of ADAMTS13 protein and VWF. This study provides the evidence that HMECs also express ADAMTS13. HMECs might also be a primary source for human plasma ADAMTS13. The overlap region for the distribution of ADAMTS13 and VWF suggests that ADAMTS13 might have a potential regulation role for VWF inside cells.

  17. Multifactorial Optimizations for Directing Endothelial Fate from Stem Cells

    PubMed Central

    Madfis, Nicole; Wong, Lian; Zamora, Jose; White, Nicholas; Reyes, Samuel; Burns, Andrew B.; Gopinathan, Ajay

    2016-01-01

    Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors—utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification. PMID:27907001

  18. Potential manipulation of endothelial progenitor cells in diabetes and its complications.

    PubMed

    Fadini, G P; Avogaro, A

    2010-07-01

    Diabetes mellitus increases cardiovascular risk through its negative impact on vascular endothelium. Although glucotoxicity and lipotoxicity account for endothelial cell damage, endothelial repair is also affected by diabetes. Endothelial progenitor cells (EPCs) are involved in the maintenance of endothelial homoeostasis and in the process of new vessel formation. For these reasons, EPCs are thought to have a protective impact within the cardiovascular system. In addition, EPCs appear to modulate the functioning of other organs, providing neurotropic signals and promoting repair of the glomerular endothelium. The exact mechanisms by which EPCs provide cardiovascular protection are unknown and the definition of EPCs is not standardized. Notwithstanding these limitations, the literature consistently indicates that EPCs are altered in type 1 and type 2 diabetes and in virtually all diabetic complications. Moreover, experimental models suggest that EPC-based therapies might help prevent or reverse the features of end-organ complications. This identifies EPCs as having a novel pathogenic role in diabetes and being a potential therapeutic target. Several ways of favourably modulating EPCs have been identified, including lifestyle intervention, commonly used medications and cell-based approaches. Herein, we provide a comprehensive overview of EPC pathophysiology and the potential for EPC modulation in diabetes.

  19. Increased circulating inflammatory endothelial cells in blacks with essential hypertension.

    PubMed

    Eirin, Alfonso; Zhu, Xiang-Yang; Woollard, John R; Herrmann, Sandra M; Gloviczki, Monika L; Saad, Ahmed; Juncos, Luis A; Calhoun, David A; Rule, Andrew D; Lerman, Amir; Textor, Stephen C; Lerman, Lilach O

    2013-09-01

    Morbidity and mortality attributable to hypertension are higher in black essential hypertensive (EH) compared with white EH patients, possibly related to differential effects on vascular injury and repair. Although circulating endothelial progenitor cells (EPCs) preserve endothelial integrity, inflammatory endothelial cells (IECs) detach from sites of injury and represent markers of vascular damage. We hypothesized that blood levels of IECs and inflammatory markers would be higher in black EH compared with white EH patients. Inferior vena cava and renal vein levels of CD34+/KDR+ (EPC) and VAP-1+ (IEC) cells were measured by fluorescence-activated cell sorting in white EH and black EH patients under fixed sodium intake and blockade of the renin-angiotensin system, and compared with systemic levels in normotensive control subjects (n=19 each). Renal vein and inferior vena cava levels of inflammatory cytokines and EPC homing factors were measured by Luminex. Blood pressure, serum creatinine, lipids, and antihypertensive medications did not differ between white and black EH patients, and EPC levels were decreased in both. Circulating IEC levels were elevated in black EH patients, and inversely correlated with EPC levels (R(2)=0.58; P=0.0001). Systemic levels of inflammatory cytokines and EPC homing factors were higher in black EH compared with white EH patients, and correlated directly with IECs. Renal vein inflammatory cytokines, EPCs, and IECs did not differ from their circulating levels. Most IECs expressed endothelial markers, fewer expressed progenitor cell markers, but none showed lymphocyte or phagocytic cell markers. Thus, increased release of cytokines and IECs in black EH patients may impair EPC reparative capacity and aggravate vascular damage, and accelerate hypertension-related complications.

  20. Fluid shear, intercellular stress, and endothelial cell alignment.

    PubMed

    Steward, Robert; Tambe, Dhananjay; Hardin, C Corey; Krishnan, Ramaswamy; Fredberg, Jeffrey J

    2015-04-15

    Endothelial cell alignment along the direction of laminar fluid flow is widely understood to be a defining morphological feature of vascular homeostasis. While the role of associated signaling and structural events have been well studied, associated intercellular stresses under laminar fluid shear have remained ill-defined and the role of these stresses in the alignment process has remained obscure. To fill this gap, we report here the tractions as well as the complete in-plane intercellular stress fields measured within the human umbilical vein endothelial cell (HUVEC) monolayer subjected to a steady laminar fluid shear of 1 Pa. Tractions, intercellular stresses, as well as their time course, heterogeneity, and anisotropy, were measured using monolayer traction microscopy and monolayer stress microscopy. Prior to application of laminar fluid flow, intercellular stresses were largely tensile but fluctuated dramatically in space and in time (317 ± 122 Pa). Within 12 h of the onset of laminar fluid flow, the intercellular stresses decreased substantially but continued to fluctuate dramatically (142 ± 84 Pa). Moreover, tractions and intercellular stresses aligned strongly and promptly (within 1 h) along the direction of fluid flow, whereas the endothelial cell body aligned less strongly and substantially more slowly (12 h). Taken together, these results reveal that steady laminar fluid flow induces prompt reduction in magnitude and alignment of tractions and intercellular stress tensor components followed by the retarded elongation and alignment of the endothelial cell body. Appreciably smaller intercellular stresses supported by cell-cell junctions logically favor smaller incidence of gap formation and thus improved barrier integrity.

  1. Pharmacologically active microcarriers for endothelial progenitor cell support and survival.

    PubMed

    Musilli, Claudia; Karam, Jean-Pierre; Paccosi, Sara; Muscari, Claudio; Mugelli, Alessandro; Montero-Menei, Claudia N; Parenti, Astrid

    2012-08-01

    The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells.

  2. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells

    PubMed Central

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence. PMID:26039751

  3. Evidence that cell surface heparan sulfate is involved in the high affinity thrombin binding to cultured porcine aortic endothelial cells.

    PubMed Central

    Shimada, K; Ozawa, T

    1985-01-01

    It has been postulated that thrombin binds to endothelial cells through, at least in part, cell surface glycosaminoglycans such as heparan sulfate, which could serve as antithrombin cofactor on the endothelium. In the present study, we have directly evaluated the binding of 125I-labeled bovine thrombin to cultured porcine aortic endothelial cells. The thrombin binding to the cell surface was rapid, reversible, and displaced by enzymatically inactive diisopropylphosphoryl-thrombin. The concentration of thrombin at half-maximal binding was approximately 20 nM. Both specific and nonspecific binding of 125I-thrombin to the endothelial cell surface was partially inhibited in the presence of protamine sulfate, after the removal of cell surface heparan sulfate by the treatment of cells with crude Flavobacterium heparinum enzyme or purified heparitinase. The binding as a function of the concentration of thrombin revealed that the maximal amount of specific binding was reduced by approximately 50% with little alteration in binding affinity by these enzymatic treatments. The reversibility and active-site independence as well as the rate of the binding did not change after heparitinase treatment. Whereas removal of chondroitin sulfates by chondroitin ABC lyase treatment of cells did not affect the binding, identical enzymatic treatments of [35S]sulfate-labeled cells showed that either heparan sulfate or chondroitin sulfate was selectively and completely removed from the cell surface by heparitinase or chondroitin ABC lyase treatment, respectively. Furthermore, proteolysis of cell surface proteins by the purified glycosaminoglycan lyases was excluded by the identical enzymatic treatments of [3H]leucine-labeled or cell surface radioiodinated cells. Our results provide the first direct evidence that heparan sulfate on the cell surface is involved in the high-affinity, active site-independent thrombin binding by endothelial cells, and also suggest the presence of thrombin

  4. Disintegrin Metalloprotease (ADAM) 10 Regulates Endothelial Permeability and T Cell Transmigration by Proteolysis of Vascular Endothelial Cadherin

    PubMed Central

    Schulz, Beate; Pruessmeyer, Jessica; Maretzky, Thorsten; Ludwig, Andreas; Blobel, Carl P.; Saftig, Paul; Reiss, Karina

    2009-01-01

    Vascular endothelial (VE)-cadherin is the major adhesion molecule of endothelial adherens junctions. It plays an essential role in controlling endothelial permeability, vascular integrity, leukocyte transmigration, and angiogenesis. Elevated levels of soluble VE-cadherin are associated with diseases like coronary atherosclerosis. Previous data showed that the extracellular domain of VE-cadherin is released by an unknown metalloprotease activity during apoptosis. In this study, we used gain of function analyses, inhibitor studies and RNA interference experiments to analyze the proteolytic release of VE-cadherin in human umbilical vein endothelial cells (HUVECs). We found that VE-cadherin is specifically cleaved by the disintegrin and metalloprotease ADAM10 in its ectodomain releasing a soluble fragment and generating a carboxyterminal membrane bound stub, which is a substrate for a subsequent γ-secretase cleavage. This ADAM10-mediated proteolysis could be induced by Ca2+-influx and staurosporine treatment, indicating that ADAM10-mediated VE-cadherin cleavage contributes to the dissolution of adherens junctions during endothelial cell activation and apoptosis, respectively. In contrast, protein kinase C activation or inhibition did not modulate VE-cadherin processing. Increased ADAM10 expression was functionally associated with an increase in endothelial permeability. Remarkably, our data indicate that ADAM10 activity also contributes to the thrombin-induced decrease of endothelial cell-cell adhesion. Moreover, knockdown of ADAM10 in HUVECs as well as in T cells by small interfering RNA impaired T cell transmigration. Taken together our data identify ADAM10 as a novel regulator of vascular permeability and demonstrate a hitherto unknown function of ADAM10 in the regulation of VE-cadherin-dependent endothelial cell functions and leukocyte transendothelial migration. PMID:18420943

  5. Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro

    PubMed Central

    Lacroix, Romaric; Sabatier, Florence; Mialhe, Agnes; Basire, Agnes; Pannell, Ralph; Borghi, Helene; Robert, Stephane; Lamy, Edouard; Plawinski, Laurent; Camoin-Jau, Laurence; Gurewich, Victor; Angles-Cano, Eduardo; Dignat-George, Francoise

    2007-01-01

    The regulation of plasmin generation on cell surfaces is of critical importance in the control of vascular homeostasis. Cell-derived microparticles participate in the dissemination of biological activities. However their capacity to promote plasmin generation has not been documented. In this study, we show that endothelial microparticles (EMP) from TNFα-stimulated endothelial cells, served as a surface for the generation of plasmin. The generation of plasmin involved expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) at the surface of EMP and was further increased by their ability to bind exogenous uPA on uPAR. Plasminogen was activated at the surface of EMP in a dose-dependent, saturable and specific manner as indicated by the inhibition of plasmin formation by ε-amino-caproic acid (ε-ACA) and carboxypeptidase B. EMP-induced plasmin generation affects tube formation mediated by endothelial progenitor cells. However, low amounts of EMP increased tube formation whereas higher concentrations inhibited it. Prevention of these effects by inhibitors of either uPA or plasmin, underscore the key role of EMP-induced plasmin generation. In conclusion, we demonstrated that EMP act as vectors supporting efficient plasmin generation and dissemination, a new pathway in the regulation of endothelial proteolytic activities with potential involvement in inflammation, angiogenesis and atherosclerosis. PMID:17606760

  6. Biomechanics and Intracellular Dynamics of Vascular Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Ou-Yang, H. Daniel

    2004-03-01

    Understanding the internal mechanical properties of living cells is essential to gain insight to basic cellular functions ranging from cellular signal transduction, intracellular traffics to cell motility. Vascular endothelial cells form a single cell layer that lines all blood vessels and serves to regulate exchanges between the blood stream and the surrounding tissues. Endothelial cells are one of the most studied cell types because of their roles in cardiovascular diseases and the linkage between their growth control and strategies of cancer treatments. This talk reports the application of a novel methodology by which scientists can explore cellular functions and study cytoskeleton dynamics of living cells at the subcellular level with minimal invasion. The methodology is based on the realization that optical tweezers can be used to measure the mechanical properties of the cytoskeleton in the vicinity of organelles and cellular structures. Optical tweezers is a technique based on the physics that dielectric materials, such as silica beads, latex particles or protein aggregates are attracted to and thus trapped at the focal point of a tightly focused laser beam in an aqueous medium. It has been shown that viscoelasticity can be determined from the movements of the trapped object in an oscillating optical tweezers. Applying the oscillating tweezers to intracellular cellular structures, we were able to determine the frequency dependent mechanical properties of the interior of cultured bovine endothelial cells. In contrast to the viscoelastic behavior expected of a network of cytoskelatal proteins, we found unusually large fluctuations in both elastic and loss moduli of the cell interior. More surprisingly, both mechanical moduli showed rhythmic behavior with a periodicity in the range of 20 - 30 seconds in healthy living cells. The rhythm could be altered by drug treatments, and the amplitude of the fluctuations diminished when cells were depleted of nutrients

  7. Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

    PubMed Central

    Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

    2016-01-01

    Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

  8. The role of scaffold microarchitecture in engineering endothelial cell immunomodulation

    PubMed Central

    Indolfi, Laura; Baker, Aaron B.; Edelman, Elazer R.

