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Sample records for azd2171 inhibits endothelial

  1. The Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor Cediranib (Recentin; AZD2171) Inhibits Endothelial Cell Function and Growth of Human Renal Tumor Xenografts

    SciTech Connect

    Siemann, Dietmar W. Brazelle, W.D.; Juergensmeier, Juliane M.

    2009-03-01

    Purpose: The goal of this study was to examine the therapeutic potential of the vascular endothelial growth factor (VEGF) signaling inhibitor cediranib in a human model of renal cell carcinoma (Caki-1). Methods and Materials: The effects of cediranib treatment on in vitro endothelial cell function (proliferation, migration, and tube formation), as well as in vivo angiogenesis and tumor growth, were determined. Results: In vitro, cediranib significantly impaired the proliferation and migration of endothelial cells and their ability to form tubes, but had no effect on the proliferation of Caki-1 tumor cells. In vivo, cediranib significantly reduced Caki-1 tumor cell-induced angiogenesis, reduced tumor perfusion, and inhibited the growth of Caki-1 tumor xenografts. Conclusions: The present results are consistent with the notion that inhibition of VEGF signaling leads to an indirect (i.e., antiangiogenic) antitumor effect, rather than a direct effect on tumor cells. These results further suggest that inhibition of VEGF signaling with cediranib may impair the growth of renal cell carcinoma.

  2. A phase II study of AZD2171 (cediranib) in the treatment of patients with acute myeloid leukemia or high-risk myelodysplastic syndrome.

    PubMed

    Mattison, Ryan; Jumonville, Alcee; Flynn, Patrick James; Moreno-Aspitia, Alvaro; Erlichman, Charles; LaPlant, Betsy; Juckett, Mark B

    2015-07-01

    Patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) not fit for intensive treatment need novel therapy options. Vascular endothelial growth factor (VEGF) receptor inhibition is one potential mechanism by which AML and MDS could be treated. The receptor tyrosine kinase inhibitor AZD2171 (cediranib) has activity against VEGF receptors KDR and FLT-1. This multicenter phase II study was designed to test cediranib's activity in patients with AML or high-risk MDS. The primary endpoint was confirmed disease response defined as a composite of complete remission, partial remission or hematologic improvement. The study enrolled 23 subjects in the AML cohort and 16 subjects in the MDS cohort. There were no confirmed responses in either group. Since the study met the stopping rule after the first stage of enrollment, the trial was closed to further accrual. Common adverse events in both cohorts included thrombocytopenia, neutropenia, anemia, fatigue, dyspnea, diarrhea, nausea and dehydration.

  3. 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.

  4. CHOP deficiency inhibits methylglyoxal-induced endothelial dysfunction.

    PubMed

    Choi, Yoon Young; Kim, Suji; Han, Jung-Hwa; Nam, Dae-Hwan; Park, Kwon Moo; Kim, Seong Yong; Woo, Chang-Hoon

    2016-11-18

    Epidemiological studies suggested that diabetic patients are susceptible to develop cardiovascular complications along with having endothelial dysfunction. It has been suggested that methylglyoxal (MGO), a glycolytic metabolite, has more detrimental effects on endothelial dysfunction rather than glucose itself. Here, we investigated the molecular mechanism by which MGO induces endothelial dysfunction via the regulation of ER stress. Biochemical data showed that 4-PBA significantly inhibited MGO-induced protein cleavages of PARP-1 and caspase-3. In addition, it was found that high glucose-induced endothelial apoptosis was enhanced in the presence of GLO1 inhibitor, suggesting the role of endogenous MGO in high glucose-induced endothelial dysfunction. MGO-induced endothelial apoptosis was significantly diminished by the depletion of CHOP with si-RNA against human CHOP, but not by SP600125, a specific inhibitor of JNK. The physiological relevance of this signaling pathway was demonstrated in CHOP deficiency mouse model, in which instillation of osmotic pump containing MGO led to aortic endothelial dysfunction. Notably, the aortic endothelial dysfunction response to MGO infusion was significantly improved in CHOP deficiency mice compared to littermate control. Taken together, these findings indicate that MGO specifically induces endothelial dysfunction in a CHOP-dependent manner, suggesting the therapeutic potential of CHOP inhibition in diabetic cardiovascular complications.

  5. 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.

  6. AZD2171 in Treating Young Patients With Recurrent, Progressive, or Refractory Primary CNS Tumors

    ClinicalTrials.gov

    2016-03-04

    Childhood Atypical Teratoid/Rhabdoid Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Cerebral Anaplastic Astrocytoma; Childhood Cerebral Astrocytoma; Childhood Grade I Meningioma; Childhood Grade II Meningioma; Childhood Grade III Meningioma; Childhood Infratentorial Ependymoma; Childhood Oligodendroglioma; Childhood Spinal Cord Neoplasm; Childhood Supratentorial Ependymoma; Recurrent Childhood Brain Neoplasm; Recurrent Childhood Brain Stem Glioma; Recurrent Childhood Cerebellar Astrocytoma; Recurrent Childhood Cerebral Astrocytoma; Recurrent Childhood Ependymoma; Recurrent Childhood Medulloblastoma; Recurrent Childhood Pineoblastoma; Recurrent Childhood Subependymal Giant Cell Astrocytoma; Recurrent Childhood Supratentorial Primitive Neuroectodermal Tumor; Recurrent Childhood Visual Pathway Glioma

  7. AZD2171 in Treating Patients With Recurrent or Stage IV Melanoma

    ClinicalTrials.gov

    2015-06-01

    Acral Lentiginous Malignant Melanoma; Ciliary Body and Choroid Melanoma, Medium/Large Size; Ciliary Body and Choroid Melanoma, Small Size; Extraocular Extension Melanoma; Intraocular Melanoma; Iris Melanoma; Lentigo Maligna Malignant Melanoma; Recurrent Melanoma; Stage, Intraocular Melanoma; Stage IV Melanoma; Superficial Spreading Malignant Melanoma

  8. Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain.

    PubMed

    Morita, Shoko; Furube, Eriko; Mannari, Tetsuya; Okuda, Hiroaki; Tatsumi, Kouko; Wanaka, Akio; Miyata, Seiji

    2015-03-01

    The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1α were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.

  9. 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.

  10. Arginase inhibition restores endothelial function in diet-induced obesity.

    PubMed

    Chung, Ji Hyung; Moon, Jiyoung; Lee, Youn Sue; Chung, Hye-Kyung; Lee, Seung-Min; Shin, Min-Jeong

    2014-08-22

    Arginase may play a major role in the regulation of vascular function in various cardiovascular disorders by impairing nitric oxide (NO) production. In the current study, we investigated whether supplementation of the arginase inhibitor N(ω)-hydroxy-nor-l-arginine (nor-NOHA) could restore endothelial function in an animal model of diet-induced obesity. Arginase 1 expression was significantly lower in the aorta of C57BL/6J mice fed a high-fat diet (HFD) supplemented with nor-NOHA (40mgkg(-1)/day) than in mice fed HFD without nor-NOHA. Arginase inhibition led to considerable increases in eNOS expression and NO levels and significant decreases in the levels of circulating ICAM-1. These findings were further confirmed by the results of siRNA-mediated knockdown of Arg in human umbilical vein endothelial cells. In conclusion, arginase inhibition can help restore dysregulated endothelial function by increasing the eNOS-dependent NO production in the endothelium, indicating that arginase could be a therapeutic target for correcting obesity-induced vascular endothelial dysfunction.

  11. 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.

  12. Endothelial proteoglycans inhibit bFGF binding and mitogenesis.

    PubMed

    Forsten, K E; Courant, N A; Nugent, M A

    1997-08-01

    Basic fibroblast growth factor (bFGF) is a known mitogen for vascular smooth muscle cells and has been implicated as having a role in a number of proliferative vascular disorders. Binding of bFGF to heparin or heparan sulfate has been demonstrated to both stimulate and inhibit growth factor activity. The activity, towards bFGF, of heparan sulfate proteoglycans present within the vascular system is likely related to the chemical characteristics of the glycosaminoglycan as well as the structure and pericellular location of the intact proteoglycans. We have previously shown that endothelial conditioned medium inhibits both bFGF binding to vascular smooth muscle cells and bFGF stimulated cell proliferation in vitro. In the present study, we have isolated proteoglycans from endothelial cell conditioned medium and demonstrated that they are responsible for the bFGF inhibitory activity. We further separated endothelial secreted proteoglycans into two fractions, PG-A and PG-B. The large sized fraction (PG-A) had greater inhibitory activity than did PG-B for both bFGF binding and bFGF stimulation of vascular smooth muscle cell proliferation. The increased relative activity of PG-A was attributed, in part, to larger heparan sulfate chains which were more potent inhibitors of bFGF binding than the smaller heparan sulfate chains on PG-B. Both proteoglycan fractions contained perlecan-like core proteins; however, PG-A contained an additional core protein (approximately 190 kDa) that was not observed in PG-B. Both proteoglycan fractions bound bFGF directly, and PG-A bound a significantly greater relative amount of bFGF than did PG-B. Thus the ability of endothelial heparan sulfate proteoglycans to bind bFGF and prevent its association with vascular smooth muscle cells appears essential for inhibition of bFGF-induced mitogenesis. The production of potent bFGF inhibitory heparan sulfate proteoglycans by endothelial cells might contribute to the maintenance of vascular homeostasis.

  13. 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

  14. Vascular endothelial-derived semaphorin 3 inhibits sympathetic axon growth.

    PubMed

    Damon, Deborah H

    2006-03-01

    Vascular sympathetic innervation is an important determinant of blood pressure and blood flow. The mechanisms that determine vascular sympathetic innervation are not well understood. Recent studies indicate that vascular endothelial cells (EC) express semaphorin 3A, a repulsive axon guidance cue. This suggests that EC would inhibit the growth of axons to blood vessels. The present study tests this hypothesis. RT-PCR and Western analyses confirmed that rat aortic vascular ECs expressed semaphorin 3A as well as other class 3 semaphorins (sema 3s). To determine the effects of EC-derived sema 3 on sympathetic axons, axon outgrowth was assessed in cultures of neonatal sympathetic ganglia grown for 72 h in the absence and presence of vascular EC. Nerve growth factor-induced axon growth in the presence of ECs was 50 +/- 4% (P < 0.05) of growth in the absence of ECs. ECs did not inhibit axon growth in the presence of an antibody that neutralized the activity of sema 3 (P > 0.05). RT-PCR and Western analyses also indicated that sema 3s were expressed in ECs of intact arteries. To assess the function of sema 3s in arteries, sympathetic ganglia were grown in the presence of arteries for 72 h, and the percentage of axons that grew toward the artery was determined: 44 +/- 4% of axons grew toward neonatal carotid arteries. Neutralization of sema 3s or removal of EC increased the percentage of axons that grew toward the artery (71 +/- 8% and 72 +/- 8%, respectively). These data indicate that vascular EC-derived sema 3s inhibit sympathetic axon growth and may thus be a determinant of vascular sympathetic innervation.

  15. Salidroside inhibits endogenous hydrogen peroxide induced cytotoxicity of endothelial cells.

    PubMed

    Zhao, Xingyu; Jin, Lianhai; Shen, Nan; Xu, Bin; Zhang, Wei; Zhu, Hongli; Luo, Zhengli

    2013-01-01

    Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., shows potent antioxidant property. Herein, we investigated the protective effects of salidroside against hydrogen peroxide (H2O2)-induced oxidative damage in human endothelial cells (EVC-304). EVC-304 cells were incubated in the presence or absence of low steady states of H2O2 (3-4 µM) generated by glucose oxidase (GOX) with or without salidroside. 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) assays were performed, together with Hoechst 33258 staining and flow cytometric analysis using Annexin-V and propidium iodide (PI) label. The results indicated that salidroside pretreatment attenuated endogenous H2O2 induced apoptotic cell death in EVC-304 cells in a dose-dependent pattern. Furthermore, Western blot data revealed that salidroside inhibited activation of caspase-3, 9 and cleavage of poly(ADP-ribose) polymerase (PARP) induced by endogenous H2O2. It also decreased the expression of Bax and rescued the balance of pro- and anti-apoptotic proteins. All these results demonstrated that salidroside may present a potential therapy for oxidative stress in cardiovascular and cerebrovascular diseases.

  16. 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.

  17. 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.

  18. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

    PubMed Central

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a

  19. 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.

  20. 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.

  1. Endothelial repair in stented arteries is accelerated by inhibition of Rho-associated protein kinase

    PubMed Central

    Hsiao, Sarah T.; Spencer, Tim; Boldock, Luke; Prosseda, Svenja Dannewitz; Xanthis, Ioannis; Tovar-Lopez, Francesco J.; Van Beusekom, Heleen M. M.; Khamis, Ramzi Y; Foin, Nicolas; Bowden, Neil; Hussain, Adil; Rothman, Alex; Ridger, Victoria; Halliday, Ian; Perrault, Cecile; Gunn, Julian; Evans, Paul C.

    2016-01-01

    Aims Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries. Methods and Results In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 μm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 μm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05). Conclusions Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK

  2. Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration

    PubMed Central

    Detaille, D; Vial, G; Borel, A-L; Cottet-Rouselle, C; Hallakou-Bozec, S; Bolze, S; Fouqueray, P; Fontaine, E

    2016-01-01

    Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of β-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy. PMID:27551496

  3. Inhibition of astroglia-induced endothelial differentiation by inorganic lead: a role for protein kinase C.

    PubMed Central

    Laterra, J; Bressler, J P; Indurti, R R; Belloni-Olivi, L; Goldstein, G W

    1992-01-01

    Microvascular endothelial function in developing brain is particularly sensitive to lead toxicity, and it has been hypothesized that this results from the modulation of protein kinase C (PKC) by lead. We examined the effects of inorganic lead on an in vitro model of central nervous system endothelial differentiation in which astroglial cells induce central nervous system endothelial cells to form capillary-like structures. Capillary-like structure formation within C6 astroglial-endothelial cocultures was inhibited by lead acetate with 50% maximal inhibition at 0.5 microM total lead. Inhibition was independent of effects on cell viability or growth. Under conditions that inhibited capillary-like structure formation, we found that lead increased membrane-associated PKC in both C6 astroglial and endothelial cells. Prolonged exposure of C6 cells to 5 microM lead for up to 16 h resulted in a time-dependent increase in membranous PKC as determined by immunoblot analysis. Membranous PKC increased after 5-h exposures to as little as 50 nM lead and was maximal at approximately 1 microM. Phorbol esters were used to determine whether PKC modulation was causally related to the inhibition of endothelial differentiation by lead. Phorbol 12-myristate 13-acetate (10 nM) inhibited capillary-like structure formation by 65 +/- 5%, whereas 4 alpha-phorbol 12,13-didecanoate was without effect. These findings suggest that inorganic lead induces cerebral microvessel dysfunction by interfering with PKC modulation in microvascular endothelial or perivascular astroglial cells. Images PMID:1438272

  4. Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.

    PubMed

    Wu, Feng; Schuster, David P; Tyml, Karel; Wilson, John X

    2007-01-01

    The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.

  5. 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

  6. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity

    PubMed Central

    Cazzaniga, Alessandra; Castiglioni, Sara; Maier, Jeanette A. M.

    2014-01-01

    Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. PMID:25210716

  7. Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion

    PubMed Central

    Zhu, Qiuyu; Yamakuchi, Munekazu; Ture, Sara; de la Luz Garcia-Hernandez, Maria; Ko, Kyung Ae; Modjeski, Kristina L.; LoMonaco, Michael B.; Johnson, Andrew D.; O’Donnell, Christopher J.; Takai, Yoshimi; Morrell, Craig N.; Lowenstein, Charles J.

    2014-01-01

    In humans, vWF levels predict the risk of myocardial infarction and thrombosis; however, the factors that influence vWF levels are not completely understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels, though the functional relationship between STXBP5 and vWF is unknown. We hypothesized that STXBP5 inhibits endothelial cell exocytosis. We found that STXBP5 is expressed in human endothelial cells and colocalizes with and interacts with syntaxin 4. In human endothelial cells reduction of STXBP5 increased exocytosis of vWF and P-selectin. Mice lacking Stxbp5 had higher levels of vWF in the plasma, increased P-selectin translocation, and more platelet-endothelial interactions, which suggests that STXBP5 inhibits endothelial exocytosis. However, Stxbp5 KO mice also displayed hemostasis defects, including prolonged tail bleeding times and impaired mesenteric arteriole and carotid artery thrombosis. Furthermore, platelets from Stxbp5 KO mice had defects in platelet secretion and activation; thus, STXBP5 inhibits endothelial exocytosis but promotes platelet secretion. Our study reveals a vascular function for STXBP5, validates the functional relevance of a candidate gene identified by GWAS, and suggests that variation within STXBP5 is a genetic risk for venous thromboembolic disease. PMID:25244095

  8. Nafamostat Mesilate Inhibits TNF-α-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production.

    PubMed

    Kang, Min-Woong; Song, Hee-Jung; Kang, Shin Kwang; Kim, Yonghwan; Jung, Saet-Byel; Jee, Sungju; Moon, Jae Young; Suh, Kwang-Sun; Lee, Sang Do; Jeon, Byeong Hwa; Kim, Cuk-Seong

    2015-05-01

    Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-α (TNF-α). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-α for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 µg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-α (3 ng/mL), and it dose dependently prevented the TNF-α-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-α-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-α-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

  9. Cell-permeable iron inhibits vascular endothelial growth factor receptor-2 signaling and tumor angiogenesis

    PubMed Central

    Kir, Devika; Saluja, Manju; Modi, Shrey; Venkatachalam, Annapoorna; Schnettler, Erica; Roy, Sabita; Ramakrishnan, Sundaram

    2016-01-01

    Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis in vivo. In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis. PMID:27589831

  10. RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

    PubMed Central

    Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C.; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C.; Alonso, Miguel A.; Correas, Isabel; Cox, Susan; Ridley, Anne J.

    2016-01-01

    Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

  11. Inhibition of matrix metalloproteinase-2 improves endothelial function and prevents hypertension in insulin-resistant rats

    PubMed Central

    Nagareddy, PR; Rajput, PS; Vasudevan, H; McClure, B; Kumar, U; MacLeod, KM; McNeill, JH

    2012-01-01

    BACKGROUND AND PURPOSE Insulin resistance is often found to be associated with high blood pressure. We propose that in insulin-resistant hypertension, endothelial dysfunction is the consequence of increased activity of vascular MMP-2. As MMP-2 proteolytically cleaves a number of extracellular matrix proteins, we hypothesized that MMP-2 impairs endothelial function by proteolytic degradation of endothelial NOS (eNOS) or its cofactor, heat shock protein 90 (HSP90). EXPERIMENTAL APPROACH We tested our hypothesis in bovine coronary artery endothelial cells and fructose-fed hypertensive rats (FHR), a model of acquired systolic hypertension and insulin resistance. KEY RESULTS Treatment of FHRs with the MMP inhibitor doxycycline, preserved endothelial function as well as prevented the development of hypertension, suggesting that MMPs impair endothelial function. Furthermore, incubating endothelial cells in vitro with a recombinant MMP-2 decreased NO production in a dose-dependent manner. Using substrate cleavage assays and immunofluorescence microscopy studies, we found that MMP-2 not only cleaves and degrades HSP90, an eNOS cofactor but also co-localizes with both eNOS and HSP90 in endothelial cells, suggesting that MMPs functionally interact with the eNOS system. Treatment of FHRs with doxycycline attenuated the decrease in eNOS and HSP90 expression but did not improve insulin sensitivity. CONCLUSIONS AND IMPLICATIONS Our data suggest that increased activity of MMP-2 in FHRs impairs endothelial function and promotes hypertension. Inhibition of MMP-2 could be a potential therapeutic strategy for the management of hypertension. PMID:21740410

  12. Inhibition of dipeptidyl peptidase 4 regulates microvascular endothelial growth induced by inflammatory cytokines

    SciTech Connect

    Takasawa, Wataru; Ohnuma, Kei; Hatano, Ryo; Endo, Yuko; Dang, Nam H.

    2010-10-08

    Research highlights: {yields} TNF-{alpha} or IL-1{beta} induces EC proliferation with reduction of CD26 expression. {yields} CD26 siRNA or DPP-4 inhibition enhances TNF-{alpha} or IL-1{beta}-induced EC proliferation. {yields} Loss of CD26/DPP-4 enhances aortic sprouting induced by TNF-{alpha} or IL-1{beta}. {yields} Capillary formation induced by TNF-{alpha} or IL-1{beta} is enahced in the CD26{sup -/-} mice. -- Abstract: CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications.