    2013-01-01

    The implantation of matrix-embedded endothelial cells (MEECs) has been reported to have great therapeutic potential in controlling the vascular response to injury and maintaining patency in arteriovenous anastomoses. While there is an appreciation of their effectiveness in clinical and animal studies, the mechanisms through which they mediate these powerful effects remain relatively unknown. In this work, we examined the hypothesis that the 3-dimensional microarchitecture of the tissue engineering scaffold was a key regulator of endothelial behavior in MEEC constructs. Notably, we found that ECs in porous collagen scaffold had a markedly altered cytoskeletal structure with oriented actin fibers and rearrangement of the focal adhesion proteins in comparison to cells grown on 2D surfaces. We examined the immunomodulatory capabilities of MEECs and discovered that they were able to reduce the recruitment of monocytes to an inflamed endothelial monolayer by 5-fold compared to EC on 2D surfaces. An analysis of secreted factors from the cells revealed an 8-fold lower release of Monocyte Chemotactic Protein-1 (MCP-1) from MEECs. Differences between 3D and 2D cultured cells were abolished in the presence of inhibitors to the focal adhesion associated signaling molecule Src suggesting that adhesion-mediated signaling is essential in controlling the potent immunomodulatory effects of MEEC. PMID:22796162

  9. Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells

    SciTech Connect

    Noguer, Oriol Villena, Joan; Lorita, Jordi; Vilaro, Senen; Reina, Manuel

    2009-03-10

    The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in 'in vitro' tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.

  10. Solid tumor therapy by selectively targeting stromal endothelial cells

    PubMed Central

    Liu, Shihui; Liu, Jie; Ma, Qian; Cao, Liu; Fattah, Rasem J.; Yu, Zuxi; Bugge, Thomas H.; Finkel, Toren; Leppla, Stephen H.

    2016-01-01

    Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors. PMID:27357689

  11. Abrupt reflow enhances cytokine-induced proinflammatory activation of endothelial cells during simulated shock and resuscitation.

    PubMed

    Li, Ranran; Zijlstra, Jan G; Kamps, Jan A A M; van Meurs, Matijs; Molema, Grietje

    2014-10-01

    Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation. Endothelial CD31 and VE-cadherin were not affected by the changes in flow in the absence or presence of TNF-α. The messenger RNA levels of proinflammatory molecules E-selectin, VCAM-1 (vascular cell adhesion molecule 1), and IL-8 (interleukin 8) were significantly induced by flow cessation respectively acute reflow, whereas ICAM-1 (intercellular adhesion molecule 1) was downregulated on flow cessation and induced by subsequent acute reflow. Flow cessation also affected the Ang/Tie2 (Angiopoietin/Tie2 receptor tyrosine kinase) system by downregulating Tie2 and inducing its endothelial ligand Ang2, an effect that was further extended on acute reflow. Furthermore, the induction of proinflammatory adhesion molecules by TNF-α under flow cessation was significantly enhanced on subsequent acute reflow. This study demonstrated that flow alterations per se during shock and resuscitation contribute to endothelial activation and that these alterations interact with proinflammatory factors coexisting in vivo such as TNF-α. The abrupt reflow-related enhancement of cytokine-induced endothelial proinflammatory activation supports the concept that sudden regain of flow during resuscitation has an aggravating effect on endothelial activation, which may play a significant role in vascular

  12. Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells

    PubMed Central

    2010-01-01

    Background Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Results Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE). There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed). These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Conclusions Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the proteomes were examined

  13. Bilirubin is an Endogenous Antioxidant in Human Vascular Endothelial Cells

    PubMed Central

    Ziberna, Lovro; Martelanc, Mitja; Franko, Mladen; Passamonti, Sabina

    2016-01-01

    Bilirubin is a standard serum biomarker of liver function. Inexplicably, it is inversely correlated with cardiovascular disease risk. Given the role of endothelial dysfunction in originating cardiovascular diseases, direct analysis of bilirubin in the vascular endothelium would shed light on these relationships. Hence, we used high-performance liquid chromatography coupled with thermal lens spectrometric detection and diode array detection for the determination of endogenous cellular IXα-bilirubin. To confirm the isomer IXα-bilirubin, we used ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer using an electrospray ionization source, as well as tandem mass spectrometric detection. We measured bilirubin in both arterial and venous rat endothelium (0.9–1.5 pmol mg−1 protein). In the human endothelial Ea.hy926 cell line, we demonstrated that intracellular bilirubin (3–5 pmol mg−1 protein) could be modulated by either extracellular bilirubin uptake, or by up-regulation of heme oxygenase-1, a cellular enzyme related to endogenous bilirubin synthesis. Moreover, we determined intracellular antioxidant activity by bilirubin, with EC50 = 11.4 ± 0.2 nM, in the range of reported values of free serum bilirubin (8.5–13.1 nM). Biliverdin showed similar antioxidant properties as bilirubin. We infer from these observations that intra-endothelial bilirubin oscillates, and may thus be a dynamic factor of the endothelial function. PMID:27381978

  14. Ca2+-mediated ascorbate release from coronary artery endothelial cells.

    PubMed

    Davis, Kim A; Samson, Sue E; Best, Kelly; Mallhi, Kanwaldeep K; Szewczyk, Magdalena; Wilson, John X; Kwan, Chiu-Yin; Grover, Ashok K

    2006-01-01

    1.--The addition of Ca(2+) ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca(2+)-mediated release. An increase in Ca(2+)-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2.--The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3.--ATP-stimulated Asc release was Ca(2+)-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca(2+)], (b) Ca(2+) ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca(2+) and chelating intracellular Ca(2+)inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4.--Accumulation of Asc by PCEC was examined at Asc concentrations of 10 microM (Na(+)-Asc symporter not saturated) and 5 mM (Na(+)-Asc symporter saturated). At 10 microM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5.--PCEC but not pig coronary artery smooth muscle cells show a Ca(2+)- mediated Asc release pathway that may be activated by agents such as ATP.

  15. Nitrones Reverse Hyperglycemia-Induced Endothelial Dysfunction in Bovine Aortic Endothelial Cells

    PubMed Central

    Headley, Colwyn A.; DiSilvestro, David; Hemann, Craig; Bryant, Kelsey E.; Chen, Chun-Aun; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A.

    2016-01-01

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse eNOS dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5 mM glucose, LG) or high glucose (50 mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC cells with nitrones for 24 h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased BH4 levels by 15% thereby decreasing NO production. Intracellular glucose transport and SOD activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC cells grown in hyperglycemic conditions resulted in in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC cells. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted. PMID:26774452

  16. Ulex europaeus I lectin as a marker for tumors derived from endothelial cells.

    PubMed

    Miettinen, M; Holthofer, H; Lehto, V P; Miettinen, A; Virtanen, I

    1983-01-01

    Some skin and soft tumors, which generally are assumed to be derived from endothelial cells or blood vessels, were characterized with fluorochrome-labeled Ulex europaeus I agglutinin (UEA I), recently shown to bind specifically to endothelial cells in various normal human tissues. The staining pattern was compared with that obtained with immunostaining using antibodies against factor-VIII-related antigen (FVIII-RAG), a known marker for endothelial cells. The results showed that UEA-I is a specific and a more sensitive marker for the endothelial cells in benign vascular lesions as compared with anti-FVIII-RAG. UEA-I also stained many neoplastic cells of endothelial sarcomas, which generally were negative for FVIII-RAG. Melanomas, anaplastic carcinomas, and other types of sarcomas were negative for both UEA-I and FVIII-RAG. The results suggest that UEA-I lectin is a specific and sensitive adjunct tool in demonstrating endothelial cells and endothelial derivation of human tumors.

  17. “Decoding” Angiogenesis: New Facets Controlling Endothelial Cell Behavior

    PubMed Central

    Sewduth, Raj; Santoro, Massimo M.

    2016-01-01

    Angiogenesis, the formation of new blood vessels, is a unique and crucial biological process occurring during both development and adulthood. A better understanding of the mechanisms that regulates such process is mandatory to intervene in pathophysiological conditions. Here we highlight some recent argument on new players that are critical in endothelial cells, by summarizing novel discoveries that regulate notorious vascular pathways such as Vascular Endothelial Growth Factor (VEGF), Notch and Planar Cell Polarity (PCP), and by discussing more recent findings that put metabolism, redox signaling and hemodynamic forces as novel unforeseen facets in angiogenesis. These new aspects, that critically regulate angiogenesis and vascular homeostasis in health and diseased, represent unforeseen new ground to develop anti-angiogenic therapies. PMID:27493632

  18. [Effects of endothelial cells on renewal and differentiation of neural stem cells].

    PubMed

    Dong, Zhiwu; Su, Le; Mino, Junying

    2007-10-01

    It is well established that neural stem cells (NSCs) are not randomly distributed throughout the brain, but rather are concentrated around blood vessels. Although NSCs lie in a vascular niche, there is no direct evidence for a functional relationship between the NSCs and blood vessel component cells. It is reported that endothelial cells release soluble factors that stimulate the self-renewal of NSCs, inhibit their differentiation, and enhance their neuron production. Endothelial coculture can activate Notch to promote self-renewal. Furthermore, vascular endothelial growth factor (VEGF) plays a significant role in neural cells; it stimulates the growth and differentiation of astrocytes in the central nervous system (CNS). Therefore, beyond their traditional role as structural components of blood vessels, endothelial cells are not only critical component of the neural stem cell niche, but they also are able to enhance neurogenesis, possibly through the secretion of brain-derived neurotrophic factor.

  19. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  20. The Interaction between Fluid Wall Shear Stress and Solid Circumferential Strain Affects Endothelial Gene Expression.

    PubMed

    Amaya, Ronny; Pierides, Alexis; Tarbell, John M

    2015-01-01

    Endothelial cells lining the walls of blood vessels are exposed simultaneously to wall shear stress (WSS) and circumferential stress (CS) that can be characterized by the temporal phase angle between WSS and CS (stress phase angle - SPA). Regions of the circulation with highly asynchronous hemodynamics (SPA close to -180°) such as coronary arteries are associated with the development of pathological conditions such as atherosclerosis and intimal hyperplasia whereas more synchronous regions (SPA closer to 0°) are spared of disease. The present study evaluates endothelial cell gene expression of 42 atherosclerosis-related genes under asynchronous hemodynamics (SPA=-180 °) and synchronous hemodynamics (SPA=0 °). This study used a novel bioreactor to investigate the cellular response of bovine aortic endothelial cells (BAECS) exposed to a combination of pulsatile WSS and CS at SPA=0 or SPA=-180. Using a PCR array of 42 genes, we determined that BAECS exposed to non-reversing sinusoidal WSS (10±10 dyne/cm2) and CS (4 ± 4%) over a 7 hour testing period displayed 17 genes that were up regulated by SPA = -180 °, most of them pro-atherogenic, including NFκB and other NFκB target genes. The up regulation of NFκB p50/p105 and p65 by SPA =-180° was confirmed by Western blots and immunofluorescence staining demonstrating the nuclear translocation of NFκB p50/p105 and p65. These data suggest that asynchronous hemodynamics (SPA=-180 °) can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA may be an important parameter characterizing arterial susceptibility to disease.

  1. ENDOTHELIAL PROGENITOR CELLS AS SHUTTLE OF ANTICANCER AGENTS.

    PubMed

    Laurenzana, Anna; Margheri, Francesca; Chilla', Anastasia; Biagioni, Alessio; Margheri, Giancarlo; Calorini, Lido; Fibbi, Gabriella; Del Rosso, Mario

    2016-08-08

    Cell therapies are treatments in which stem or progenitor cells are induced to differentiate into the specific cell type required to repair damaged or destroyed tissues. Following their discovery, endothelial progenitor cells (EPCs) have stimulated a worldwide interest as possible vehicles to perform an autologous cell-therapy of tumors. Taking into account the tumor-homing properties of EPCs, two different approaches to control cancer progression have been pursued by combining the cell-based therapy with gene therapy or with nanomedicine. The first one is based on the possibility to engineer EPCs to express different transgenes, the second one on the capacity of EPCs to uptake nanomaterials. Here we will review the most important progresses covering the following issues: the characterization of bona fide endothelial progenitor cells, their role in tumor vascularisation and metastasis, and preclinical data about their use in cell-based tumor therapy, considering anti-angiogenic, suicide, immune-stimulating and oncolytic virus gene-therapy. The mixed approach of EPC cell therapy and nanomedicine will be discussed in terms of plasmonic-dependent thermoablation and molecular imaging.

  2. Endothelial cell sensing, restructuring, and invasion in collagen hydrogel structures.

    PubMed

    Hosseini, Y; Agah, M; Verbridge, S S

    2015-11-01

    Experimental tools to model cell-tissue interactions will likely lead to new ways to both understand and treat cancer. While the mechanical properties and regulation of invasion have been recently studied for tumor cells, they have received less attention in the context of tumor vascular dynamics. In this article, we have investigated the interaction between the surfaces of structures encountered by endothelial cells invading their surrounding extracellular matrix (ECM) during angiogenesis. For this purpose, we have fabricated round and sharp geometries with various curvature and sharpness indices in collagen hydrogel over a wide range of stiffness to mimic different microenvironments varying from normal to tumor tissues. We have then cultured endothelial cells on these structures to investigate the bi-directional interaction between the cells and ECM. We have observed that cell invasion frequency is higher from the structures with the highest sharpness and curvature index, while interestingly the dependence of invasion on the local micro-geometry is strongest for the highest density matrices. Notably, structures with the highest invasion length are linked with higher deformation of side structures, which may be related to traction force-activated signaling suggesting further investigation. We have noted that round structures are more favorable for cell adhesion and in some cases round structures drive cell invasion faster than sharp ones. These results highlight the ability of endothelial cells to sense small variations in ECM geometry, and respond with a balance of matrix invasion as well as deformation, with potential implications for feedback mechanisms that may enhance vascular abnormality in response to tumor-induced ECM alterations.