  13. Adiporedoxin suppresses endothelial activation via inhibiting MAPK and NF-κB signaling

    PubMed Central

    He, Hui; Guo, Fang; Li, Yong; Saaoud, Fatma; Kimmis, Brooks D.; Sandhu, Jeena; Fan, Michelle; Maulik, Dev; Lessner, Susan; Papasian, Christopher J.; Fan, Daping; Jiang, Zhisheng; Fu, Mingui

    2016-01-01

    Adiporedoxin (Adrx) is a recently discovered redox regulatory protein that is preferentially expressed in adipose tissue and plays a critical role in the regulation of metabolism via its modulation of adipocyte protein secretion. We here report that Adrx suppresses endothelial cell activation via inhibiting MAPK and NF-kB signaling pathways. Adrx is constitutively expressed in human vascular endothelial cells, and significantly induced by a variety of stimuli such as TNFα, IL-1β, H2O2 and OxLDL. Overexpression of Adrx significantly attenuated TNFα-induced expression of VCAM-1 and ICAM-1, and thus reduced monocyte adherence to human umbilical vein endothelial cells (HUVECs). Conversely, siRNA-mediated knockdown of Adrx increased TNFα-induced expression of adhesion molecules and monocyte adherence to HUVECs. Furthermore, forced expression of Adrx decreased TNFα-induced activation of ERK1/2, JNK, p38 and IKKs in HUVECs. Adrx mutant in the CXXC motif that lost its anti-redox activity is less efficient than the wild-type Adrx, suggesting that Adrx-mediated inhibition of endothelial activation is partially dependent on its antioxidant activity. Finally, Adrx expression was markedly increased in human atheroma compared with normal tissue from the same carotid arteries. These results suggest that Adrx is an endogenous inhibitor of endothelial activation, and might be a therapeutic target for vascular inflammatory diseases. PMID:27941911

  14. Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole

    PubMed Central

    Nacev, Benjamin A.; Liu, Jun O.

    2011-01-01

    Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC50 dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy. PMID:21969860

  15. Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo

    PubMed Central

    Li, Peng; Yin, Ya-Ling; Guo, Tao; Sun, Xue-Ying; Ma, Hui; Zhu, Mo-Li; Zhao, Fan-Rong; Xu, Ping; Chen, Yuan; Wan, Guang-Rui; Jiang, Fan; Peng, Qi-Sheng; Liu, Chao; Liu, Li-Ying

    2016-01-01

    Background: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation. Methods: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization. Results: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin. Conclusions: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases. PMID:27765794

  16. Curcumin protects endothelial cells against homocysteine induced injury through inhibiting inflammation

    PubMed Central

    Li, Jian; Luo, Ming; Xie, Nanzi; Wang, Jianxin; Chen, Li

    2016-01-01

    Objective: This study aimed to investigate the protective effects of curcumin on the homocysteine (HCY) induced injury to the endothelial cells. Methods: Endothelial cells were treated with HCY at different concentrations, and MTT assay was employed to determine an optimal concentration of HCY. Cells were divided into 3 groups: normal control group, HCY group and HCY + curcumin group. In curcumin group, cells were pretreated with 2.5 mmol/L HCY for 2 h and then incubated with curcumin at different concentrations. MTT assay was employed to detect the cell viability. ELISA was performed to detect the content of IL-8 in the supernatant. Western blotting was used to detect NF-κB expression in cells. Results: (1) Endothelial cells were polygonal or stone-like, or aggregated to form masses, and then gradually became long spindle shaped, cell body enlarged, cells were rich in cytoplasm, and immunohistochemistry for factor VIII showed positive. (2) MTT assay showed HCY at ≥2.5 mmol/L caused significant damage to endothelial cells as compared to control group. Thus, 2.5 mmol/L HCY was used in following experiments. (3) ELISA showed IL-8 in the supernatant increased significantly in a time dependent manner after HCY treatment (P<0.01), but curcumin could significantly inhibit the IL-8 secretion in endothelial cells after HCY treatment. (4) Western blotting showed HCY was able to markedly increase NF-κB expression, which, however, was significantly inhibited by curcumin. Conclusion: Curcumin is able to protect the endothelial cells against HCY induced injury through inhibiting NF-κB activation and down-regulating IL-8 expression. PMID:27904665

  17. The Antifungal Drug Itraconazole Inhibits Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, Trafficking, and Signaling in Endothelial Cells*

    PubMed Central

    Nacev, Benjamin A.; Grassi, Paola; Dell, Anne; Haslam, Stuart M.; Liu, Jun O.

    2011-01-01

    Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2. PMID:22025615

  18. Phloroglucinol Inhibits the in vitro Differentiation Potential of CD34 Positive Cells into Endothelial Progenitor Cells

    PubMed Central

    Kwon, Yi-Hong; Lee, Jun-Hee; Jung, Seok-Yun; Kim, Jae-Won; Lee, Sang-Hun; Lee, Dong-Hyung; Lee, Kyu-Sup; Lee, Boo-Yong; Kwon, Sang-Mo

    2012-01-01

    Inhibiting the bioactivities of circulating endothelial progenitor cells (EPCs) results in significant inhibition of neovessel formation during tumor angiogenesis. To investigate the potential effect of phloroglucinol as an EPC inhibitor, we performed several in vitro functional assays using CD34+ cells isolated from human umbilical cord blood (HUCB). Although a high treatment dose of phloroglucinol did not show any cell toxicity, it specifically induced the cell death of EPCs under serum free conditions through apoptosis. In the EPC colony-forming assay (EPC-CFA), we observed a significant decreased in the small EPC-CFUs for the phloroglucinol group, implying that phloroglucinol inhibited the early stage of EPC commitment. In addition, in the in vitro expansion assay using CD34+ cells, treatment with phloroglucinol was shown to inhibit endothelial lineage commitment, as demonstrated by the decrease in endothelial surface markers of EPCs including CD34+, CD34+/CD133+, CD34+/CD31+ and CD34+/CXCR4+. This is the first report to demonstrate that phloroglucinol can inhibit the functional bioactivities of EPCs, indicating that phloroglucinol may be used as an EPC inhibitor in the development of biosafe anti-tumor drugs that target tumor angiogenesis. PMID:24116289

  19. 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.

  20. [Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

    PubMed

    Liu, Yanhua; Li, Bingong; Wang, Yuqin; Wang, Delong; Zou, Jin; Ke, Xuan; Hao, Yanqin

    2016-12-01

    Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-β2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-β2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-β and Notch signaling pathway.

  1. TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor

    SciTech Connect

    Bruneau, Sarah; Datta, Dipak; Flaxenburg, Jesse A.; Pal, Soumitro; Briscoe, David M.

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer TNF-receptor associated factors (TRAFs) function in the angiogenesis response. Black-Right-Pointing-Pointer TRAF6 regulates basal and inducible expression of VEGF in endothelial cells (EC). Black-Right-Pointing-Pointer TRAF6 is an endogenous inhibitor of EC proliferation and migration in EC. Black-Right-Pointing-Pointer TRAF6 inhibits VEGF expression in part via its ability to regulate Src signaling. -- Abstract: TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4-6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P < 0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [{sup 3}H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling

  2. Linagliptin but not Sitagliptin inhibited transforming growth factor-β2-induced endothelial DPP-4 activity and the endothelial-mesenchymal transition.

    PubMed

    Shi, Sen; Kanasaki, Keizo; Koya, Daisuke

    2016-02-26

    Dipeptidyl peptidase (DPP)-4 plays an important role in endothelial cell biology. We have shown that the DPP-4 inhibitor Linagliptin can inhibit the endothelial-mesenchymal transition (EndMT) and ameliorate diabetic kidney fibrosis associated with the suppression of DPP-4 protein levels via the induction of miR-29. The current study demonstrated that such effects of Linagliptin on endothelial cell profibrotic programs were drug-specific but not class effects. In the cell-free system, both Linagliptin and Sitagliptin inhibited recombinant DPP-4 activity in a concentration-dependent manner. Linagliptin can inhibit all of the following: DPP-4 activity and protein level, integrin β1 protein levels, EndMT, and DPP-4 3'UTR activity; Sitagliptin, however, inhibited none of these in the current study. Additionally, TGF-β2 induced both the induction of VEGF-R1 and the suppression of VEGF-R2 levels in endothelial cells, and both were inhibited by Linagliptin but not by Sitagliptin. miR-29, the miR that negatively regulates the 3'UTR of DPP-4 mRNA, was suppressed by TGF-β2 and restored by Linagliptin but not by Sitagliptin. Following the overexpression of pCMV-DPP-4-GFP and pCMV6-Myc-DPP-4 in endothelial cells, the proximity of Myc-DPP-4 and DPP-4-GFP was suppressed by Linagliptin but not by Sitagliptin, suggesting that only Linagliptin inhibited the homo-dimer formation of DPP-4 in endothelial cells; this difference in activity between the two gliptins could explain their diverse effects on endothelial cell biology. In conclusion, each of the DPP-4 inhibitors may have unique drug-specific effects.

  3. Ascorbate inhibits platelet-endothelial adhesion in an in-vitro model of sepsis via reduced endothelial surface P-selectin expression.

    PubMed

    Secor, Dan; Swarbreck, Scott; Ellis, Christopher G; Sharpe, Michael D; Feng, Qingping; Tyml, Karel

    2017-01-01

    Plugging of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous ascorbate injection reduces platelet adhesion to the capillary wall and capillary plugging in septic mice. Both platelet adhesion and capillary plugging require P-selectin, a key adhesion molecule. To elucidate the beneficial effect of ascorbate, we hypothesized that ascorbate reduces platelet-endothelial adhesion by reducing P-selectin surface expression in endothelial cells. We used mouse platelets, and monolayers of cultured microvascular endothelial cells (mouse skeletal muscle origin) stimulated with lipopolysaccharide, to examine platelet-endothelial adhesion. P-selectin mRNA expression in endothelial cells was determined by real-time PCR and P-selectin protein expression at the surface of these cells by immunofluorescence. Secretion of von Willebrand factor from cells into the supernatant (a measure of P-selectin-containing granule exocytosis) was determined by ELISA. Lipopolysaccharide (10 μg/ml, 1 h) increased platelet-endothelial adhesion. P-selectin-blocking antibody inhibited this adhesion. Lipopolysaccharide also increased P-selectin mRNA in endothelial cells, P-selectin expression at the endothelial surface, and von Willebrand factor secretion. Ascorbate pretreatment (100 μmol/l, 4 h) inhibited the increased platelet adhesion, surface expression of P-selectin, and von Willebrand factor secretion, but not the increase in P-selectin mRNA. The lipopolysaccharide-induced increase in platelet-endothelial adhesion requires P-selectin presence at the endothelial surface. Ascorbate's ability to reduce this presence could be important in reducing both platelet adhesion to the capillary wall and capillary plugging in sepsis.

  4. 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.