  3. A coculture system of cavernous endothelial and smooth muscle cells.

    PubMed

    Ning, H; Lin, G; Lue, T F; Lin, C-S

    2013-01-01

    In erectile dysfunction (ED) research, monocultures of cavernous endothelial cells (CECs) and smooth muscle cells (CSMCs) have been reported, but a CEC-CSMC coculture system is still lacking. In the present study, we wished to investigate the feasibility of setting up such a system and test whether it can be used for diabetic ED research. Cavernous tissues were obtained from patients undergoing surgery for penile prosthesis. CSMCs were isolated by explant culture and verified by calponin staining. CECs were isolated by binding to CD31 antibody, followed by magnetic capture. These CECs were nearly 100% pure endothelial cells as determined by flow cytometric analysis for endothelial markers CD31, vWF and eNOS. Functional analyses, that is, low-density lipoprotein (LDL) uptake and capillary tube formation, also confirmed their endothelial phenotype. When cocultured with CSMCs, CECs formed capillary-like structures, and based on the extent of this capillary-like network, it was determined that a ratio of 1:4 in cell number between CECs and CSMCs was better than ratios of 1:1 and 1:9. It was also found that direct contact between CECs and CSMCs was necessary and a coculture period of 3 weeks was optimal. Autologous CSMCs were better than allogeneic CSMCs, and fibroblasts were completely incompetent. When treated with high glucose (25 mM), the CEC-CSMC coculture expressed significantly lower level of CD31 but significantly higher level of collagen-IV (Col-IV), and the diameter of the capillaries increased significantly, when compared with normal glucose (5 mM)-treated cocultures. These data are consistent with previously observed changes in the cavernous tissues of diabetic patients and thus suggest that the coculture system could be utilized for diabetic ED research.

  4. Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells.

    PubMed

    Watanabe, Takuya; Yasutaka, Yuki; Nishioku, Tsuyoshi; Kusakabe, Sae; Futagami, Koujiro; Yamauchi, Atsushi; Kataoka, Yasufumi

    2011-06-01

    The conversion of cellular prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion disease. Although PrP(C) is a ubiquitous glycoprotein that is present in various cell types, the physiological role of PrP(C) remains obscure. The present study aimed to determine whether PrP(C) mediates migration of brain microvascular endothelial cells. Small interfering RNAs (siRNAs) targeting PrP(C) were transfected into a mouse brain microvascular endothelial cell line (bEND.3 cells). siPrP1, selected among three siRNAs, reduced mRNA and protein levels of PrP(C) in bEND.3 cells. Cellular migration was evaluated with a scratch-wound assay. siPrP1 suppressed migration without significantly affecting cellular proliferation. This study provides the first evidence that PrP(C) may be necessary for brain microvascular endothelial cells to migrate into damaged regions in the brain. This function of PrP(C) in the brain endothelium may be a mechanism by which the neurovascular unit recovers from an injury such as an ischemic insult.

  5. Homocysteine injures vascular endothelial cells by inhibiting mitochondrial activity

    PubMed Central

    Yang, Fengyong; Qi, Xiujing; Gao, Zheng; Yang, Xingju; Zheng, Xingfeng; Duan, Chonghao; Zheng, Jian

    2016-01-01

    The aim of the present study was to investigate the role of homocysteine (Hcy) in the pathogenesis of pulmonary embolism (PE) and the associated molecular mechanisms in human umbilical vein endothelial cells (HUVECs). Hcy contents were detected with high-performance liquid chromatography. Apoptosis was detected by flow cytometry using Annexin-V staining. Cytochrome c oxidase (COX) activity was assessed with an enzyme activity assay, and the expression levels of COX 17 were determined by western blot analysis. Intracellular reactive oxygen species levels were measured using a microplate reader with a fluorescence probe. The results demonstrated that, compared with the control group, the serum Hcy levels were significantly elevated in the PE group, suggesting that Hcy may be an indicator for PE. Following treatment with Hcy, the apoptosis rate was markedly elevated in HUVECs. Moreover, Hcy decreased COX activity and downregulated the expression of COX 17 in HUVECs. Furthermore, Hcy increased the ROS levels in these endothelial cells. However, all the above-mentioned physiopathological changes induced by Hcy in HUVECs could be restored by folic acid. In conclusion, the results of the present study demonstrated that Hcy inhibited COX activity, downregulated COX 17 expression, increased intracellular ROS levels and enhanced apoptosis in endothelial cells. PMID:27698720

  6. Endothelial cell metabolism in normal and diseased vasculature

    PubMed Central

    Eelen, Guy; de Zeeuw, Pauline; Simons, Michael; Carmeliet, Peter

    2015-01-01

    Higher organisms rely on a closed cardiovascular circulatory system with blood vessels supplying vital nutrients and oxygen to distant tissues. Not surprisingly, vascular pathologies rank among the most life-threatening diseases. At the crux of most of these vascular pathologies are (dysfunctional) endothelial cells (ECs), the cells lining the blood vessel lumen. ECs display the remarkable capability to switch rapidly from a quiescent state to a highly migratory and proliferative state during vessel sprouting. This angiogenic switch has long been considered to be dictated by angiogenic growth factors (eg vascular endothelial growth factor; VEGF) and other signals (eg Notch) alone, but recent findings show that it is also driven by a metabolic switch in ECs. Furthermore, these changes in metabolism may even override signals inducing vessel sprouting. Here, we review how EC metabolism differs between the normal and dysfunctional/diseased vasculature and how it relates to or impacts the metabolism of other cell types contributing to the pathology. We focus on the biology of ECs in tumor blood vessel and diabetic ECs in atherosclerosis as examples of the role of endothelial metabolism in key pathological processes. Finally, current as well as unexplored ‘EC metabolism’-centric therapeutic avenues are discussed. PMID:25814684

  7. Single-cell analysis of endothelial morphogenesis in vivo.

    PubMed

    Yu, Jianxin A; Castranova, Daniel; Pham, Van N; Weinstein, Brant M

    2015-09-01

    Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.

  8. Early responses of vascular endothelial cells to topographic cues.

    PubMed

    Dreier, Britta; Gasiorowski, Joshua Z; Morgan, Joshua T; Nealey, Paul F; Russell, Paul; Murphy, Christopher J

    2013-08-01

    Vascular endothelial cells in vivo are exposed to multiple biophysical cues provided by the basement membrane, a specialized extracellular matrix through which vascular endothelial cells are attached to the underlying stroma. The importance of biophysical cues has been widely reported, but the signaling pathways that mediate cellular recognition and response to these cues remain poorly understood. Anisotropic topographically patterned substrates with nano- through microscale feature dimensions were fabricated to investigate cellular responses to topographic cues. The present study focuses on early events following exposure of human umbilical vein endothelial cells (HUVECs) to these patterned substrates. In serum-free medium and on substrates without protein coating, HUVECs oriented parallel to the long axis of underlying ridges in as little as 30 min. Immunocytochemistry showed clear differences in the localization of the focal adhesion proteins Src, p130Cas, and focal adhesion kinase (FAK) in HUVECs cultured on topographically patterned surfaces and on planar surfaces, suggesting involvement of these proteins in mediating the response to topographic features. Knockdown experiments demonstrated that FAK was not necessary for HUVEC alignment in response to topographic cues, although FAK knockdown did modulate HUVEC migration. These data identify key events early in the cellular response to biophysical stimuli.

  9. Interaction of recombinant octameric hemoglobin with endothelial cells.

    PubMed

    Gaucher, Caroline; Domingues-Hamdi, Élisa; Prin-Mathieu, Christine; Menu, Patrick; Baudin-Creuza, Véronique

    2015-02-01

    Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells. The rHbβG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbβG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb βG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

  10. Decrease of fibrinolytic activity in human endothelial cells by arsenite.

    PubMed

    Jiang, Shinn-Jong; Lin, Tsun-Mei; Wu, Hua-Lin; Han, Huai-Song; Shi, Guey-Yueh

    2002-01-01

    Blackfoot disease (BFD) is an endemic peripheral vascular occlusive disease that occurred in the southwest coast of Taiwan. It is believed that arsenic in the drinking water from artesian wells plays an important role in the development of the disease. We have previously shown that BFD patients had significant lower tissue-type plasminogen activator (t-PA) antigen level and higher plasminogen activator inhibitor, Type 1 (PAI-1) antigen level than normal controls. The purpose of this study was to investigate the effects of arsenite on the fibrinolytic and anticoagulant activities of cultured macrovascular and microvascular endothelial cells. Incubation of human microvascular endothelial cells (HMEC-1), but not human umbilical vein endothelial cells (HUVECs), with arsenite caused a decrease of t-PA mRNA level, a rise of both PAI-1 mRNA level and PAI activity. Arsenite could also inhibit the thrombomodulin (TM) mRNA expression and reduce the TM antigen level in HMEC-1. In conclusion, arsenite had a greater effect on HMEC-1 as compared to HUVECs in lowering the fibrinolytic activity and may be responsible for the reduced capacity of fibrinolysis associated with BFD.

  11. Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation

    PubMed Central

    Gama Sosa, Miguel A.; De Gasperi, Rita; Hof, Patrick R.; Elder, Gregory A.

    2016-01-01

    Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1−/− embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1−/− cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1−/− cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1−/− cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1−/− cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1−/− cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway. PMID:27443835

  12. Ultrasound fails to induce proliferation of human brain and mouse endothelial cell lines

    NASA Astrophysics Data System (ADS)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    Both in vitro and in vivo studies suggest that ultrasound (US) is capable of inducing angiogenesis. There is no information, however, on whether ultrasound can induce proliferation of brain endothelial cells. We therefore explored the angiogenic potential of ultrasound on a novel immortalised human brain endothelial cell line (hCMEC/D3) and on mouse brain microvascular endothelial cells (bEND3). Ultrasound failed to enhance cell proliferation in both cell lines at all acoustic pressures studied. Endothelial cell damage occurred at 0.24 MPa with significantly slower proliferation. Cells growing in Opticell{trade mark, serif} dishes did not show damage or reduced proliferation at these pressures.

  13. Microtopography and flow modulate the direction of endothelial cell migration.

    PubMed

    Uttayarat, P; Chen, M; Li, M; Allen, F D; Composto, R J; Lelkes, P I

    2008-02-01

    The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 microm in width at a constant depth of 1 microm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm(2)), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm(2)) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.

  14. Human endothelial cell responses to cardiovascular inspired pulsatile shear stress

    NASA Astrophysics Data System (ADS)

    Watson, Matthew; Baugh, Lauren; Black, Lauren, III; Kemmerling, Erica

    2016-11-01

    It is well established that hemodynamic shear stress regulates blood vessel structure and the development of vascular pathology. This process can be studied via in vitro models of endothelial cell responses to pulsatile shear stress. In this study, a macro-scale cone and plate viscometer was designed to mimic various shear stress waveforms found in the body and apply these stresses to human endothelial cells. The device was actuated by a PID-controlled DC gear-motor. Cells were exposed to 24 hours of pulsatile shear and then imaged and stained to track their morphology and secretions. These measurements were compared with control groups of cells exposed to constant shear and no shear. The results showed that flow pulsatility influenced levels of secreted proteins such as VE-cadherin and neuroregulin IHC. Cell morphology was also influenced by flow pulsatility; in general cells exposed to pulsatile shear stress developed a higher aspect ratio than cells exposed to no flow but a lower aspect ratio than cells exposed to steady flow.

  15. Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

    NASA Technical Reports Server (NTRS)

    Love, Felisha D.; Melhado, Caroline D.; Bosah, Francis N.; Harris-Hooker, Sandra A.; Sanford, Gary L.

    1998-01-01

    Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

  16. Deleterious effects of endotoxin on cultured endothelial cells: an in vitro model of vascular injury

    SciTech Connect

    Yamada, O.; Moldow, C.F.; Sacks, T.; Craddock, P.R.; Boogaerts, M.A.; Jacob, H.S.

    1981-06-01

    The effects of endotoxin-triggered granulocytes on the viability of endothelial cells in vitro was investigated. Endotoxin or its lipid A component caused granulocytes to adhere to and significantly damage cultured endothelial cells. Fresh serum is not necessary but does amplify both adherence and endothelial injury. Much of the endothelial injury was inhibited by free-radical scavengers or by blocking granulocyte adhesion to endothelial cells and appears to result from free radical production by the stimulated granulocyte. Studies in this model suggest a pathogenic role for the endotoxin-triggered granulocyte in the Shwartzman reaction and perhaps related clinical disorders.

  17. CTC-Endothelial Cell Interactions during Metastasis

    DTIC Science & Technology

    2013-04-01

    selectin under physiological blood flow using a parallel flow chamber system. Prostate cancer cells, MDAPCa 2b were first labeled with anti- PSMA ...monoclonal antibody J591 conjugated with alexa fluor 488 (J591-488) that recognizes prostate specific membrane antigen ( PSMA ) and is internalized...following binding to PSMA . We observed that the mean rolling velocity of MDAPCa 2b cells on HUVECs ranged from 4.2-6 µm/s at 0.5-4 dyn/cm2 shear stress

  18. Cerebral endothelial expression of Robo1 affects brain infiltration of polymorphonuclear neutrophils during mouse stroke recovery.

    PubMed

    Gangaraju, Sandhya; Sultan, Khadeejah; Whitehead, Shawn N; Nilchi, Ladan; Slinn, Jacqueline; Li, Xuesheng; Hou, Sheng T

    2013-06-01

    Increased brain infiltration of polymorphonuclear neutrophils (PMNs) occurs early after stroke and is important in eliciting brain inflammatory response during stroke recovery. In order to understand the molecular mechanism of PMN entry, we investigated the expression and requirement for Slit1, a chemorepulsive guidance cue, and its cognate receptor, Robo1, in a long-term recovery mouse model of cerebral ischemia. The expression levels of Robo1 were significantly decreased bilaterally at 24h following reperfusion. Robo1 expression levels remained suppressed in the ipsilateral cortex until 28d post MCAO-reperfusion, while the levels of Robo1 in the contralateral cortex recovered to the level of sham-operated mouse by 7d reperfusion. Circulating PMNs express high levels of Slit1, but not Robo1. Influx of PMNs into the ischemic core area occurred early (24h) after cerebral ischemia, when endothelial Robo1 expression was significantly reduced in the ischemic brain, indicating that Robo1 may form a repulsive barrier to PMN entry into the brain parenchyma. Indeed, blocking Slit1 on PMNs in a transwell migration assay in combination with an antibody blocking of Robo1 on human umbilical vein endothelial cells (HUVEC) significantly increased PMN transmigration during oxygen glucose deprivation, an in vitro model of ischemia. Collectively, in the normal brain, the presence of Slit1 on PMNs, and Robo1 on cerebral endothelial cells, generated a repulsive force to prevent the infiltration of PMNs into the brain. During stroke recovery, a transient reduction in Robo1 expression on the cerebral endothelial cells allowed the uncontrolled infiltration of Slit1-expressing PMNs into the brain causing inflammatory reactions.

  19. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation

    PubMed Central

    McCabe, Kathryn L.; Kunzevitzky, Noelia J.; Chiswell, Brian P.; Xia, Xin; Goldberg, Jeffrey L.; Lanza, Robert

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. Results hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. Conclusion hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. PMID:26689688

  20. Endothelial Cell Toxicity of Vancomycin Infusion Combined with Other Antibiotics.

    PubMed

    Drouet, Maryline; Chai, Feng; Barthélémy, Christine; Lebuffe, Gilles; Debaene, Bertrand; Décaudin, Bertrand; Odou, Pascal

    2015-08-01

    French guidelines recommend central intravenous (i.v.) infusion for high concentrations of vancomycin, but peripheral intravenous (p.i.v.) infusion is often preferred in intensive care units. Vancomycin infusion has been implicated in cases of phlebitis, with endothelial toxicity depending on the drug concentration and the duration of the infusion. Vancomycin is frequently infused in combination with other i.v. antibiotics through the same administrative Y site, but the local toxicity of such combinations has been poorly evaluated. Such an assessment could improve vancomycin infusion procedures in hospitals. Human umbilical vein endothelial cells (HUVEC) were challenged with clinical doses of vancomycin over 24 h with or without other i.v. antibiotics. Cell death was measured with the alamarBlue test. We observed an excess cellular death rate without any synergistic effect but dependent on the numbers of combined infusions when vancomycin and erythromycin or gentamicin were infused through the same Y site. Incompatibility between vancomycin and piperacillin-tazobactam was not observed in our study, and rinsing the cells between the two antibiotic infusions did not reduce endothelial toxicity. No endothelial toxicity of imipenem-cilastatin was observed when combined with vancomycin. p.i.v. vancomycin infusion in combination with other medications requires new recommendations to prevent phlebitis, including limiting coinfusion on the same line, reducing the infusion rate, and choosing an intermittent infusion method. Further studies need to be carried out to explore other drug combinations in long-term vancomycin p.i.v. therapy so as to gain insight into the mechanisms of drug incompatibility under multidrug infusion conditions.

  1. Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

    PubMed Central

    Uthamaraj, Susheil; Tefft, Brandon J.; Hlinomaz, Ota; Sandhu, Gurpreet S.; Dragomir-Daescu, Dan

    2015-01-01

    Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing. PMID:26436434

  2. Hypoxia and Reoxygenation Induce Endothelial Nitric Oxide Synthase Uncoupling in Endothelial Cells through Tetrahydrobiopterin Depletion and S-Glutathionylation

    PubMed Central

    2015-01-01

    Ischemia-reperfusion injury is accompanied by endothelial hypoxia and reoxygenation that trigger oxidative stress with enhanced superoxide generation and diminished nitric oxide (NO) production leading to endothelial dysfunction. Oxidative depletion of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin can trigger eNOS uncoupling, in which the enzyme generates superoxide rather than NO. Recently, it has also been shown that oxidative stress can induce eNOS S-glutathionylation at critical cysteine residues of the reductase site that serves as a redox switch to control eNOS coupling. While superoxide can deplete tetrahydrobiopterin and induce eNOS S-glutathionylation, the extent of and interaction between these processes in the pathogenesis of eNOS dysfunction in endothelial cells following hypoxia and reoxygenation remain unknown. Therefore, studies were performed on endothelial cells subjected to hypoxia and reoxygenation to determine the severity of eNOS uncoupling and the role of cofactor depletion and S-glutathionylation in this process. Hypoxia and reoxygenation of aortic endothelial cells triggered xanthine oxidase-mediated superoxide generation, causing both tetrahydrobiopterin depletion and S-glutathionylation with resultant eNOS uncoupling. Replenishing cells with tetrahydrobiopterin along with increasing intracellular levels of glutathione greatly preserved eNOS activity after hypoxia and reoxygenation, while targeting either mechanism alone only partially ameliorated the decrease in NO. Endothelial oxidative stress, secondary to hypoxia and reoxygenation, uncoupled eNOS with an altered ratio of oxidized to reduced glutathione inducing eNOS S-glutathionylation. These mechanisms triggered by oxidative stress combine to cause eNOS dysfunction with shift of the enzyme from NO to superoxide production. Thus, in endothelial reoxygenation injury, normalization of both tetrahydrobiopterin levels and the glutathione pool are needed for maximal

  3. Endothelialization of Magnetic Graft Materials using SPION-labeled Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Newman, Brant R.; Dragomir-Daescu, Dan; Harbuzariu, Adriana; McIntosh, Malcolm; Harburn, J. Jonathan; Parakka, Anthony; Kalra, Manju; Holmes, David; Simari, Robert D.; Sandhu, Gurpreet S.

    2010-12-01

    Seeding vascular grafts with autologous endothelial cells (EC) has been shown to improve in vivo patency, but high cost and development time have prevented widespread clinical use. A technique for loading EC with superparamagnetic iron-oxide nanospheres (SPIONs) was recently described. SPION-loaded EC experience magnetic attractive forces in the presence of sufficient magnetic field gradients. Using a multi-factorial design of experiments approach, the quantity and spatial distribution of magnetizable metal particles within a poly (ether urethane) matrix were systematically varied to produce unique material specimens. Specimens were seeded with SPION-loaded ECs, and cell coverage was quantified at various post-seeding time intervals using micrographic image analysis. The effects of changing design parameters on cell capture and sustained cell viability on magnetic substrates were statistically examined. Magnetized ferrites and samarium cobalt demonstrated cell capture, though cytotoxicity prevented sustained cell growth. Cobalt chromium substrates showed effective cell capture and growth to near complete confluence for up to one month.

  4. Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1.

    PubMed

    Hawkins, Brian T; Grego, Sonia; Sellgren, Katelyn L

    2015-05-22

    Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.

  5. Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

    PubMed Central

    Hida, Yasuhiro; Maishi, Nako; Towfik, Alam Mohammad; Inoue, Nobuo; Shindoh, Masanobu; Hida, Kyoko

    2013-01-01

    There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment. PMID:24260373

  6. Effects of verteporfin-mediated photodynamic therapy on endothelial cells

    NASA Astrophysics Data System (ADS)

    Kraus, Daniel; Chen, Bin

    2015-03-01

    Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.

  7. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    NASA Astrophysics Data System (ADS)

    Strobel, Claudia; Oehring, Hartmut; Herrmann, Rudolf; Förster, Martin; Reller, Armin; Hilger, Ingrid

    2015-05-01

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO2) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO2 nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO2 nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  8. Roles of cell confluency and fluid shear in 3-dimensional intracellular forces in endothelial cells.

    PubMed

    Hur, Sung Sik; del Álamo, Juan C; Park, Joon Seok; Li, Yi-Shuan; Nguyen, Hong A; Teng, Dayu; Wang, Kuei-Chun; Flores, Leona; Alonso-Latorre, Baldomero; Lasheras, Juan C; Chien, Shu

    2012-07-10

    We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell-cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell-cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially non-uniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell-cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemo-mechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.

  9. Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity.

    PubMed

    Lopes Bezerra, Leila M; Filler, Scott G

    2004-03-15

    Invasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity.

  10. Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells.

    PubMed

    Nojiri, Takashi; Hosoda, Hiroshi; Tokudome, Takeshi; Miura, Koichi; Ishikane, Shin; Otani, Kentaro; Kishimoto, Ichiro; Shintani, Yasushi; Inoue, Masayoshi; Kimura, Toru; Sawabata, Noriyoshi; Minami, Masato; Nakagiri, Tomoyuki; Funaki, Soichiro; Takeuchi, Yukiyasu; Maeda, Hajime; Kidoya, Hiroyasu; Kiyonari, Hiroshi; Shioi, Go; Arai, Yuji; Hasegawa, Takeshi; Takakura, Nobuyuki; Hori, Megumi; Ohno, Yuko; Miyazato, Mikiya; Mochizuki, Naoki; Okumura, Meinoshin; Kangawa, Kenji

    2015-03-31

    Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A-nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells.

  11. Atrial natriuretic peptide prevents cancer metastasis through vascular endothelial cells

    PubMed Central

    Nojiri, Takashi; Hosoda, Hiroshi; Tokudome, Takeshi; Miura, Koichi; Ishikane, Shin; Otani, Kentaro; Kishimoto, Ichiro; Shintani, Yasushi; Inoue, Masayoshi; Kimura, Toru; Sawabata, Noriyoshi; Minami, Masato; Nakagiri, Tomoyuki; Funaki, Soichiro; Takeuchi, Yukiyasu; Maeda, Hajime; Kidoya, Hiroyasu; Kiyonari, Hiroshi; Shioi, Go; Arai, Yuji; Hasegawa, Takeshi; Takakura, Nobuyuki; Hori, Megumi; Ohno, Yuko; Miyazato, Mikiya; Mochizuki, Naoki; Okumura, Meinoshin; Kangawa, Kenji

    2015-01-01

    Most patients suffering from cancer die of metastatic disease. Surgical removal of solid tumors is performed as an initial attempt to cure patients; however, surgery is often accompanied with trauma, which can promote early recurrence by provoking detachment of tumor cells into the blood stream or inducing systemic inflammation or both. We have previously reported that administration of atrial natriuretic peptide (ANP) during the perioperative period reduces inflammatory response and has a prophylactic effect on postoperative cardiopulmonary complications in lung cancer surgery. Here we demonstrate that cancer recurrence after curative surgery was significantly lower in ANP-treated patients than in control patients (surgery alone). ANP is known to bind specifically to NPR1 [also called guanylyl cyclase-A (GC-A) receptor]. In mouse models, we found that metastasis of GC-A–nonexpressing tumor cells (i.e., B16 mouse melanoma cells) to the lung was increased in vascular endothelium-specific GC-A knockout mice and decreased in vascular endothelium-specific GC-A transgenic mice compared with control mice. We examined the effect of ANP on tumor metastasis in mice treated with lipopolysaccharide, which mimics systemic inflammation induced by surgical stress. ANP inhibited the adhesion of cancer cells to pulmonary arterial and micro-vascular endothelial cells by suppressing the E-selectin expression that is promoted by inflammation. These results suggest that ANP prevents cancer metastasis by inhibiting the adhesion of tumor cells to inflamed endothelial cells. PMID:25775533

  12. Endothelial cells regulate the proliferation of monocytes in vitro.

    PubMed

    Pakala, R; Benedict, C R

    1999-11-01

    Monocytes (MPhis) are among the first cells to accumulate in early atherosclerotic lesions and generally are believed to be incapable of proliferation. However, recent studies indicate that the number of MPhis in atherosclerotic lesion may increase due to induction of local proliferation. Since proliferation of hematopoietic lineage cells is strongly influenced by interaction with neighboring cell types, we examined the ability of vascular endothelial cells (EC), smooth muscle cells or fibroblasts to stimulate MPhi proliferation. In this study, we show that only when seeded at high densities MPhis could proliferate in culture. However, when contact co-cultured with EC, MPhis proliferated at a higher rate (260% on day 6) than those cultured alone or co-cultured with smooth muscle cells or fibroblasts. Endothelial cells could stimulate the proliferation of MPhis even at non-proliferating densities. Only EC that were growth arrested or in lag phase could induce MPhi proliferation, whereas those in the exponential proliferating phase were non-stimulatory. Conditioned medium prepared from EC in growth arrested or lag phase failed to stimulate MPhi proliferation. Similarly physical separation of MPhis from EC also resulted in no proliferation. These results suggest that EC induced MPhi proliferation is contact dependent and no soluble factors are involved in this induction. This EC induced MPhi proliferation may have a profound effect on the rate of progression of atherosclerosis.

  13. Activation of endothelial nitric oxide synthase is dependent on its interaction with globular actin in human umbilical vein endothelial cells.

    PubMed

    Mi, Qiongyu; Chen, Nan; Shaifta, Yasin; Xie, Liping; Lu, Hui; Liu, Zhen; Chen, Qi; Hamid, Colleen; Becker, Silke; Ji, Yong; Ferro, Albert

    2011-09-01

    Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.

  14. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  15. A Comparison of Endothelial Cell Loss in Combined Cataract and MIGS (Hydrus) Procedure to Phacoemulsification Alone: 6-Month Results

    PubMed Central

    Fea, Antonio M.; Consolandi, Giulia; Pignata, Giulia; Cannizzo, Paola Maria Loredana; Lavia, Carlo; Billia, Filippo; Rolle, Teresa; Grignolo, Federico M.