  5. TSG attenuates LPC-induced endothelial cells inflammatory damage through notch signaling inhibition.

    PubMed

    Zhao, Jing; Liang, Yuan; Song, Fan; Xu, Shouzhu; Nian, Lun; Zhou, Xuanxuan; Wang, Siwang

    2016-01-01

    Lysophosphatidylcholine (LPC) induces inflammation in endothelial cells (ECs) but the mechanism is not fully understood. The Notch signaling pathway is involved in chronic EC inflammation, but its functions in LPC-induced endothelial inflammatory damage and 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside's (TSG) protective effect during LPC-induced inflammatory damage in human umbilical vein endothelial cells (HUVECs) is largely unknown. We report that Notch signaling activation contributed to LPC-induced injury in HUVECs, and that TSG protected HUVECs from LPC-induced injury by antagonizing Notch signaling activation by LPC. γ-secretase inhibitor (DAPT), a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to inhibit Notch activity. HUVECs were exposed to LPC in the presence or absence of TSG, DAPT, and Notch1 siRNA. LPC treatment of HUVECs resulted in reduced cell viability, and Notch1 and Hes1 upregulation. Either silencing of Notch1 by siRNA or pharmacological inhibition of Notch signaling by DAPT prevented the loss of cell viability, and induction of apoptosis, and enhanced expression Notch1, Hes1 and MCP-1 by LPC in HUVECs. Similarly, TSG reduced LPC stimulation of Notch1, Hes1, and MCP-1 expression, prevented the release of IL-6 and CRP and rescued HUVECs from LPC-induced cell damage. Our data indicate that the Notch signaling pathway is a crucial mediator of endothelial inflammatory damage and that TSG protects against endothelial inflammatory damage by inhibiting the Notch signaling pathway. Our findings suggest that targeting Notch signaling by natural products such as TSG is a promising strategy for the prevention and treatment of chronic inflammation associated diseases, including atherosclerosis. © 2015 IUBMB Life, 68(1):37-50, 2016.

  6. Copper induces--and copper chelation by tetrathiomolybdate inhibits--endothelial activation in vitro.

    PubMed

    Wei, Hao; Zhang, Wei-Jian; Leboeuf, Renee; Frei, Balz

    2014-01-01

    Endothelial activation with increased expression of cellular adhesion molecules and chemokines critically contributes to vascular inflammation and atherogenesis. Redox-active transition metal ions play an important role in vascular oxidative stress and inflammation. Therefore, the goal of the present study was to investigate the role of copper in endothelial activation and the potential anti-inflammatory effects of copper chelation by tetrathiomolybdate (TTM) in human aortic endothelial cells (HAECs). Incubating HAECs with cupric sulfate dose- and time-dependently increased mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1). Copper also activated the redox-sensitive transcription factors, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1), which was inhibited by pretreatment of the cells with TTM. Furthermore, TTM dose-dependently inhibited tumor necrosis factor α (TNFα)-induced activation of NF-κB and AP-1, as well as mRNA and protein expression of VCAM-1, ICAM-1, and MCP-1, which was abolished by preincubating the cells with 5 µM TTM and 15 µM cupric sulfate. The inhibitory effect of TTM on TNFα-induced NF-κB activation was associated with decreased phosphorylation and degradation of IκBα. These data suggest that intracellular copper causes activation of redox-sensitive transcription factors and upregulation of inflammatory mediators in endothelial cells. Copper chelation by TTM may attenuate TNFα-induced endothelial activation and, hence, inhibit vascular inflammation and atherosclerosis.

  7. Inhibition of NADPH oxidase activation in endothelial cells by ortho-methoxy-substituted catechols.

    PubMed

    Johnson, David K; Schillinger, Kurt J; Kwait, David M; Hughes, Chambers V; McNamara, Erin J; Ishmael, Fauod; O'Donnell, Robert W; Chang, Ming-Mei; Hogg, Michael G; Dordick, Jonathan S; Santhanam, Lakshmi; Ziegler, Linda M; Holland, James A

    2002-01-01

    NADPH oxidase is a major enzymatic source of oxygen free radicals in stimulated endothelial cells (ECs). The ortho-methoxy-substituted catechol, apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, inhibits the release of superoxide anion (O2*-) by this enzyme. The compound acts by blocking the assembly of a functional NADPH oxidase complex. The underlying chemistry of this inhibitory activity, and its physiological significance to EC proliferation, have been investigated. A critical event is the reaction of ortho-methoxy-substituted catechols with reactive oxygen species (ROS) and peroxidase. Analysis of this reaction reveals that apocynin is converted to a symmetrical dimer through the formation of a 5,5' carbon-carbon bond. Both reduced glutathione and L-cysteine inhibit this dimerization process. Catechols without the ortho-methoxy-substituted group do not undergo this chemical reaction. Superoxide production by an endothelial cell-free system incubated with apocynin was nearly completely inhibited after a lagtime for inhibition of ca. 2 min. Conversely, O2*- production was nearly completely inhibited, without a lagtime, by incubation with the dimeric form of apocynin. The apocynin dimer undergoes a two-electron transfer reaction with standard redox potentials of -0.75 and -1.34 V as determined by cyclic voltammetry. Inhibition of endothelial NADPH oxidase by apocynin caused a dose-dependent inhibition of cell proliferation. These findings identify a metabolite of an ortho-methoxy-substituted catechol, which may be the active compound formed within stimulated ECs that prevents NADPH oxidase complex assembly and activation.

  8. α-1 Antitrypsin Inhibits Caspase-3 Activity, Preventing Lung Endothelial Cell Apoptosis

    PubMed Central

    Petrache, Irina; Fijalkowska, Iwona; Medler, Terry R.; Skirball, Jarrett; Cruz, Pedro; Zhen, Lijie; Petrache, Horia I.; Flotte, Terence R.; Tuder, Rubin M.

    2006-01-01

    α-1 Antitrypsin (A1AT) is an abundant circulating serpin with a postulated function in the lung of potently inhibiting neutrophil-derived proteases. Emphysema attributable to A1AT deficiency led to the concept that a protease/anti-protease imbalance mediates cigarette smoke-induced emphysema. We hypothesized that A1AT has other pathobiological relevant functions in addition to elastase inhibition. We demonstrate a direct prosurvival effect of A1AT through inhibition of lung alveolar endothelial cell apoptosis. Primary pulmonary endothelial cells internalized human A1AT, which co-localized with and inhibited staurosporine-induced caspase-3 activation. In cell-free studies, native A1AT, but not conformers lacking an intact reactive center loop, inhibited the interaction of recombinant active caspase-3 with its specific substrate. Furthermore, overexpression of human A1AT via replication-deficient adeno-associated virus markedly attenuated alveolar wall destruction and oxidative stress caused by caspase-3 instillation in a mouse model of apoptosis-dependent emphysema. Our findings suggest that direct inhibition of active caspase-3 by A1AT may represent a novel anti-apoptotic mechanism relevant to disease processes characterized by excessive structural cell apoptosis, oxidative stress, and inflammation, such as pulmonary emphysema. PMID:17003475

  9. Flavonoids from Citrus unshiu Marc. inhibit cancer cell adhesion to endothelial cells by selective inhibition of VCAM-1.

    PubMed

    Jin, Hana; Lee, Won Sup; Yun, Jeong Won; Jung, Ji Hyun; Yi, Sang Mi; Kim, Hye Jung; Choi, Yung Hyun; Kim, Gonsup; Jung, Jin-Myung; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan

    2013-11-01

    Citrus fruits have been used as edible fruit and a component of traditional medicine for various diseases including cancer since ancient times. Herein, we investigated the anticancer activity of flavonoids of Citrus unshiu Marc. (FCM) focusing on anti-metastatic effects. We prepared FCM and performed experiments using MDA-MB-231 human breast cancer cells. FCM inhibited TNF-induced cancer cell adhesion to human umbilical vein endothelial cells (HUVECs) without showing any toxicity. FCM inhibited the expression of VCAM-1, but not of ICAM-1, on MDA-MB-231 cells as well as HUVECs. FCM inhibited protein kinase C (PKC) phosphorylation, but not Akt phosphorylation. FCM also inhibited cancer cell invasion in a dose-dependent manner, but not MMP-9 expression. In conclusion, this study suggested that FCM inhibits TNF-induced cancer cell adhesion to HUVECs by inhibiting VCAM-1 through inhibition of PKC, providing evidence that FCM have anti-metastatic activity by inhibiting adhesion molecules and invasion on human breast cancer cells.

  10. Mechanism study of endothelial protection and inhibits platelet activation of low molecular weight fucoidan from Laminaria japonica

    NASA Astrophysics Data System (ADS)

    Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue; Yan, Meixing

    2016-10-01

    Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline (0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. vWF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce vWF level in vascular endothelial injury rats and also significantly reduce vWF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and vWF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.

  11. 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.

  12. Rapamycin antagonizes TNF induction of VCAM-1 on endothelial cells by inhibiting mTORC2

    PubMed Central

    Wang, Chen; Qin, Lingfeng; Manes, Thomas D.; Kirkiles-Smith, Nancy C.; Tellides, George

    2014-01-01

    Recruitment of circulating leukocytes into inflamed tissues depends on adhesion molecules expressed by endothelial cells (ECs). Here we report that rapamycin pretreatment reduced the ability of TNF-treated ECs to capture T cells under conditions of venular flow. This functional change was caused by inhibition of TNF-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and could be mimicked by knockdown of mammalian target of rapamycin (mTOR) or rictor, but not raptor, implicating mTORC2 as the target of rapamycin for this effect. Mechanistically, mTORC2 acts through Akt to repress Raf1-MEK1/2-ERK1/2 signaling, and inhibition of mTORC2 consequently results in hyperactivation of ERK1/2. Increased ERK1/2 activity antagonizes VCAM-1 expression by repressing TNF induction of the transcription factor IRF-1. Preventing activation of ERK1/2 reduced the ability of rapamycin to inhibit TNF-induced VCAM-1 expression. In vivo, rapamycin inhibited mTORC2 activity and potentiated activation of ERK1/2. These changes correlated with reduced endothelial expression of TNF-induced VCAM-1, which was restored via pharmacological inhibition of ERK1/2. Functionally, rapamycin reduced infiltration of leukocytes into renal glomeruli, an effect which was partially reversed by inhibition of ERK1/2. These data demonstrate a novel mechanism by which rapamycin modulates the ability of vascular endothelium to mediate inflammation and identifies endothelial mTORC2 as a potential therapeutic target. PMID:24516119

  13. Inhibition of proliferation of retinal vascular endothelial cells more effectively than choroidal vascular endothelial cell proliferation by bevacizumab

    PubMed Central

    Mynampati, Bharani Krishna; Sambhav, Kumar; Grover, Sandeep; Chalam, Kakarla V.