    2015-01-01

    Purpose. To compare the corneal endothelial cell loss after phacoemulsification, alone or combined with microinvasive glaucoma surgery (MIGS), in nonglaucomatous versus primary open angle glaucoma (POAG) eyes affected by age-related cataract. Methods. 62 eyes of 62 patients were divided into group 1 (n = 25, affected by age-related cataract) and group 2 (n = 37, affected by age-related cataract and POAG). All patients underwent cataract surgery. Group 2 was divided into subgroups A (n = 19, cataract surgery alone) and B (n = 18, cataract surgery and MIGS). Prior to and 6 months after surgery the patients' endothelium was studied. Main outcomes were CD (cell density), SD (standard deviation), CV (coefficient of variation), and 6A (hexagonality coefficient) variations after surgeries. Results. There were no significant differences among the groups concerning preoperative endothelial parameters. The differences in CD before and after surgery were significant in all groups: 9.1% in group 1, 17.24% in group 2A, and 11.71% in group 2B. All endothelial parameters did not significantly change after surgery. Conclusions. Phacoemulsification determined a loss of endothelial cells in all groups. After surgery the change in endothelial parameters after MIGS was comparable to the ones of patients who underwent cataract surgery alone. PMID:26664740

  16. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    PubMed Central

    Kerasioti, Efthalia; Stagos, Dimitrios; Georgatzi, Vasiliki; Bregou, Erinda; Priftis, Alexandros; Kafantaris, Ioannis; Kouretas, Dimitrios

    2016-01-01

    Excessive production of reactive oxygen species (ROS) may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP) from tert-butyl hydroperoxide- (tBHP-) induced oxidative stress in endothelial cells (EA.hy926) were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH) and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS), protein carbonyls (CARB), and oxidized glutathione (GSSG) were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress. PMID:27127549

  17. Nitric Oxide Inhibits Hetero-adhesion of Cancer Cells to Endothelial Cells: Restraining Circulating Tumor Cells from Initiating Metastatic Cascade

    NASA Astrophysics Data System (ADS)

    Lu, Yusheng; Yu, Ting; Liang, Haiyan; Wang, Jichuang; Xie, Jingjing; Shao, Jingwei; Gao, Yu; Yu, Suhong; Chen, Shuming; Wang, Lie; Jia, Lee

    2014-03-01

    Adhesion of circulating tumor cells (CTCs) to vascular endothelial bed becomes a crucial starting point in metastatic cascade. We hypothesized that nitric oxide (NO) may prevent cancer metastasis from happening by its direct vasodilation and inhibition of cell adhesion molecules (CAMs). Here we show that S-nitrosocaptopril (CAP-NO, a typical NO donor) produced direct vasorelaxation that can be antagonized by typical NO scavenger hemoglobin and guanylate cyclase inhibitor. Cytokines significantly stimulated production of typical CAMs by the highly-purified human umbilical vein endothelial cells (HUVECs). CAP-NO inhibited expression of the stimulated CAMs (particularly VCAM-1) and the resultant hetero-adhesion of human colorectal cancer cells HT-29 to the HUVECs in a concentration-dependent manner. The same concentration of CAP-NO, however, did not significantly affect cell viability, cell cycle and mitochondrial membrane potential of HT-29, thus excluding the possibility that inhibition of the hetero-adhesion was caused by cytotoxicity by CAP-NO on HT-29. Hemoglobin reversed the inhibition of CAP-NO on both the hetero-adhesion between HT-29 and HUVECs and VCAM-1 expression. These data demonstrate that CAP-NO, by directly releasing NO, produces vasorelaxation and interferes with hetero-adhesion of cancer cells to vascular endothelium via down-regulating expression of CAMs. The study highlights the importance of NO in cancer metastatic prevention.

  18. Lipopolysaccharide-induced apoptosis in transformed bovine brain endothelial cells and human dermal microvessel endothelial cells: the role of JNK.

    PubMed

    Karahashi, Hisae; Michelsen, Kathrin S; Arditi, Moshe

    2009-06-01

    Stimulation of transformed bovine brain endothelial cells (TBBEC) with LPS leads to apoptosis while human microvessel endothelial cells (HMEC) need the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of MAPK and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation of caspase 3, caspase 6, and caspase 8 after the stimulation of LPS, followed by poly(ADP-ribose) polymerase cleavage and lactate dehydrogenase release. We also observed DNA cleavage determined by TUNEL staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a JNK inhibitor, suppressed the activation of caspases and lactate dehydrogenase release. Moreover, a PI3K inhibitor (LY294002) suppressed activation of caspases and combined treatment with both SP600125 and LY294002 completely inhibited the activation of caspases. These results suggest that the JNK signaling pathway through the tyrosine kinase and PI3K pathways is involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither ERK1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase 3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells.

  19. Arginase inhibition enhances angiogenesis in endothelial cells exposed to hypoxia.

    PubMed

    Wang, Lin; Bhatta, Anil; Toque, Haroldo A; Rojas, Modesto; Yao, Lin; Xu, Zhimin; Patel, Chintan; Caldwell, Ruth B; Caldwell, R William

    2015-03-01

    Hypoxia-induced arginase elevation plays an essential role in several vascular diseases but influence of arginase on hypoxia-mediated angiogenesis is completely unknown. In this study, in vitro network formation in bovine aortic endothelial cells (BAEC) was examined after exposure to hypoxia for 24h with or without arginase inhibition. Arginase activity, protein levels of the two arginase isoforms, eNOS, and VEGF as well as production of NO and ROS were examined to determine the involvement of arginase in hypoxia-mediated angiogenesis. Hypoxia elevated arginase activity and arginase 2 expression but reduced active p-eNOS(Ser1177) and NO levels in BAEC. In addition, both VEGF protein levels and endothelial elongation and network formation were reduced with continued hypoxia, whereas ROS levels increased and NO levels decreased. Arginase inhibition limited ROS, restored NO formation and VEGF expression, and prevented the reduction of angiogenesis. These results suggest a fundamental role of arginase activity in regulating angiogenic function.

  20. Salvianolic acid B improves vascular endothelial function in diabetic rats with blood glucose fluctuations via suppression of endothelial cell apoptosis.

    PubMed

    Ren, Younan; Tao, Shanjun; Zheng, Shuguo; Zhao, Mengqiu; Zhu, Yuanmei; Yang, Jieren; Wu, Yuanjie

    2016-11-15

    Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).

  1. Liver cyst cytokines promote endothelial cell proliferation and development.

    PubMed

    Brodsky, Kelley S; McWilliams, Ryan R; Amura, Claudia R; Barry, Nicholas P; Doctor, R Brian

    2009-10-01

    Autosomal dominant polycystic kidney (ADPKD) is highly prevalent genetic disease. Liver cyst disease is the most common extrarenal manifestation in ADPKD and accounts for up to 10% of ADPKD morbidity and mortality. The clinical features of ADPKD liver disease arise from dramatic increases in liver cyst volumes. To identify mechanisms that promote liver cyst growth, the present study characterized the degree of vascularization of liver cyst walls and determined that cyst-specific cytokines and growth factors can drive endothelial cell proliferation and development. Microscopic techniques demonstrated liver cyst walls are well vascularized. A comparative analysis found the vascular density in free liver cyst walls was greater in mice than in humans. Treatment of human micro-vascular endothelial cells (HMEC-1) with human liver cyst fluid (huLCF) induced a rapid increase in vascular endothelium growth factor receptor 2 (VEGFR2) phosphorylation that persisted for 45-60 min and was blocked by 20 microM SU5416, a VEGFR tyrosine kinase inhibitor. Similarly, huLCF treatment of HMEC-1 cells induced an increase in the cell proliferation rate (131 +/- 6% of control levels; P > 0.05) and the degree of vascular development ('tube' diameter assay: 92 +/- 14 microm for huLCF vs. 12 +/- 7 microm for vehicle); P > 0.05). Both cell proliferation and vascular development were sensitive to SU5416. These studies indicate that factors secreted by liver cyst epithelia can activate VEGF signaling pathways and induce endothelial cell proliferation and differentiation. The present studies suggest that targeting VEGFR2-dependent angiogenesis may be an effective therapeutic strategy in blocking ADPKD liver cyst vascularization and growth.

  2. PC12 Cells Differentiate into Chromaffin Cell-Like Phenotype in Coculture with Adrenal Medullary Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Mizrachi, Yaffa; Naranjo, Jose R.; Levi, Ben-Zion; Pollard, Harvey B.; Lelkes, Peter I.

    1990-08-01

    Previously we described specific in vitro interactions between PC12 cells, a cloned, catecholamine-secreting pheochromocytoma cell line derived from the rat adrenal medulla, and bovine adrenal medullary endothelial cells. We now demonstrate that these interactions induce the PC12 cells to acquire physical and biochemical characteristics reminiscent of chromaffin cells. Under coculture conditions involving direct cell-cell contact, the endothelial cells and the PC12 cells reduced their rates of proliferation; upon prolonged coculture PC12 cells clustered into nests of cells similar to the organization of chromaffin cells seen in vivo. Within 3 days in coculture with endothelial cells, but not with unrelated control cells, PC12 cells synthesized increased levels of [Met]enkephalin. In addition, PC12 cells, growing on confluent endothelial monolayers, failed to extend neurites in response to nerve growth factor. Neither medium conditioned by endothelial cells nor fixed endothelial cells could by themselves induce all of these different phenomena in the PC12 cells. These results suggest that under coculture conditions PC12 cells change their state of differentiation toward a chromaffin cell-like phenotype. The rapid, transient increase in the expression of the protooncogene c-fos suggests that the mechanism(s) inducing the change in the state of differentiation in PC12 cells in coculture with the endothelial cells may be distinct from that described for the differentiation of PC12 cells--e.g., by glucocorticoids. We propose that similar interactions between endothelial cells and chromaffin cell precursors may occur during embryonic development and that these interactions might be instrumental for the organ-specific differentiation of the adrenal medulla in vivo.

  3. Glycoproteomic Analysis of the Secretome of Human Endothelial Cells*

    PubMed Central

    Yin, Xiaoke; Bern, Marshall; Xing, Qiuru; Ho, Jenny; Viner, Rosa; Mayr, Manuel

    2013-01-01

    Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H218O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation (HCD) with electron-transfer dissociation (ETD) for a hybrid linear ion trap–orbitrap mass spectrometer. After deglycosylation with PNGase F in the presence of H218O, 123 unique peptides displayed 18O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted, and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples. PMID:23345538

  4. SECs (Sinusoidal Endothelial Cells), Liver Microenvironment, and Fibrosis

    PubMed Central

    Natarajan, Vaishaali; Harris, Edward N.

    2017-01-01

    Liver fibrosis is a wound-healing response to chronic liver injury such as alcoholic/nonalcoholic fatty liver disease and viral hepatitis with no FDA-approved treatments. Liver fibrosis results in a continual accumulation of extracellular matrix (ECM) proteins and paves the way for replacement of parenchyma with nonfunctional scar tissue. The fibrotic condition results in drastic changes in the local mechanical, chemical, and biological microenvironment of the tissue. Liver parenchyma is supported by an efficient network of vasculature lined by liver sinusoidal endothelial cells (LSECs). These nonparenchymal cells are highly specialized resident endothelial cell type with characteristic morphological and functional features. Alterations in LSECs phenotype including lack of LSEC fenestration, capillarization, and formation of an organized basement membrane have been shown to precede fibrosis and promote hepatic stellate cell activation. Here, we review the interplay of LSECs with the dynamic changes in the fibrotic liver microenvironment such as matrix rigidity, altered ECM protein profile, and cell-cell interactions to provide insight into the pivotal changes in LSEC physiology and the extent to which it mediates the progression of liver fibrosis. Establishing the molecular aspects of LSECs in the light of fibrotic microenvironment is valuable towards development of novel therapeutic and diagnostic targets of liver fibrosis. PMID:28293634

  5. 3D map of the human corneal endothelial cell

    PubMed Central

    He, Zhiguo; Forest, Fabien; Gain, Philippe; Rageade, Damien; Bernard, Aurélien; Acquart, Sophie; Peoc’h, Michel; Defoe, Dennis M.; Thuret, Gilles

    2016-01-01

    Corneal endothelial cells (CECs) are terminally differentiated cells, specialized in regulating corneal hydration and transparency. They are highly polarized flat cells that separate the cornea from the aqueous humor. Their apical surface, in contact with aqueous humor is hexagonal, whereas their basal surface is irregular. We characterized the structure of human CECs in 3D using confocal microscopy of immunostained whole corneas in which cells and their interrelationships remain intact. Hexagonality of the apical surface was maintained by the interaction between tight junctions and a submembraneous network of actomyosin, braced like a drum. Lateral membranes, which support enzymatic pumps, presented complex expansions resembling interdigitated foot processes at the basal surface. Using computer-aided design and drafting software, we obtained a first simplified 3D model of CECs. By comparing their expression with those in epithelial, stromal and trabecular corneal cells, we selected 9 structural or functional proteins for which 3D patterns were specific to CECs. This first 3D map aids our understanding of the morphologic and functional specificity of CECs and could be used as a reference for characterizing future cell therapy products destined to treat endothelial dysfunctions. PMID:27381832

  6. In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

    NASA Astrophysics Data System (ADS)

    Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

    2016-03-01

    The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

  7. A microfluidic cell culture system for monitoring of sequential changes in endothelial cells after heat stress.

    PubMed

    Tazawa, Hidekatsu; Sato, Kenjiro; Tsutiya, Atsuhiro; Tokeshi, Manabu; Ohtani-Kaneko, Ritsuko

    2015-08-01

    Endothelial damage induced by a highly elevated body temperature is crucial in some diseases including viral hemorrhagic fevers. Here, we report the heat-induced sequential changes of endothelial cells under shear stress, which were determined with a microfluidic culture system. Although live cell imaging showed only minor changes in the appearance of heat-treated cells, Hsp70 mRNA expression analysis demonstrated that the endothelial cells in channels of the system responded well to heat treatment. F-actin staining also revealed clear changes in the bundles of actin filaments after heat treatment. Well-organized bundles of actin filaments in control cells disappeared in heat-treated cells cultured in the channel. Furthermore, the system enabled detection of sequential changes in plasminogen activator inhibitor-1 (PAI-1) secretion from endothelial cells. PAI-1 concentration in the effluent solution was significantly elevated for the first 15min after initiation of heat treatment, and then decreased subsequently. This study provides fundamental information on heat-induced endothelial changes under shear stress and introduces a potent tool for analyzing endothelial secretions.

  8. Mechanotransductional Basis of Endothelial Cell Response to Intravascular Bubbles

    PubMed Central

    Klinger, Alexandra L.; Pichette, Benjamin; Sobolewski, Peter; Eckmann, David M.

    2011-01-01

    Vascular air embolism resulting from too rapid decompression is a well-known risk in deep-sea diving, aviation and space travel. It is also a common complication during surgery or other medical procedures when air or other endogenously administered gas is entrained in the circulation. Preventive and post-event treatment options are extremely limited for this dangerous condition, and none of them address the poorly understood pathophysiology of endothelial response to intravascular bubble presence. Using a novel apparatus allowing precise manipulation of microbubbles in real time fluorescence microscopy studies, we directly measure human umbilical vein endothelial cell responses to bubble contact. Strong intracellular calcium transients requiring extracellular calcium are observed upon cell-bubble interaction. The transient is eliminated both by the presence of the stretch activated channel inhibitor, gadolinium, and the transient receptor potential vanilliod family inhibitor, ruthenium red. No bubble induced calcium upsurge occurs if the cells are pretreated with an inhibitor of actin polymerization, cytochalasin-D. This study explores the biomechanical mechanisms at play in bubble interfacial interactions with endothelial surface layer (ESL) macromolecules, reassessing cell response after selective digestion of glycocalyx glycosoaminoglycans, hyaluran (HA) and heparin sulfate (HS). HA digestion causes reduction of cell-bubble adherence and a more rapid induction of calcium influx after contact. HS depletion significantly decreases calcium transient amplitudes, as does pharmacologically induced sydencan ectodomain shedding. The surfactant perfluorocarbon oxycyte abolishes any bubble induced calcium transient, presumably through direct competition with ESL macromolecules for interfacial occupancy, thus attenuating the interactions that trigger potentially deleterious biochemical pathways. PMID:21931900

  9. The chemokine CXCL13 (BCA-1) inhibits FGF-2 effects on endothelial cells.

    PubMed

    Spinetti, G; Camarda, G; Bernardini, G; Romano Di Peppe, S; Capogrossi, M C; Napolitano, M

    2001-11-23

    Several chemokines, belonging to both the CXC and CC classes, act as positive or negative regulators of angiogenesis. We sought to investigate the role of CXCL13, B cell-attracting chemokine 1 (BCA-1), also known as B-lymphocyte chemoattractant (BLC), on endothelial cell functions. We tested the effect of CXCL13 on HUVEC chemotaxis and proliferation in the presence of fibroblast growth factor (FGF)-2 and found that such chemokine inhibits FGF-2-induced functions, while is not active by itself. To test whether other FGF-2-mediated biological activities may be affected, we evaluated the ability of CXCL13 to rescue HUVEC from starvation-induced apoptosis, as FGF-2 is a survival factor for endothelial cells, and found that CXCL13 partially inhibits such rescue. Multiple mechanisms may be responsible for these biological activities as CXCL13 displaces FGF-2 binding to endothelial cells, inhibits FGF-2 homodimerization, and induces the formation of CXCL13-FGF-2 heterodimers. Our data suggest that CXCL13 may modulate angiogenesis by interfering with FGF-2 activity.

  10. Cell-Based Screening Identifies Paroxetine as an Inhibitor of Diabetic Endothelial Dysfunction

    PubMed Central

    Gerö, Domokos; Szoleczky, Petra; Suzuki, Kunihiro; Módis, Katalin; Oláh, Gabor; Coletta, Ciro; Szabo, Csaba

    2013-01-01

    We have conducted a phenotypic screening in endothelial cells exposed to elevated extracellular glucose (an in vitro model of hyperglycemia) to identify compounds that prevent hyperglycemia-induced reactive oxygen species (ROS) formation without adversely affecting cell viability. From a focused library of >6,000 clinically used drug-like and pharmacologically active compounds, several classes of active compounds emerged, with a confirmed hit rate of <0.5%. Follow-up studies focused on paroxetine, a clinically used antidepressant compound that has not been previously implicated in the context of hyperglycemia or diabetes. Paroxetine reduced hyperglycemia-induced mitochondrial ROS formation, mitochondrial protein oxidation, and mitochondrial and nuclear DNA damage, without interfering with mitochondrial electron transport or cellular bioenergetics. The ability of paroxetine to improve hyperglycemic endothelial cell injury was unique among serotonin reuptake blockers and can be attributed to its antioxidant effect, which primarily resides within its sesamol moiety. Paroxetine maintained the ability of vascular rings to respond to the endothelium-dependent relaxant acetylcholine, both during in vitro hyperglycemia and ex vivo, in a rat model of streptozotocin-induced diabetes. Thus, the current work identifies a novel pharmacological action of paroxetine as a protector of endothelial cells against hyperglycemic injury and raises the potential of repurposing of this drug for the experimental therapy of diabetic cardiovascular complications. PMID:23223176

  11. Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells.

    PubMed Central

    Morita, T; Kurihara, H; Maemura, K; Yoshizumi, M; Yazaki, Y

    1993-01-01

    Hemodynamic shear stress alters the architecture and functions of vascular endothelial cells. We have previously shown that the synthesis of endothelin-1 (ET-1) in endothelial cells is increased by exposure to shear stress. Here we examined whether shear stress-induced alterations in cytoskeletal structures are responsible for increases in ET-1 synthesis in cultured porcine aortic endothelial cells. Exposure of endothelial cells to 5 dyn/cm2 of low shear stress rapidly increased monomeric G-actin contents within 5 min without changing total actin contents. The ratio of G- to total actin, 54 +/- 0.8% in quiescent endothelial cells, increased to 87 +/- 4.2% at 6 h and then decreased. Following the disruption of filamentous (F)-actin into G-actin, ET-1 mRNA levels in endothelial cells also increased within 30 min and reached a peak at 6 h. The F-actin stabilizer, phalloidin, abolished shear stress-induced increases in ET-1 mRNA; however, it failed to inhibit increases in ET-1 mRNA secondary to other stimulants. This indicates that shear stress-induced increases in ET-1 mRNA levels may be mediated by the disruption of actin fibers. Furthermore, increases in ET-1 gene expression can be induced by actin-disrupting agents, cytochalasin B and D. Another cytoskeleton-disrupting agent, colchicine, which inhibits dimerization of tubulin, did not affect the basal level of ET-1 mRNA. However, colchicine completely inhibited shear stress- and cytochalasin B-induced increases in ET-1 mRNA levels. These results suggest that shear stress-induced ET-1 gene expression in endothelial cells is mediated by the disruption of actin cytoskeleton and this induction is dependent on the integrity of microtubules. Images PMID:8408624

  12. Carbamylated Low-Density Lipoprotein (cLDL)-Mediated Induction of Autophagy and Its Role in Endothelial Cell Injury

    PubMed Central

    Bose, Chhanda; Shah, Sudhir V.; Karaduta, Oleg K.

    2016-01-01

    Patients with chronic kidney disease (CKD) have high risk of cardiovascular complications. Plasma levels of carbamylated proteins produced by urea-derived isocyanate or thiocyanate are elevated in CKD patients and that they are significant predictors of cardiovascular events and all-cause mortality. Carbamylated LDL (cLDL) has pro-atherogenic properties and is known to affect major biological processes relevant to atherosclerosis including endothelial cell injury. The underlying mechanisms of cLDL-induced endothelial cell injury are not well understood. Although autophagy has been implicated in atherosclerosis, cLDL-mediated induction of autophagy and its role in endothelial cell injury is unknown. Our studies demonstrate that human coronary artery endothelial cells (HCAECs) respond to cLDL by specific induction of key autophagy proteins including LC3-I, beclin-1, Atg5, formation of lipid-conjugated LC3-II protein, and formation of punctate dots of autophagosome-associated LC3-II. We demonstrated that autophagy induction is an immediate response to cLDL and occurred in a dose and time-dependent manner. Inhibition of cLDL-induced autophagy by a specific siRNA to LC3 as well as by an autophagy inhibitor provided protection from cLDL-induced cell death and DNA fragmentation. Our studies demonstrate that autophagy plays an important role in cLDL-mediated endothelial cell injury and may provide one of the underlying mechanisms for the pathogenesis of cLDL-induced atherosclerosis in CKD patients. PMID:27973558

  13. Specific binding of angiogenin to calf pulmonary artery endothelial cells.

    PubMed

    Badet, J; Soncin, F; Guitton, J D; Lamare, O; Cartwright, T; Barritault, D

    1989-11-01

    Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4 degrees C of 125I-labeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by 80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10(-9) M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10(-6) M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 125I-labeled human recombinant ANG binding only in the absence of copper.

  14. Vascular endothelial cells minimize the total force on their nuclei.

    PubMed Central

    Hazel, A L; Pedley, T J

    2000-01-01

    The vascular endothelium is a cellular monolayer that lines the arterial walls. It plays a vital role in the initiation and development of atherosclerosis, an occlusive arterial disease responsible for 50% of deaths in the Western world. The focal nature of the disease suggests that hemodynamic forces are an important factor in its pathogenesis. This has led to the investigation of the effects of mechanical forces on the endothelial cells themselves. It has been found that endothelial cells do respond to stresses induced by the flowing blood; in particular, they elongate and align with an imposed flow direction. In this paper, we calculate the distribution of force exerted on a three-dimensional hump, representing the raised cell nucleus, by a uniform shear flow. It is found that, for a nonaxisymmetric ellipsoidal hump, the least total force is experienced when the hump is aligned with the flow. Furthermore, for a hump of fixed volume, there is a specific aspect ratio combination that results in the least total force upon the hump, (0.38:2.2:1.0; height:length:width). This is approximately the same as the average aspect ratio taken up by the cell nuclei in vivo (0.27:2.23:1.0). It is possible, therefore, that the cells respond to the flow in such a way as to minimize the total force on their nuclei. PMID:10620272

  15. Small caliber arterial endothelial cells calcium signals elicited by PAR2 are preserved from endothelial dysfunction

    PubMed Central

    Hennessey, John C; Stuyvers, Bruno D; McGuire, John J

    2015-01-01

    Endothelial cell (EC)-dependent vasodilation by proteinase-activated receptor 2 (PAR2) is preserved in small caliber arteries in disease states where vasodilation by muscarinic receptors is decreased. In this study, we identified and characterized the PAR2-mediated intracellular calcium (Ca2+)-release mechanisms in EC from small caliber arteries in healthy and diseased states. Mesenteric arterial EC were isolated from PAR2 wild-type (WT) and null mice, after saline (controls) or angiotensin II (AngII) infusion, for imaging intracellular calcium and characterizing the calcium-release system by immunofluorescence. EC Ca2+ signals comprised two forms of Ca2+-release events that had distinct spatial-temporal properties and occurred near either the plasmalemma (peripheral) or center of EC. In healthy EC, PAR2-dependent increases in the densities and firing rates of both forms of Ca2+-release were abolished by inositol 1,4,5- trisphosphate receptor (IP3R) inhibitor, but partially reduced by transient potential vanilloid channels inhibitor ruthenium red (RR). Acetylcholine (ACh)-induced less overall Ca2+-release than PAR2 activation, but enhanced selectively the incidence of central events. PAR2-dependent Ca2+-activity, inhibitors sensitivities, IP3R, small- and intermediate-conductance Ca2+-activated potassium channels expressions were unchanged in EC from AngII WT. However, the same cells exhibited decreases in ACh-induced Ca2+-release, RR sensitivity, and endothelial nitric oxide synthase expression, indicating AngII-induced dysfunction was differentiated by receptor, Ca2+-release, and downstream targets of EC activation. We conclude that PAR2 and muscarinic receptors selectively elicit two elementary Ca2+ signals in single EC. PAR2-selective IP3R-dependent peripheral Ca2+-release mechanisms are identical between healthy and diseased states. Further study of PAR2-selective Ca2+-release for eliciting pathological and/or normal EC functions is warranted. PMID:25729579

  16. Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells.

    PubMed

    Santos, Lívia; Fuhrmann, Gregor; Juenet, Maya; Amdursky, Nadav; Horejs, Christine-Maria; Campagnolo, Paola; Stevens, Molly M

    2015-08-13

    Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

  17. PDGFRβ and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells.

    PubMed

    Plantamura, Ilaria; Casalini, Patrizia; Dugnani, Erica; Sasso, Marianna; D'Ippolito, Elvira; Tortoreto, Monica; Cacciatore, Matilde; Guarnotta, Carla; Ghirelli, Cristina; Barajon, Isabella; Bianchi, Francesca; Triulzi, Tiziana; Agresti, Roberto; Balsari, Andrea; Campiglio, Manuela; Tripodo, Claudio; Iorio, Marilena V; Tagliabue, Elda

    2014-07-01

    Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes. In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation. Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRβ and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo. In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRβ and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.

  18. Growth factor- and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

    PubMed Central

    Peplow, Philip V.