    2017-01-01

    AIM To evaluate the differential inhibitory effects of bevacizumab on cell proliferation of vascular endothelial growth factor (VEGF)-stimulated choroidal vascular endothelial cells (CVECs) and retinal vascular endothelial cells (RVECs) in vitro. METHODS VEGF (400 ng/mL) enriched CVECs and RVECs were treated with escalating doses of bevacizumab (0.1, 0.5, 1, 1.5 and 2 mg/mL). Cell proliferation changes were analyzed with WST-1 assay and trypan blue exclusion assay at 48, 72h and 1wk. Morphological changes were recorded with bright field microscopy. RESULTS VEGF enriched RVECs showed significantly more decline of cell viability than CVECs after bevacizumab treatment. One week after treatment, RVEC cell proliferation decreased by 29.7%, 37.5%, 52.8%, 35.9% and 45.6% at 0.1, 0.5, 1.0, 1.5 and 2 mg/mL bevacizumab respectively compared to CVEC proliferation decrease of 4.1%, 7.7%, 2.4%, 4.1% and 17.7% (P<0.05) by WST-1 assay. Trypan blue exclusion assay also revealed similar decrease in RVEC proliferation of 20%, 60%, 73.3%, 80% and 93.3% compared to CVEC proliferation decrease of 4%, 12%, 22.9%, 16.7% and 22.2% respectively (P<0.05). The maximum differential effect between the two cell types was observed at bevacizumab doses of 1.0 and 1.5 mg/mL at all time points. RVECs were 22 fold more sensitive (P<0.01) compared to CVECs (52.8% vs 2.4%) at concentration of 1.0 mg/mL, and 8.7 fold more at 1.5 mg/mL (35.9% vs 4.1%) 1wk after treatment (P<0.05 respectively). CONCLUSION VEGF-enriched RVECs are more susceptible to bevacizumab inhibition than CVECs at clinically used dosage of 1.25 mg and this differential sensitivity between two cell types should be taken into consideration in dosage selection. PMID:28149771

  14. ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

    SciTech Connect

    Inagaki, Junko; Takahashi, Katsuyuki; Ogawa, Hiroko; Asano, Keiichi; Faruk Hatipoglu, Omer; Zeynel Cilek, Mehmet; Obika, Masanari; Ohtsuki, Takashi; Hofmann, Matthias; Kusachi, Shozo; Ninomiya, Yoshifumi; Hirohata, Satoshi

    2014-05-01

    Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy. - Highlights: • ADAMTS1 significantly inhibited tube formation and cell proliferation in HMVEC-dLy. • Reduced lymph endothelial cell migration in ADAMTS1 transduced co-culture systems. • VEGFC-stimulated phosphorylation of VEGFR3 is attenuated by ADAMTS1. • Reduced phosphorylation of Akt and ERK1/2 in ADAMTS1 treated HMVEC-dLy. • ADAMTS1 binds directly to VEGFC.

  15. Tissue-engineered endothelial cell layers on surface-modified Ti for inhibiting in vitro platelet adhesion

    NASA Astrophysics Data System (ADS)

    Wang, Xiupeng; He, Fupo; Li, Xia; Ito, Atsuo; Sogo, Yu; Maruyama, Osamu; Kosaka, Ryo; Ye, Jiandong

    2013-06-01

    A tissue-engineered endothelial layer was prepared by culturing endothelial cells on a fibroblast growth factor-2 (FGF-2)-l-ascorbic acid phosphate magnesium salt n-hydrate (AsMg)-apatite (Ap) coated titanium plate. The FGF-2-AsMg-Ap coated Ti plate was prepared by immersing a Ti plate in supersaturated calcium phosphate solutions supplemented with FGF-2 and AsMg. The FGF-2-AsMg-Ap layer on the Ti plate accelerated proliferation of human umbilical vein endothelial cells (HUVECs), and showed slightly higher, but not statistically significant, nitric oxide release from HUVECs than on as-prepared Ti. The endothelial layer maintained proper function of the endothelial cells and markedly inhibited in vitro platelet adhesion. The tissue-engineered endothelial layer formed on the FGF-2-AsMg-Ap layer is promising for ameliorating platelet activation and thrombus formation on cardiovascular implants.

  16. Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta.

    PubMed

    Soucy, Kevin G; Lim, Hyun Kyo; Attarzadeh, David O; Santhanam, Lakshmi; Kim, Jae Hyung; Bhunia, Anil K; Sevinc, Baris; Ryoo, Sungwoo; Vazquez, Marcelo E; Nyhan, Daniel; Shoukas, Artin A; Berkowitz, Dan E

    2010-05-01

    Radiation exposure is associated with the development of various cardiovascular diseases. Although irradiation is known to cause elevated oxidant stress and chronic inflammation, both of which are detrimental to vascular function, the molecular mechanisms remain incompletely understood. We previously demonstrated that radiation causes endothelial dysfunction and increased vascular stiffness by xanthine oxidase (XO) activation. In this study, we investigated whether dietary inhibition of XO protects against radiation-induced vascular injury. We exposed 4-mo-old rats to a single dose of 0 or 5 Gy gamma radiation. These rats received normal drinking water or water containing 1 mM oxypurinol, an XO inhibitor. We measured XO activity and superoxide production in rat aorta and demonstrated that both were significantly elevated 2 wk after radiation exposure. However, oxypurinol treatment in irradiated rats prevented aortic XO activation and superoxide elevation. We next investigated endothelial function through fluorescent measurement of nitric oxide (NO) and vascular tension dose responses. Radiation reduced endothelium-dependent NO production in rat aorta. Similarly, endothelium-dependent vasorelaxation in the aorta of irradiated rats was significantly attenuated compared with the control group. Dietary XO inhibition maintained NO production at control levels and prevented the development of endothelial dysfunction. Furthermore, pulse wave velocity, a measure of vascular stiffness, increased by 1 day postirradiation and remained elevated 2 wk after irradiation, despite unchanged blood pressures. In oxypurinol-treated rats, pulse wave velocities remained unchanged from baseline throughout the experiment, signifying preserved vascular health. These findings demonstrate that XO inhibition can offer protection from radiation-induced endothelial dysfunction and cardiovascular complications.

  17. Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

    PubMed Central

    Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

    2015-01-01

    Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function. PMID:26029925

  18. Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

    PubMed

    Jeong, Ye-Ji; Jung, Myung Gu; Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

    2015-01-01

    Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

  19. Targeting endothelial connexin40 inhibits tumor growth by reducing angiogenesis and improving vessel perfusion

    PubMed Central

    Alonso, Florian; Domingos-Pereira, Sonia; Le Gal, Loïc; Derré, Laurent; Meda, Paolo; Jichlinski, Patrice; Nardelli-Haefliger, Denise; Haefliger, Jacques-Antoine

    2016-01-01

    Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40−/−), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40−/− but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40−/− mice. As a result, Cx40−/− mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment. PMID:26883111

  20. Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin.

    PubMed

    Chiesi, M; Schwaller, R

    1995-02-14

    The effect of natural polyphenols on three isoforms of NO-synthase was investigated. Among the compounds tested, tannin was the most potent, inhibiting endothelial constitutive NO synthase (eNOS) with an IC50 of 2.2 microM. Other NOS isoforms (i.e. neuronal constitutive NOS and smooth muscle inducible NOS) were also inhibited but at much higher concentrations (selectivity ratio of approx. 20-30). Quercetin was also an effective but less potent inhibitor of eNOS (IC50 = 220 microM). The kinetics of tannin inhibition were investigated to gather information on the mechanism of action. Tannin did not interfere with the interaction of the enzyme with the co-substrates L-arginine and NADPH nor with the cofactor tetrahydrobiopterin. The inhibition level was also independent of free Ca2+ concentration as well as of the presence of high exogenous calmodulin concentrations.

  1. Jin Fu Kang Oral Liquid Inhibits Lymphatic Endothelial Cells Formation and Migration

    PubMed Central

    Wang, Dan; Tang, Jie

    2016-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Jin Fu Kang (JFK), an oral liquid prescription of Chinese herbal drugs, has been clinically available for the treatment of non-small cell lung cancer (NSCLC). Lymphangiogenesis is a primary event in the process of cancer development and metastasis, and the formation and migration of lymphatic endothelial cells (LECs) play a key role in the lymphangiogenesis. To assess the activity of stromal cell-derived factor-1 (SDF-1) and the coeffect of SDF-1 and vascular endothelial growth factor-C (VEGF-C) on the formation and migration of LECs and clarify the inhibitory effects of JFK on the LECs, the LECs were differentiated from CD34+/VEGFR-3+ endothelial progenitor cells (EPCs), and JFK-containing serums were prepared from rats. SDF-1 and VEGF-C both induced the differentiation of CD34+/VEGFR-3+ EPCs towards LECs and enhanced the LECs migration. Couse of SDF-1 and VEGF-C displayed an additive effect on the LECs formation but not on their migration. JFK inhibited the formation and migration of LECs, and the inhibitory effects were most probably via regulation of the SDF-1/CXCR4 and VEGF-C/VEGFR-3 axes. The current finding suggested that JFK might inhibit NSCLC through antilymphangiogenesis and also provided a potential to discover antilymphangiogenesis agents from natural resources. PMID:27698675

  2. G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition.

    PubMed

    Zhang, Panpan; He, Qiuping; Chen, Dongbo; Liu, Weixiao; Wang, Lu; Zhang, Chunxia; Ma, Dongyuan; Li, Wei; Liu, Bing; Liu, Feng

    2015-10-01

    In vertebrates, embryonic hematopoietic stem and progenitor cells (HSPCs) are derived from a subset of endothelial cells, the hemogenic endothelium (HE), through the endothelial-to-hematopoietic transition (EHT). Notch signaling is essential for HSPC development during embryogenesis across vertebrates. However, whether and how it regulates EHT remains unclear. Here, we show that G protein-coupled receptor 183 (Gpr183) signaling serves as an indispensable switch for HSPC emergence by repressing Notch signaling before the onset of EHT. Inhibition of Gpr183 significantly upregulates Notch signaling and abolishes HSPC emergence. Upon activation by its ligand 7α-25-OHC, Gpr183 recruits β-arrestin1 and the E3 ligase Nedd4 to degrade Notch1 in specified HE cells and then facilitates the subsequent EHT. Importantly, 7α-25-OHC stimulation promotes HSPC emergence in vivo and in vitro, providing an attractive strategy for enhancing the in vitro generation of functional HSPCs.

  3. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation

    SciTech Connect

    Lamy, Sylvie Ouanouki, Amira; Béliveau, Richard; Desrosiers, Richard R.

    2014-03-10

    Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention. - Highlights: • We investigated five compounds contained in extra virgin olive oil on angiogenesis. • Hydroxytyrosol, taxifolin and oleic acid are the best angiogenesis inhibitors. • Olive oil compounds affect endothelial cell functions essential for

  4. Benidipine, a dihydropyridine-calcium channel blocker, inhibits lysophosphatidylcholine-induced endothelial injury via stimulation of nitric oxide release.