    2014-01-01

    Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes specific growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endothelial progenitor cells migrate and home to specific sites following ischemic stroke via growth factor/cytokine gradients. Some growth factors are less stable under acidic conditions of tissue ischemia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovascularization following ischemic stroke. PMID:25317152

  19. Oxytocin stimulates migration and invasion in human endothelial cells

    PubMed Central

    Cattaneo, M G; Chini, B; Vicentini, L M

    2007-01-01

    Background and purpose: It has recently been reported that oxytocin is produced by some tumour cell types, and that oxytocin receptors, belonging to the G-protein-coupled receptor (GPCR) family, are expressed in a variety of cell types. Among these, human umbilical vein endothelial cells (HUVECs) respond to oxytocin with an increased proliferation, suggesting a possible role for the hormone in the regulation of angiogenesis. Experimental approach: We employed chemotaxis and chemoinvasion assays to characterize the effect of oxytocin on HUVEC motility, and immunoblot analysis to study its molecular mechanisms of action. Key results: We showed that oxytocin stimulates migration and invasion in HUVECs via oxytocin receptor activation. Searching for the molecular mechanism(s) responsible for oxytocin's pro-migratory effect, we identified the Gq coupling of oxytocin receptors and phospholipase C (PLC) as the main effectors of oxytocin's action in HUVECs. We also found that oxytocin stimulates the phosphorylation of endothelial nitric oxide synthase (eNOS) via the phosphatidylinositol-3-kinase (PI-3-K)/AKT pathway, and that the activation of PI-3-K and formation of nitric oxide (NO) are required for the pro-migratory effect of oxytocin. Conclusions and implications: The ability of oxytocin to stimulate HUVEC motility and invasion suggests that the hormone can participate in physiopathological processes where activation of endothelial cells plays an important role, for example, in angiogenesis. Interestingly, both the AKT and eNOS phosphorylation induced by oxytocin receptor activation depended on PLC activity, thus suggesting the existence of a still undefined mechanism connecting PLC to the PI-3-K/AKT pathway, upon oxytocin stimulation. PMID:18059319

  20. Role of NADPH Oxidase-4 in Human Endothelial Progenitor Cells

    PubMed Central

    Hakami, Nora Y.; Ranjan, Amaresh K.; Hardikar, Anandwardhan A.; Dusting, Greg J.; Peshavariya, Hitesh M.

    2017-01-01

    Introduction: Endothelial progenitor cells (EPCs) display a unique ability to promote angiogenesis and restore endothelial function in injured blood vessels. NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) serves as a signaling molecule and promotes endothelial cell proliferation and migration as well as protecting against cell death. However, the role of NOX4 in EPC function is not completely understood. Methods: EPCs were isolated from human saphenous vein and mammary artery discarded during bypass surgery. NOX4 gene and protein expression in EPCs were measured by real time-PCR and Western blot analysis respectively. NOX4 gene expression was inhibited using an adenoviral vector expressing human NOX4 shRNA (Ad-NOX4i). H2O2 production was measured by Amplex red assay. EPC migration was evaluated using a transwell migration assay. EPC proliferation and viability were measured using trypan blue counts. Results: Inhibition of NOX4 using Ad-NOX4i reduced Nox4 gene and protein expression as well as H2O2 formation in EPCs. Inhibition of NOX4-derived H2O2 decreased both proliferation and migration of EPCs. Interestingly, pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) decreased NOX4 expression and reduced survival of EPCs. However, the survival of EPCs was further diminished by TNF-α in NOX4-knockdown cells, suggesting that NOX4 has a protective role in EPCs. Conclusion: These findings suggest that NOX4-type NADPH oxidase is important for proliferation and migration functions of EPCs and protects against pro-inflammatory cytokine induced EPC death. These properties of NOX4 may facilitate the efficient function of EPCs which is vital for successful neovascularization. PMID:28386230

  1. Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

    PubMed Central

    1995-01-01

    Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a

  2. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  3. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    PubMed

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction.

  4. The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

    SciTech Connect

    Delaney, Christie E.; Weagant, Brodie T.; Addison, Christina L. . E-mail: caddison@ohri.ca

    2006-08-01

    We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.

  5. In vitro induction of human adipose-derived stem cells into lymphatic endothelial-like cells.

    PubMed

    Yang, Yi; Chen, Xiao-hu; Li, Fu-gui; Chen, Yun-xian; Gu, Li-qiang; Zhu, Jia-kai; Li, Ping

    2015-02-01

    Human adipose-derived stem cells (hADSCs) may provide a suitable number of progenitors for the treatment of lymphatic edema; however, to date the protocols for inducing hADSCs into this tissue type have not been standardized. We wished to investigate the induction of hADSCs into lymphatic endothelial-like cells using vascular endothelial growth factor-C156S (VEGF-C156S) and other growth factors in vitro. hADSCs from healthy adult adipose tissue were purified using enzyme digestion. Differentiation was induced using medium containing VEGF-C156S and bovine fibroblast growth factor (bFGF). Differentiation was confirmed using immunostaining for lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and fms-related tyrosine kinase 4 (FLT-4), two lymphatic endothelial cell markers. The expression levels of LYVE-1, prospero homeobox 1 (PROX-1), and FLT-4 throughout induction were assessed using reverse transcriptase quantitative polymerase chain reaction. hADSCs were successfully obtained by trypsin digest and purification. Flow cytometry showed these cells were similar to mesenchymal stem cells, with a high positive rate of CD13, CD29, CD44, and CD105, and a low positive rate of CD31, CD34, CD45, and HLA-DR. Induction to lymphatic endothelial-like cells was successful, with cells expressing high levels of LYVE-1, PROX-1, and FLT-4. Adipose-derived stem cells can be induced to differentiate into lymphatic endothelial-like cells using a medium containing VEGF-C156S, bFGF, and other growth factors. This population of lymphatic endothelial-like cells may be useful for lymphatic reconstruction in the future.

  6. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells

    PubMed Central

    Vemula, Sasidhar; Meador, Jonathan Luke; Yoshimoto, Momoko; Ferkowicz, Michael J; Fett, Alexa; Gupta, Manav; Rapp, Brian M; Saadatzadeh, Mohammad Reza; Ginsberg, Michael; Elemento, Olivier; Lee, Younghee; Voytik-Harbin, Sherry L; Chung, Hyung Min; Hong, Ki Sung; Reid, Emma; O'Neill, Christina L; Medina, Reinhold J; Stitt, Alan W; Murphy, Michael P; Rafii, Shahin; Broxmeyer, Hal E; Yoder, Mervin C

    2015-01-01

    The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony–forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel–forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >108 ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells. PMID:25306246

  7. Transcript Analysis Reveals a Specific HOX Signature Associated with Positional Identity of Human Endothelial Cells

    PubMed Central

    Toshner, Mark; Dunmore, Benjamin J.; McKinney, Eoin F.; Southwood, Mark; Caruso, Paola; Upton, Paul D.; Waters, John P.; Ormiston, Mark L.; Skepper, Jeremy N.; Nash, Gerard; Rana, Amer A.; Morrell, Nicholas W.

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole “HOX transcriptome” of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity. PMID:24651450

  8. Transcript analysis reveals a specific HOX signature associated with positional identity of human endothelial cells.

    PubMed

    Toshner, Mark; Dunmore, Benjamin J; McKinney, Eoin F; Southwood, Mark; Caruso, Paola; Upton, Paul D; Waters, John P; Ormiston, Mark L; Skepper, Jeremy N; Nash, Gerard; Rana, Amer A; Morrell, Nicholas W

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole "HOX transcriptome" of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity.

  9. Maslinic Acid Inhibits Proliferation of Renal Cell Carcinoma Cell Lines and Suppresses Angiogenesis of Endothelial Cells

    PubMed Central

    Thakor, Parth; Song, Wenzhe; Subramanian, Ramalingam B.; Thakkar, Vasudev R.; Vesey, David A.

    2017-01-01

    Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC) remains a treatment-resistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1), endothelial cells (human umbilical vein endothelial cell line [HUVEC]), and primary cultures of kidney proximal tubular epithelial cells (PTEC) were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

  10. Polyphenols action against oxidative stress formation in endothelial cells.

    PubMed

    Łuczaj, Wojciech; Zapora, Ewa; Szczepański, Marek; Wnuczko, Krzysztof; Skrzydlewska, Elzbieta

    2009-01-01

    The aim of this study was to investigate the influence of epigallocatechin-3-gallate (EGCG), theaflavins (TFs) and black tea extract (BTE) on oxidative stress formation as well as on antioxidant system of human vein endothelial cells (HUVEC). HUVEC were incubated for 0,5 h with 100 mM tert-butyl hydroperoxide (t-BHP) for oxidative stress formation. The influence of EGCG, TFs, and BTE on oxidative stress and antioxidant system parameters was investigated by the pre-incubation for 2 h with 50 mg/mL of each compound. Half hour exposure to t-BHP caused statistically significant decrease in GSH-Px activity and in the content of GSH, vitamin A, vitamin E as well as tryptophan. Moreover, pretreatment of cells with t-BHP caused statistically significant increase in activities of Cu,Zn-SOD, GSSG-R and in the level of MDA and dityrosine. Pretreated with t-BHP endothelial cells, subjected to EGCG, TFs and black tea extract, are partially protected against oxidative activity of t-BHP causing statistically significant increase in GSH-Px activity, GSH and tryptophan level and decrease in MDA and dityrosine level in comparison with HUVEC pretreated with t-BHP group. These results indicate the beneficial effect of tea polyphenolic compounds on HUVEC antioxidant abilities and, in consequence, their protective effect in cell components.

  11. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    NASA Technical Reports Server (NTRS)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1-14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10-50 microM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular

  12. Influence of the coating with extracellular matrix and the number of cell passages on the endothelialization of a polystyrene surface.

    PubMed

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

    2015-01-01

    The proper morphology and function of the vascular endothelium are prerequisites for a sufficient supply of the tissues. Endothelial cell (EC) dysfunction can lead to circulatory disorders and the development of cardiovascular diseases. The endothelialization of cardiovascular implants is a sophisticated task since EC miss their natural environment and physiological stimuli in vitro. In addition, different studies revealed that the EC behavior and morphology depended on the substrate and the passage number of the EC. Therefore, the comparison of endothelialization studies is very difficult, when passage and substrate are unknown.The aim of this study was to investigate the growth potential and cell morphology of human venous endothelial cells (HUVEC) as a function of different cell passages and different substrates (pristine polystyrene, tissue-typical ECM-coated polystyrene). The study revealed that HUVEC morphology and growth potential were significantly different on pristine polystyrene compared to the basal lamina-like ECM-coated polystyrene surface. Furthermore, it became obvious that the passage of the cells affected the endothelialization of the polystyrene surface significantly. In conclusion, this study emphasized the need for a critical consideration of EC data whereas a simple comparison of results is not possible if EC age and passage is unknown.

  13. Synergism of matrix stiffness and vascular endothelial growth factor on mesenchymal stem cells for vascular endothelial regeneration.

    PubMed

    Wingate, Kathryn; Floren, Michael; Tan, Yan; Tseng, Pi Ou Nancy; Tan, Wei

    2014-09-01

    Mesenchymal stem cells (MSCs) hold tremendous potential for vascular tissue regeneration. Research has demonstrated that individual factors in the cell microenvironment such as matrix elasticity and growth factors regulate MSC differentiation to vascular lineage. However, it is not well understood how matrix elasticity and growth factors combine to direct the MSC fate. This study examines the combined effects of matrix elasticity and vascular endothelial growth factor (VEGF) on both MSC differentiation into endothelial lineage and MSC paracrine signaling. MSCs were seeded in soft nanofibrous matrices with or without VEGF, and in Petri dishes with or without VEGF. Only MSCs seeded in three-dimensional soft matrices with VEGF showed significant increases in the expression of endothelial markers (vWF, eNOS, Flt-1, and Flk-1), while eliminating the expression of smooth muscle marker (SM-α-actin). MSCs cultured in VEGF alone on two-dimensional dishes showed increased expression of both early-stage endothelial and smooth muscle markers, indicating immature vascular differentiation. Furthermore, MSCs cultured in soft matrices with VEGF showed faster upregulation of endothelial markers compared with MSCs cultured in VEGF alone. Paracrine signaling studies found that endothelial cells cultured in the conditioned media from MSCs differentiated in the soft matrix and VEGF condition exhibited increased migration and formation of capillary-like structures. These results demonstrate that VEGF and soft matrix elasticity act synergistically to guide MSC differentiation into mature endothelial phenotype while enhancing paracrine signaling. Therefore, it is critical to control both mechanical and biochemical factors to safely regenerate vascular tissues with MSCs.

  14. Scanning electron microscopic analysis of endothelial cell coverage and quality in large vessels from multi-organ donors: effects of preservation on endothelial cell integrity.

    PubMed

    van Leeuwen, E B; Molema, G; van Luyn, M J; de Jong, K P; Dijk, F; Slooff, M J; Ruiters, M H; van der Meer, J

    2000-06-01

    Endothelial cell integrity (coverage and quality) of large donor vessels is important because these vessels are used for vascular reconstructions in solid-organ transplantation. Disruption of the endothelial cell monolayer will initiate blood coagulation and may lead to thrombosis of large vessels, often resulting in the loss of the transplanted organ. Iliac arteries and veins, removed from 10 heart-beating multi-organ donors at the end of the donor procedure, were analyzed using scanning electron microscopy at three different time points of preservation. Endothelial cell coverage and quality were determined immediately after removal from the donor, after 10 h (time of transplantation) and 7 d storage in 'University of Wisconsin' cold preservation solution (UW). Endothelial cell coverage decreased during the preservation of arteries, but was maintained in veins. Storage of the veins for 7 d in plastic bags showed a decreased endothelial cell coverage compared to storage in glass vials. Early removal of the blood vessels and proper storage, free floating and in clean UW, may improve maintenance of the endothelial cell integrity. These findings may be important in order to reduce the risk of thrombosis and, consequently, organ failure after transplantation. Furthermore, vessels with maintained endothelial cell integrity after 7 d may be used for in vitro research.