    PubMed

    Matsubara, Masahiro; Yao, Kozo; Hasegawa, Kazuhide

    2006-01-01

    Benidipine hydrochloride (benidipine), which is a long-lasting dihydropyridine calcium channel blocker, exerts antihypertensive action via inhibition of Ca(2+) influx through L-type voltage-dependent calcium channels. In addition, benidipine is shown to restore endothelial function. However, the mechanisms whereby benidipine has protective effects on endothelium are poorly defined. Nitric oxide (NO), which is produced by endothelial NO synthase (eNOS), plays important roles in endothelial function. In this study, we examined effects of benidipine on NO production from human umbilical vein endothelial cells. Benidipine (0.3-10 microM) augmented eNOS expression and total eNOS enzymatic activities. Benidipine also promoted the production of NO and the accumulation of cGMP, a second messenger of NO. Lysophosphatidylcholine (lysoPC), a component of oxidized low-density lipoproteins, induced caspase-3 activation followed by apoptosis of endothelial cells. Benidipine (0.3-10 microM) prevented lysoPC-induced caspase-3 activation, which was canceled by Nomega-nitro-L-arginine-methyl ester (L-NAME) (250-2500 microM), an inhibitor of NOS. Moreover, diethylenetetraamine NONOate (30-100 microM), a NO donor, inhibited the caspase-3 activation. These results suggested that the increase in NO production by benidipine might be involved in the inhibition of caspase induction. The direct enhancement of endothelial NO release by benidipine may be in part responsible for amelioration of endothelial dysfunction.

  5. Selective HDAC6 inhibition prevents TNF-α-induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema.

    PubMed

    Yu, Jinyan; Ma, Zhongsen; Shetty, Sreerama; Ma, Mengshi; Fu, Jian

    2016-07-01

    Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate nonnuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by tubastatin A blocked TNF-α-induced lung endothelial cell hyperpermeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pretreatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by small interfering RNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by tubastatin A is a potent approach against lung endothelial barrier dysfunction.

  6. PGC-1-related coactivator (PRC) negatively regulates endothelial adhesion of monocytes via inhibition of NF κB activity

    SciTech Connect

    Chengye, Zhan; Daixing, Zhou Qiang, Zhong; Shusheng, Li

    2013-09-13

    Highlights: •First time to display that LPS downregulate the expression of PRC. •First time to show that PRC inhibits the induction of VCAM-1 and E-selectin. •First time to show that PRC inhibit monocytes attachment to endothelial cells. •First time to display that PRC inhibits transcriptional activity of NF-κB. •PRC protects the respiration rate and suppresses the glycolysis rate against LPS. -- Abstract: PGC-1-related coactivator (PRC) is a growth-regulated transcriptional cofactor known to activate many of the nuclear genes specifying mitochondrial respiratory function. Endothelial dysfunction is a prominent feature found in many inflammatory diseases. Adhesion molecules, such as VCAM-1, mediate the attachment of monocytes to endothelial cells, thereby playing an important role in endothelial inflammation. The effects of PRC in regards to endothelial inflammation remain unknown. In this study, our findings show that PRC can be inhibited by the inflammatory cytokine LPS in cultured human umbilical vein endothelial cells (HUVECs). In the presence of LPS, the expression of endothelial cell adhesion molecular, such as VCAM1 and E-selectin, is found to be increased. These effects can be negated by overexpression of PRC. Importantly, monocyte adhesion to endothelial cells caused by LPS is significantly attenuated by PRC. In addition, overexpression of PRC protects mitochondrial metabolic function and suppresses the rate of glycolysis against LPS. It is also found that overexpression of PRC decreases the transcriptional activity of NF-κB. These findings suggest that PRC is a negative regulator of endothelial inflammation.

  7. Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats and involves signaling through RBP-Jkappa.

    PubMed

    MacKenzie, Farrell; Duriez, Patrick; Larrivée, Bruno; Chang, Linda; Pollet, Ingrid; Wong, Fred; Yip, Calvin; Karsan, Aly

    2004-09-15

    Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases the intracellular domain of the receptor that translocates to the nucleus and regulates transcription through the DNA-binding protein RBP-Jkappa. Previously, it has been shown that the Notch4 intracellular region (N4IC) can inhibit endothelial sprouting and angiogenesis. Here, N4IC deletion mutants were assessed for their ability to inhibit human microvascular endothelial cell (HMEC) sprouting with the use of a quantitative endothelial sprouting assay. Deletion of the ankyrin repeats, but not the RAM (RBP-Jkappa associated module) domain or C-terminal region (CT), abrogated the inhibition of fibroblast growth factor 2 (FGF-2)- and vascular endothelial growth factor (VEGF)-induced sprouting by Notch4, whereas the ankyrin repeats alone partially blocked sprouting. The ankyrin repeats were also the only domain required for up-regulation of RBP-Jkappa-dependent gene expression. Interestingly, enforced expression of the ankyrin domain alone was sufficient to up-regulate some, but not all, RBP-Jkappa-dependent genes. Although N4IC reduced VEGF receptor-2 (VEGFR-2) and vascular endothelial (VE)-cadherin expression, neither of these events is necessary and sufficient to explain N4IC-mediated inhibition of sprouting. A constitutively active RBP-Jkappa mutant significantly inhibited HMEC sprouting but not as strongly as N4IC. Thus, Notch4-induced inhibition of sprouting requires the ankyrin repeats and appears to involve RBP-Jkappa-dependent and -independent signaling.

  8. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    SciTech Connect

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. )

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  9. Curcumin attenuates endothelial cell oxidative stress injury through Notch signaling inhibition.

    PubMed

    Yang, Yang; Duan, Weixun; Liang, Zhenxin; Yi, Wei; Yan, Juanjuan; Wang, Ning; Li, Yue; Chen, Wensheng; Yu, Shiqiang; Jin, Zhenxiao; Yi, Dinghua

    2013-03-01

    Previous studies have demonstrated that Notch signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, our aim was to explore the role of the Notch signaling pathway in hydrogen peroxide (H(2)O(2))-induced OSI and the protective effect of curcumin during (H(2)O(2))-induced injury in human umbilical vein endothelial cells (HUVECs). DAPT, a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to study Notch activity. Further, HUVECs were exposed to H(2)O(2) in the absence or presence of curcumin. DAPT and Notch1 siRNA significantly inhibited OSI and the expression of Notch1 and Hes1. Curcumin conferred a protective effect on the HUVECs against H(2)O(2), which was evidenced by improved cell viability, adhesive ability and migratory ability and a decreased apoptotic index, decreased production of reactive oxygen species (ROS) and a reduction in several biochemical parameters. Immunofluorescence and Western blotting analyses demonstrated that H(2)O(2) treatment upregulated the expression of Notch1, Hes1, Caspase3, Bax and cytochrome c downregulated the expression of Bcl2, and treatment with curcumin reversed these effects. We demonstrated for the first time that the inhibition of Notch signaling pathway imparts a protective effect against endothelial OSI. The protective effects of curcumin against OSI are at least in part dependent on Notch1 inhibition.

  10. Dual mechanisms of NF-kappaB inhibition in carnosol-treated endothelial cells

    SciTech Connect

    Lian, K.-C.; Chuang, J.-J.; Hsieh, C.-W.; Wung, B.-S.; Huang, G.-D.; Jian, T.-Y.; Sun, Y.-W.

    2010-05-15

    The increased adhesion of monocytes to injured endothelial layers is a critical early event in atherogenesis. Under inflammatory conditions, there is increased expression of specific cell adhesion molecules on activated vascular endothelial cells, which increases monocyte adhesion. In our current study, we demonstrate a putative mechanism for the anti-inflammatory effects of carnosol, a diterpene derived from the herb rosemary. Our results show that both carnosol and rosemary essential oils inhibit the adhesion of TNFalpha-induced monocytes to endothelial cells and suppress the expression of ICAM-1 at the transcriptional level. Moreover, carnosol was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein IkappaBalpha in short term pretreatments but not in 12 h pretreatments. Our data show that carnosol reduces IKK-beta phosphorylation in pretreatments of less than 3 h. In TNFalpha-treated ECs, NF-kappaB nuclear translocation and transcriptional activity was abolished by up to 12 h of carnosol pretreatment and this was blocked by Nrf-2 siRNA. The long-term inhibitory effects of carnosol thus appear to be mediated through its induction of Nrf-2-related genes. The inhibition of ICAM-1 expression and p65 translocation is reversed by HO-1 siRNA. Carnosol also upregulates the Nrf-2-related glutathione synthase gene and thereby increases the GSH levels after 9 h of exposure. Treating ECs with a GSH synthesis inhibitor, BSO, blocks the inhibitory effects of carnosol. In addition, carnosol increases p65 glutathionylation. Hence, our present findings indicate that carnosol suppresses TNFalpha-induced singling pathways through the inhibition of IKK-beta activity or the upregulation of HO-1 expression. The resulting GSH levels are dependent, however, on the length of the carnosol pretreatment period.

  11. Degradable poly(apigenin) polymer inhibits tumor cell adhesion to vascular endothelial cells.

    PubMed

    Cochran, David B; Gray, Lindsay N; Anderson, Kimberly W; Dziubla, Thomas D

    2016-10-01

    Cancer and the inflammatory system share a complex intertwined relationship. For instance, in response to an injury or stress, vascular endothelial cells will express cell adhesion molecules as a means of recruiting leukocytes. However, circulating tumor cells (CTCs) have been shown to highjack this expression for the adhesion and invasion during the metastatic cascade. As such, the initiation of endothelial cell inflammation, either by surgical procedures (cancer resection) or chemotherapy can inadvertently increase the metastatic potential of CTCs. Yet, systemic delivery of anti-inflammatories, which weaken the entire immune system, may not be preferred in some treatment settings. In this work, we demonstrate that a long-term releasing flavone-based polymer and subsequent nanoparticle delivery system can inhibit tumor cell adhesion, through the suppression of endothelial cell adhesion molecule expression. The degradation of a this anti-inflammatory polymer provides longer term, localized release profile of active therapeutic drug in nanoparticle form as compared with that of the free drug, permitting more targeted anti-metastatic therapies. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1438-1447, 2016.

  12. Phagocytosis by macrophages and endothelial cells inhibits procoagulant and fibrinolytic activity of acute promyelocytic leukemia cells.

    PubMed

    Xie, Rui; Gao, Chunyan; Li, Wen; Zhu, Jiuxin; Novakovic, Valerie; Wang, Jing; Ma, Ruishuang; Zhou, Jin; Gilbert, Gary E; Shi, Jialan

    2012-03-08

    The coagulopathy of acute promyelocytic leukemia (APL) is mainly related to procoagulant substances and fibrinolytic activators of APL blasts, but the fate of these leukemic cells is unknown. The aim of this study was to investigate the removal of APL blasts by macrophages and endothelial cells in vitro and consequent procoagulant and fibrinolytic activity of APL cells. We found that human umbilical vein endothelial cells as well as THP-1 and monocyte-derived macrophages bound, engulfed, and subsequently degraded immortalized APL cell line NB4 and primary APL cells. Lactadherin promoted phagocytosis of APL cells in a time-dependent fashion. Furthermore, factor Xa and prothrombinase activity of phosphatidylserine-exposed target APL cells was time-dependently decreased after incubation with phagocytes (THP-1-derived macrophages or HUVECs). Thrombin production on target APL cells was reduced by 40%-45% after 2 hours of coincubation with phagocytes and 80% by a combination of lactadherin and phagocytes. Moreover, plasmin generation of target APL cells was inhibited 30% by 2 hours of phagocytosis and ∼ 50% by lactadherin-mediated engulfment. These results suggest that engulfment by macrophages and endothelial cells reduce procoagulant and fibrinolytic activity of APL blasts. Lactadherin and phagocytosis could cooperatively ameliorate the clotting disorders in APL.

  13. CLIC1 Inhibition Attenuates Vascular Inflammation, Oxidative Stress, and Endothelial Injury

    PubMed Central

    Hu, Xiao; Wang, Cui; Lu, Dezhao; Gong, Chenxue; Yang, Jinhuan; Zong, Lei

    2016-01-01

    Endothelial dysfunction, which includes endothelial oxidative damage and vascular inflammation, is a key initiating step in the pathogenesis of atherosclerosis (AS) and an independent risk factor for this disorder. Intracellular chloride channel 1 (CLIC1), a novel metamorphic protein, acts as a sensor of cell oxidation and is involved in inflammation. In this study, we hypothesize that CLIC1 plays an important role in AS. Apolipoprotein E-deficient mice were supplied with a normal diet or a high-fat and high-cholesterol diet for 8 weeks. Overexpressed CLIC1 was associated with the accelerated atherosclerotic plaque development, amplified oxidative stress, and in vivo release of inflammatory cytokines. We subsequently examined the underlying molecular mechanisms through in vitro experiments. Treatment of cultured human umbilical vein endothelial cells (HUVECs) with H2O2 induced endothelial oxidative damage and enhanced CLIC1 expression. Suppressing CLIC1 expression through gene knocked-out (CLIC1−/−) or using the specific inhibitor indanyloxyacetic acid-94 (IAA94) reduced ROS production, increased SOD enzyme activity, and significantly decreased MDA level. CLIC1−/− HUVECs exhibited significantly reduced expression of TNF-α and IL-1β as well as ICAM-1 and VCAM-1 at the protein levels. In addition, H2O2 promoted CLIC1 translocation to the cell membrane and insertion into lipid membranes, whereas IAA94 inhibited CLIC1 membrane translocation induced by H2O2. By contrast, the majority of CLIC1 did not aggregate on the cell membrane in normal HUVECs, and this finding is consistent with the changes in cytoplasmic chloride ion concentration. This study demonstrates for the first time that CLIC1 is overexpressed during AS development both in vitro and in vivo and can regulate the accumulation of inflammatory cytokines and production of oxidative stress. Our results also highlight that deregulation of endothelial functions may be associated with the membrane

  14. 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.

  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. Salvianolic acid B inhibits platelets-mediated inflammatory response in vascular endothelial cells.

    PubMed

    Xu, Shixin; Zhong, Aiqin; Bu, Xiaokun; Ma, Huining; Li, Wei; Xu, Xiaomin; Zhang, Junping

    2015-01-01

    Salvianolic acid B (SAB) is a hydrophilic component isolated from the Chinese herb Salviae miltiorrhizae, which has been used clinically for the treatment of ischemic cardiovascular and cerebrovascular diseases. Platelets-mediated vascular inflammatory response contributes to the initiation and progression of atherosclerosis. In this paper, we focus on the modulating effects of SAB on the inflammatory reaction of endothelial cells triggered by activated platelets. Human umbilical vein endothelial cells (EA.hy926) were pretreated with SAB followed by co-culture with ADP-activated platelets. Adhesion of platelets to endothelial cells was observed by amorphological method. The activation of nuclear factor-kappa B was evaluated by NF-κB p65 nuclear translocation and the protein phosphorylation. A determination of the pro-inflammatory mediators (ICAM-1, IL-1β, IL-6, IL-8, MCP-1) mRNA and protein were also conducted. In addition, the inhibitory effects of SAB on platelets activation were also evaluated using a platelet aggregation assay and assessing the release level of soluble P-selectin. The results showed that SAB dose-dependently inhibited ADP- or α-thrombin-induced human platelets aggregation in platelet rich plasma (PRP) samples, and significantly decreased soluble P-selectin release from both agonists stimulated washed platelets. It was also found that pre-treatment with SAB reduced adhesion of ADP-activated platelets to EA.hy926 cells and inhibited NF-κB activation. In addition, SAB significantly suppressed pro-inflammatory mediators mRNA and protein in EA.hy926 cells in a dose-dependent manner. These results indicated that, in addition to its inhibitory effects on platelets activation, SAB was able to attenuate platelets-mediated inflammatory responses in endothelial cells even if the platelets had already been activated. This anti-inflammatory effect was related to the inhibition of NF-κB activation. Our findings suggest that SAB may be a potential

  17. Activation of Protein Kinase G (PKG) Reduces Neointimal Hyperplasia, Inhibits Platelet Aggregation, and Facilitates Re-endothelialization

    PubMed Central

    Kim, Ju-Young; Yang, Han-Mo; Lee, Joo-Eun; Kim, Baek-Kyung; Jin, Sooryeonhwa; Lee, Jaewon; Park, Kyung-Woo; Cho, Hyun-Jai; Kwon, Yoo-Wook; Lee, Hae-Young; Kang, Hyun-Jae; Oh, Byung-Hee; Park, Young-Bae; Kim, Hyo-Soo

    2016-01-01

    In spite of its great success in reducing restenosis, drug-eluting stent (DES) has unfavorable aspects such as stent thrombosis and delayed re-endothelialization. We examined the effects of PKG activation by Exisulind on neointimal formation, platelet aggregation, and re-endothelialization. Exisulind significantly reduced VSMCs viability, cell cycle progression, migration, and neointimal hyperplasia after vascular injury in rat carotid arteries. Interestingly, in contrast to the effect on VSMC viability, Exisulind did not reduce the viability of endothelial cells. Increased PKG activity by Exisulind inhibited PDGF-stimulated phenotype change of VSMCs from a contractile to a synthetic form. Conversely, the use of PKG inhibitor or gene transfer of dominant-negative PKG reversed the effects of Exisulind, resulting in the increased viability of VSMCs and neointimal formation. In addition, Exisulind facilitated the differentiation of peripheral blood mononuclear cells to endothelial lineage via PKG pathway, while inhibiting to VSMCs lineage, which was correlated with the enhanced re-endothelialization in vivo. Finally, Exisulind reduced platelet aggregation, which was mediated via PKG activation. This study demonstrated that Exisulind inhibits neointimal formation and platelet aggregation while increasing re-endothelialization via PKG pathway. These findings suggest that Exisulind could be a promising candidate drug of DES for the prevention of restenosis without other complications. PMID:27833146

  18. Phentolamine inhibits angiogenesis in vitro: Suppression of proliferation migration and differentiation of human endothelial cells.

    PubMed

    Pan, Liangli; Liu, Chenyang; Kong, Yanan; Piao, Zhengguo; Cheng, Biao

    2016-06-16

    It is widely known that the β-adrenergic receptor (AR) blocker (propranolol) inhibits human endothelial cell (EC) angiogenesis in vitro, but how the α-AR antagonist (phentolamine) affects human EC angiogenesis has not yet been studied. Here, we show for the first time that both human dermal microvascular ECs (HDMECs) and human brain microvascular ECs (HBMECs) express α-ARs. Moreover, our results indicate that phentolamine inhibits the proliferation, migration, and tubulogenesis of HDMECs and HBMECs. Finally, VEGFR-2 and Ang1/2 expression of HDMECs was suppressed by phentolamine. Together, these results indicate that phentolamine impairs several critical events of neovascularization, and α-ARs, as well as the VEGF/VEGFR-2 and Ang/Tie-2 signaling pathways, may be involved in these processes. Our results suggest a novel therapeutic strategy for the use of α-blockers in the treatment of human angiogenesis-dependent diseases.

  19. Deoxynivalenol inhibits proliferation and induces apoptosis in human umbilical vein endothelial cells.

    PubMed

    Deng, Chao; Ji, Changyun; Qin, Weisen; Cao, Xifeng; Zhong, Jialian; Li, Yugu; Srinivas, Swaminath; Feng, Youjun; Deng, Xianbo

    2016-04-01

    Deoxynivalenol (DON) is a stable mycotoxins found in cereals infected by certain fungal species and causes adverse health effects in animals and human such as vomiting, diarrhea and reproductive toxicity. In this study, we investigated the toxic and apoptotic effects of DON in human umbilical vein endothelial cells (HUVECs), a good model for studying inflammation. The results show that DON significantly inhibited the viability of HUVECs. DON could also inhibit the proliferation of HUVECs through G2/M phase arrest in cell cycle progression. Moreover, oxidative stress induced by DON was indicated by observations of increased levels of reactive oxygen species (ROS). In addition, DON also causes mitochondrial damage by decreasing the mitochondrial membrane potential and inducing apoptosis by up-regulation of apoptosis-related genes like caspase-3, caspase-9, and Bax genes, and down-regulation of Bcl-2 gene. These results together suggest that DON could induce cell cycle arrest, oxidative stress, and apoptosis in HUVECs.

  20. Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

    PubMed Central

    Poulos, Michael G.; Ramalingam, Pradeep; Gutkin, Michael C.; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J. P.; Elemento, Olivier; Levine, Ross L.; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B.; Shim, Jae-Hyuck; Butler, Jason M.

    2016-01-01

    Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens. PMID:28000664

  1. Olive oil compounds inhibit vascular endothelial growth factor receptor-2 phosphorylation.

    PubMed

    Lamy, Sylvie; Ouanouki, Amira; Béliveau, Richard; Desrosiers, Richard R

    2014-03-10

    Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention.