  15. A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

    PubMed Central

    Gabriele, Morena; Del Prato, Stefano; Pucci, Laura

    2017-01-01

    Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS) orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs), bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG), on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER) stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation. PMID:28386305

  16. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors

    PubMed Central

    Bai, Huai; Forrester, John V.; Zhao, Min

    2015-01-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24 h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors. PMID:21524919

  17. Uptake and transport of superparamagnetic iron oxide nanoparticles through human brain capillary endothelial cells.

    PubMed

    Thomsen, L B; Linemann, T; Pondman, K M; Lichota, J; Kim, K S; Pieters, R J; Visser, G M; Moos, T

    2013-10-16

    The blood-brain barrier (BBB) formed by brain capillary endothelial cells (BCECs) constitutes a firm physical, chemical, and immunological barrier, making the brain accessible to only a few percent of potential drugs intended for treatment inside the central nervous system. With the purpose of overcoming the restraints of the BBB by allowing the transport of drugs, siRNA, or DNA into the brain, a novel approach is to use superparamagnetic iron oxide nanoparticles (SPIONs) as drug carriers. The aim of this study was to investigate the ability of fluorescent SPIONs to pass through human brain microvascular endothelial cells facilitated by an external magnet. The ability of SPIONs to penetrate the barrier was shown to be significantly stronger in the presence of an external magnetic force in an in vitro BBB model. Hence, particles added to the luminal side of the in vitro BBB model were found in astrocytes cocultured at a remote distance on the abluminal side, indicating that particles were transported through the barrier and taken up by astrocytes. Addition of the SPIONs to the culture medium did not negatively affect the viability of the endothelial cells. The magnetic force-mediated dragging of SPIONs through BCECs may denote a novel mechanism for the delivery of drugs to the brain.

  18. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

    PubMed

    Bai, Huai; Forrester, John V; Zhao, Min

    2011-07-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors.

  19. An assay for macrophage-mediated regulation of endothelial cell proliferation.

    PubMed

    Khan, Aslam Ali; Apte, Rajendra S

    2008-01-01

    We have developed an assay that quantifies the potential of macrophages to regulate proliferation of endothelial cells. We show that young mice macrophages can be distinguished from old mice macrophages by their ability to inhibit vascular endothelial cell proliferation. While young mice macrophages robustly inhibit proliferation, old mice macrophages fail to do so and actually promote the proliferation of endothelial cells. In this report, we outline a technique that directly assesses the effect of macrophages on modulation of endothelial cell proliferation. This assay will help us in understanding the mechanisms of macrophage function in several disease states characterized by abnormal angiogenesis including cancers, angiogenic eye disease and atherosclerotic heart disease.

  20. Intestinal and peri-tumoral lymphatic endothelial cells are resistant to radiation-induced apoptosis

    SciTech Connect

    Sung, Hoon Ki; Morisada, Tohru; Cho, Chung-Hyun; Oike, Yuichi; Lee, Jayhun; Sung, Eon Ki; Chung, Jae Hoon; Suda, Toshio; Koh, Gou Young . E-mail: gykoh@kaist.ac.kr

    2006-06-30

    Radiation therapy is a widely used cancer treatment, but it is unable to completely block cancer metastasis. The lymphatic vasculature serves as the primary route for metastatic spread, but little is known about how lymphatic endothelial cells respond to radiation. Here, we show that lymphatic endothelial cells in the small intestine and peri-tumor areas are highly resistant to radiation injury, while blood vessel endothelial cells in the small intestine are relatively sensitive. Our results suggest the need for alternative therapeutic modalities that can block lymphatic endothelial cell survival, and thus disrupt the integrity of lymphatic vessels in peri-tumor areas.

  1. Effects of Blood Products on Inflammatory Response in Endothelial Cells In Vitro

    PubMed Central

    Buddeberg, Felix; Schuppli, Caroline; Roth Z'graggen, Birgit; Hasler, Melanie; Schanz, Urs; Mehr, Manuela; Spahn, Donat R.; Beck Schimmer, Beatrice

    2012-01-01

    Background Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. Methods The inflammatory response from pre-activated (endotoxin-stimulated) and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC), platelet concentrates (PC) and fresh frozen plasma (FFP) was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. Results Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. Conclusion Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells. PMID:22438924

  2. Arginine deiminase modulates endothelial tip cells via excessive synthesis of reactive oxygen species.

    PubMed

    Zhuo, Wei; Song, Xiaomin; Zhou, Hao; Luo, Yongzhang

    2011-10-01

    ADI (arginine deiminase), an enzyme that hydrolyses arginine, has been reported as an anti-angiogenesis agent. However, its molecular mechanism is unclear. We have demonstrated for the first time that ADI modulates the angiogenic activity of endothelial tip cells. By arginine depletion, ADI disturbs actin filament in endothelial tip cells, causing disordered migratory direction and decreased migration ability. Furthermore, ADI induces excessive synthesis of ROS (reactive oxygen species), and activates caspase 8-, but not caspase 9-, dependent apoptosis in endothelial cells. These findings provide a novel mechanism by which ADI inhibits tumour angiogenesis through modulating endothelial tip cells.

  3. Polyploidy impairs human aortic endothelial cell function and is prevented by nicotinamide phosphoribosyltransferase.

    PubMed

    Borradaile, Nica M; Pickering, J Geoffrey

    2010-01-01

    Polyploid endothelial cells are found in aged and atherosclerotic arteries. However, whether increased chromosome content has an impact on endothelial cell function is unknown. We show here that human aortic endothelial cells become tetraploid as they approach replicative senescence. Furthermore, accumulation of tetraploid endothelial cells was accelerated during growth in high glucose. Interestingly, induction of polyploidy was completely prevented by modest overexpression of the NAD+ regenerating enzyme, nicotinamide phosphoribosyltransferase (Nampt). To determine the impact of polyploidy on endothelial cell function, independent of replicative senescence, we induced tetraploidy using the spindle poison, nocodazole. Global gene expression analyses of tetraploid endothelial cells revealed a dysfunctional phenotype characterized by a cell cycle arrest profile (decreased CCNE2/A2, RBL1, BUB1B; increased CDKN1A) and increased expression of genes involved in inflammation (IL32, TNFRSF21/10C, PTGS1) and extracellular matrix remodeling (COL5A1, FN1, MMP10/14). The protection from polyploidy conferred by Nampt was not associated with enhanced poly(ADP-ribose) polymerase-1 or sirtuin (SIRT) 2 activity, but with increased SIRT1 activity, which reduced cellular reactive oxygen species and the associated oxidative stress stimulus for the induction of polyploidy. We conclude that human aortic endothelial cells are prone to chromosome duplication that, in and of itself, can induce characteristics of endothelial dysfunction. Moreover, the emergence of polyploid endothelial cells during replicative aging and glucose overload can be prevented by optimizing the Nampt-SIRT1 axis.

  4. Subcellular characterization of glucose uptake in coronary endothelial cells.

    PubMed

    Gaudreault, N; Scriven, D R L; Laher, I; Moore, E D W

    2008-01-01

    Despite all the evidence linking glucose toxicity to an increased risk of cardiovascular diseases, very little is known about the regulation of glucose uptake in endothelial cells. We have previously reported an asymmetric distribution of the GLUTs (1-5) and SGLT-1 in en face preparations of rat coronary artery endothelia [Gaudreault N., Scriven D.R., Moore E.D., 2004. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 47(12),2081-2092]. We assessed this time, through immunocytochemistry and wide field fluorescence microscopy coupled to deconvolution, the presence and subcellular distribution of glucose transporters in cultures of human coronary artery endothelial cells (HCAECs). HCAECs express GLUT-1 to 5 and SGLT-1, but their subcellular distribution lacks the luminal/abluminal asymmetry and the proximity to cell-to-cell junctions observed in intact endothelium. To determine the impact of the transporters' distribution on intracellular glucose accumulation, a fluorescent glucose analog (2-NBDG) was used in conjunction with confocal microscopy to monitor uptake in individual cells; the arteries were mounted in an arteriograph chamber with physiological flow rates. The uptake in both preparations was inhibited by cytochalasin-B and d-glucose and stimulated by insulin, but the distribution of the incorporated 2-NBDG mirrored that of the transporters. In HCAEC it was distributed throughout the cell and in the intact arterial endothelium it was restricted to the narrow cytosolic volume adjacent to the cell-to-cell junctions. We suggest that the latter subcellular organization and compartmentalization may facilitate transendothelial transport of glucose in intact coronary artery.

  5. Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40 peptide in brain endothelial cells.

    PubMed

    Fonseca, Ana Catarina R G; Ferreiro, Elisabete; Oliveira, Catarina R; Cardoso, Sandra M; Pereira, Cláudia F

    2013-12-01

    Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1-40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca(2+) homeostasis due to the release of Ca(2+) from this intracellular store. Finally, Aβ1-40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1-40 concomitantly with caspase-12 activation. Furthermore, Aβ1-40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1-40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration.

  6. iNOS Activation Regulates β-catenin Association with Its Partners in Endothelial Cells

    PubMed Central

    Gonzalez, Deyarina; Rojas, Armando; Herrera, Maria Beatriz; Conlan, R. Steven

    2012-01-01

    Background Signals that disrupt β-catenin association to cadherins may influence the translocation of β-catenin to the nucleus to regulate transcription. Post-translational modification of proteins is a signalling event that may lead to changes in structural conformation, association or function of the target proteins. NO and its derivatives induce nitration of proteins during inflammation. It has been described that animals treated with NO donors showed increased permeability due to modulation of VE-cadherin/catenin complex. We, therefore, aim to evaluate the effect of iNOS activation on the expression, nuclear localisation and function of β-catenin in endothelial cells. Methodology/Principal Findings Expression, nuclear localisation, post-translational modifications and function of β-catenin was analysed by cell fractionation, immunoprecipitation, immunoblots, QRT-PCR and permeability assays in murine endothelial cells (H5V). Influence of macrophage activation on expression of VE-cadherin/p120-catenin/β-catenin complex in co-cultured H5V cells was also assessed. Activation of macrophages to produce NO provoked a decrease in VE-cadherin/p120-catenin/β-catenin expression in H5V cells. Phosphorylation of β-catenin, p120-catenin and VE-cadherin, and reduction in the barrier properties of the cell monolayer was associated with iNOS induction. Moreover, high NO levels provoked nitration of β-catenin, and induced its translocation to the nucleus. In the nucleus of NOS activated cells, nitration levels of β-catenin influenced its association with TCF4 and p65 proteins. High levels of NO altered β-catenin mediated gene expression of NFκB and Wnt target genes without affecting cell viability. Conclusions NOS activity modulates β-catenin post-translational modifications, function and its association with different partners to promote endothelial cell survival. Therapeutic manipulation of iNOS levels may remove a critical cytoprotective mechanism of importance in

  7. Shear stress-induced redistribution of the glycocalyx on endothelial cells in vitro.

    PubMed

    Bai, Ke; Wang, Wen

    2014-04-01

    The glycocalyx is the inner most layer of the endothelium that is in direct contact with the circulating blood. Shear stress affects its synthesis and reorganization. This study focuses on changes in the spatial distribution of the glycocalyx caused by shear stimulation and its recovery following the removal of the shear stress. Sialic acid components of the glycocalyx on human umbilical vain endothelial cells are observed using confocal microscopy. The percentage area of the cell membrane covered by the glycocalyx, as well as the average fluorescence intensity ratio between the apical and edge areas of the cell is used to assess the spatial distribution of the glycocalyx on the cell membrane. Our results show that following 24 h shear stimulation, the glycocalyx relocates near the edge of endothelial cells (i.e., cell-cell junction regions). Following the removal of the shear stress, the glycocalyx redistributes and gradually appears in the apical region of the cell membrane. This redistribution is faster in the early hours (<4 h) after shear stimulation than that in the later stage (e.g., between 8 and 24 h). We further investigate the recovery of the glycocalyx after its enzyme degradation under either static or shear flow conditions. Our results show that following 24 h recovery under shear flow, the glycocalyx reappears predominantly near the edge of endothelial cells. Static and shear flow conditions result in notable changes in the spatial recovery of the glycocalyx, but the difference is not statistically significant. We hypothesize that newly synthesized glycocalyx is not structurally well developed. Its weak interaction with flow results in less than significant redistribution, contrary to what has been observed for a well-developed glycocalyx layer.

  8. Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas

    PubMed Central

    Fontanini, G; Boldrini, L; Chinè, S; Pisaturo, F; Basolo, F; Calcinai, A; Lucchi, M; Mussi, A; Angeletti, C A; Bevilacqua, G

    1999-01-01

    The vascular endothelial growth factor (VEGF) has been shown to be strictly related to vascular permeability and endothelial cell growth under physiological and pathological conditions. In tumour development and progression, VEGF plays a pivotal role in the development of the tumoral vascular network, and useful information in the progression of human cancer can be obtained by analysing the vascular endothelial growth factor expression of the tumours. In this study, we investigated the vascular endothelial growth factor transcript expression in non-small-cell lung carcinomas to evaluate the significance of this factor in a group of cancers in which the vascular pattern has been shown to significantly affect progression. Surgical samples of 42 patients with NSCLC were studied using reverse transcription polymerase chain reaction (PCR) analysis and in situ hybridization. Thirty-three out of 42 cases (78.6%) showed VEGF transcript expression predominantly as transcripts for the secretory forms of VEGF (isoforms 121 and 165). In situ hybridization, performed on 24 out of 42 samples, showed that the VEGF transcript expression was in several cases present in the cytoplasm both of neoplastic and normal cells, even if the VEGF mRNA was less expressed in the corresponding non-tumoral part. The VEGF 121 expression was associated with hilar and/or mediastinal nodal involvement (P = 0.02), and, taken together, the VEGF isoforms were shown to significantly influence overall (P = 0.02) and disease-free survival (P = 0.03). As a regulator of tumour angiogenesis, VEGF may represent a useful indicator of progression and poor prognosis in non-small-cell lung carcinomas. © 1999 Cancer Research Campaign PMID:9888482