  2. Fucoidan extract derived from Undaria pinnatifida inhibits angiogenesis by human umbilical vein endothelial cells.

    PubMed

    Liu, Fang; Wang, Jia; Chang, Alan K; Liu, Bing; Yang, Lili; Li, Qiaomei; Wang, Peisheng; Zou, Xiangyang

    2012-06-15

    In recent years, anti-angiogenic therapy has become an effective strategy for inhibiting tumor growth. Fucoidan is a class of fucose-enriched sulfated polysaccharides found in brown algae, and it is known to have strong anti-tumor property. Using a human umbilical vein endothelial cells (HUVEC)-based cell culture model, the present study investigated the anti-angiogenic activity of fucoidan extracted from the brown seaweed Undaria pinnatifida. Treatment of HUVECs with various concentrations of fucoidan resulted in significant inhibition of cell proliferation, cell migration, tube formation and vascular network formation. However, significant inhibition of cell proliferation only occurred with longer treatment time (48 h instead of 24h or less). About 40% of cell proliferation and cell migration and 61% of tube formation by HUVECs were inhibited by 400 μg/ml fucoidan, the maximum concentration tested. These results appeared to suggest that modulation of angiogenesis by fucoidan might not occur through growth inhibition and apoptosis. Ex vivo angiogenesis assay demonstrated that at 100 μg/ml, fucoidan caused significant reduction in microvessel outgrowth. Western blot and RT-PCR analyses indicated that at 400 μg/ml, fucoidan significantly reduced the expression of the angiogenesis factor VEGF-A in the suppression of angiogenesis activity. Our results showed that fucoidan isolated from U. pinnatifida may have a new therapeutic potential in the prevention angiogenesis-related diseases.

  3. SIRT1 inhibits inflammatory response partly through regulation of NLRP3 inflammasome in vascular endothelial cells.

    PubMed

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

    2016-09-01

    Emerging evidence has indicated that vascular endothelial cells (ECs) not only form the barrier between blood and the vessel wall but also serve as conditional innate immune cells. Our previous study found that SIRT1, a class III histone deacetylase, inhibits the inflammatory response in ECs. Recent studies revealed that SIRT1 also participates in the modulation of immune responses. Although the NLRP3 inflammasome is known to be a crucial component of the innate immune system, there is no direct evidence demonstrating the anti-inflammatory effect of SIRT1 on ECs through the NLRP3 inflammasome. In this study, we observed that lipopolysaccharide (LPS) and adenosine triphosphate (ATP) triggered the activation of NLRP3 inflammasome in human umbilical vein ECs (HUVECs). Moreover, SIRT1 expression was reduced in HUVECs stimulated with LPS and ATP. SIRT1 activator inhibited the expression of monocyte chemotactic protein-1 (MCP-1) and C-reactive protein (CRP), whereas SIRT1 knockdown resulted in significant increases in MCP-1 and CRP levels in HUVECs stimulated with LPS and ATP. Importantly, the lack of SIRT1 enhanced NLRP3 inflammasome activation and subsequent caspase-1 cleavage. On the other hand, NLRP3 siRNA blocked the activation of the NLRP3 inflammasome in HUVECs stimulated with LPS plus ATP. Further study revealed that NLRP3 inflammasome blockade significantly reduced MCP-1 and CRP production in HUVECs. In vivo studies indicated that implantation of the periarterial carotid collar inhibited arterial SIRT1 expression in rabbits. Meanwhile, treatment with a SIRT1 activator decreased the expression levels of MCP-1 and CRP in collared arteries and the interleukin (IL)-1β level in serum. Taken together, these findings indicate that NLRP3 inflammasome activation promoted endothelial inflammation and that SIRT1 inhibits the inflammatory response partly through regulation of the NLRP3 inflammasome in ECs.

  4. Cocaine Inhibits Store-Operated Ca2+ Entry in Brain Microvascular Endothelial Cells: Critical Role for Sigma-1 Receptors

    PubMed Central

    Brailoiu, G. Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2015-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum. Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism promoted by depletion of intracellular Ca2+ stores, in rat brain microvascular endothelial cells. Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies provide an unprecedented role for Sig-1R as a SOCE inhibitor. PMID:26467159

  5. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

    PubMed

    Brailoiu, G Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2016-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

  6. Infantile hemangioma-derived stem cells and endothelial cells are inhibited by class 3 semaphorins

    SciTech Connect

    Nakayama, Hironao; Huang, Lan; Kelly, Ryan P.; Oudenaarden, Clara R.L.; Dagher, Adelle; Hofmann, Nicole A.; Moses, Marsha A.; Bischoff, Joyce; Klagsbrun, Michael

    2015-08-14

    Class 3 semaphorins were discovered as a family of axon guidance molecules, but are now known to be involved in diverse biologic processes. In this study, we investigated the anti-angiogenic potential of SEMA3E and SEMA3F (SEMA3E&F) in infantile hemangioma (IH). IH is a common vascular tumor that involves both vasculogenesis and angiogenesis. Our lab has identified and isolated hemangioma stem cells (HemSC), glucose transporter 1 positive (GLUT1{sup +}) endothelial cells (designated as GLUT1{sup sel} cells) based on anti-GLUT1 magnetic beads selection and GLUT1-negative endothelial cells (named HemEC). We have shown that these types of cells play important roles in hemangiogenesis. We report here that SEMA3E inhibited HemEC migration and proliferation while SEMA3F was able to suppress the migration and proliferation in all three types of cells. Confocal microscopy showed that stress fibers in HemEC were reduced by SEMA3E&F and that stress fibers in HemSC were decreased by SEMA3F, which led to cytoskeletal collapse and loss of cell motility in both cell types. Additionally, SEMA3E&F were able to inhibit vascular endothelial growth factor (VEGF)-induced sprouts in all three types of cells. Further, SEMA3E&F reduced the level of p-VEGFR2 and its downstream p-ERK in HemEC. These results demonstrate that SEMA3E&F inhibit IH cell proliferation and suppress the angiogenic activities of migration and sprout formation. SEMA3E&F may have therapeutic potential to treat or prevent growth of highly proliferative IH. - Highlights: • SEMA3E&F reduce actin stress fibers and induce cytoskeletal collapse in HemEC. • SEMA3E&F inhibit angiogenic activities of HemEC. • SEMA3E&F can interrupt the VEGF-A-VEGFR2-ERK signaling pathway in HemEC. • Plexin D1 and NRP2 are induced during HemSC/GLUT1{sup sel}-to-EC differentiation.

  7. Effects of Vascular-Endothelial Protein Tyrosine Phosphatase Inhibition on Breast Cancer Vasculature and Metastatic Progression

    PubMed Central

    2013-01-01

    Background The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. Methods AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)–deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. Results AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ2 test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel–Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. Conclusions Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor

  8. Lithium prevents early cytosolic calcium increase and secondary injurious calcium overload in glycolytically inhibited endothelial cells

    SciTech Connect

    Bosche, Bert; Schäfer, Matthias; Graf, Rudolf; Härtel, Frauke V.; Schäfer, Ute; Noll, Thomas

    2013-05-03

    Highlights: •We investigate free calcium as a central signalling element in endothelial cells. •Inhibition of glycolysis with 2-deoxy-D-glucose reduces cellular ATP. •This manoeuvre leads to a biphasic increase and overload of free calcium. •Pre-treatment with lithium for 24 h abolishes both phases of the calcium increase. •This provides a new strategy to protect endothelial calcium homeostasis and barrier function. -- Abstract: Cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) is a central signalling element for the maintenance of endothelial barrier function. Under physiological conditions, it is controlled within narrow limits. Metabolic inhibition during ischemia/reperfusion, however, induces [Ca{sup 2+}]{sub i} overload, which results in barrier failure. In a model of cultured porcine aortic endothelial monolayers (EC), we addressed the question of whether [Ca{sup 2+}]{sub i} overload can be prevented by lithium treatment. [Ca{sup 2+}]{sub i} and ATP were analysed using Fura-2 and HPLC, respectively. The combined inhibition of glycolytic and mitochondrial ATP synthesis by 2-desoxy-D-glucose (5 mM; 2-DG) plus sodium cyanide (5 mM; NaCN) caused a significant decrease in cellular ATP content (14 ± 1 nmol/mg protein vs. 18 ± 1 nmol/mg protein in the control, n = 6 culture dishes, P < 0.05), an increase in [Ca{sup 2+}]{sub i} (278 ± 24 nM vs. 71 ± 2 nM in the control, n = 60 cells, P < 0.05), and the formation of gaps between adjacent EC. These observations indicate that there is impaired barrier function at an early state of metabolic inhibition. Glycolytic inhibition alone by 10 mM 2-DG led to a similar decrease in ATP content (14 ± 2 nmol/mg vs. 18 ± 1 nmol/mg in the control, P < 0.05) with a delay of 5 min. The [Ca{sup 2+}]{sub i} response of EC was biphasic with a peak after 1 min (183 ± 6 nM vs. 71 ± 1 nM, n = 60 cells, P < 0.05) followed by a sustained increase in [Ca{sup 2+}]{sub i}. A 24-h pre-treatment with 10 mM of lithium

  9. Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate.

    PubMed

    Hovinga, Koos E; Shimizu, Fumiko; Wang, Rong; Panagiotakos, Georgia; Van Der Heijden, Maartje; Moayedpardazi, Hamideh; Correia, Ana Sofia; Soulet, Denis; Major, Tamara; Menon, Jayanthi; Tabar, Viviane

    2010-06-01

    Glioblastoma multiforme (GBM) is a highly heterogeneous malignant tumor. Recent data suggests the presence of a hierarchical organization within the GBM cell population that involves cancer cells with stem-like behavior, capable of repopulating the tumor and contributing to its resistance to therapy. Tumor stem cells are thought to reside within a vascular niche that provides structural and functional support. However, most GBM studies involve isolated tumor cells grown under various culture conditions. Here, we use a novel three-dimensional organotypic "explant" system of surgical GBM specimens that preserves cytoarchitecture and tumor stroma along with tumor cells. Notch inhibition in explants results in decreased proliferation and self-renewal of tumor cells but is also associated with a decrease in endothelial cells. When endothelial cells are selectively eliminated from the explants via a toxin conjugate, we also observed a decrease in self-renewal of tumor stem cells. These findings support a critical role for tumor endothelial cells in GBM stem cell maintenance, mediated at least in part by Notch signaling. The explant system further highlighted differences in the response to radiation between explants and isolated tumor neurospheres. Combination treatment with Notch blockade and radiation resulted in a substantial decrease in proliferation and in self-renewal in tumor explants while radiation alone was less effective. This data suggests that the Notch pathway plays a critical role in linking angiogenesis and cancer stem cell self-renewal and is thus a potential therapeutic target. Three-dimensional explant systems provide a novel approach for the study of tumor and microenvironment interactions.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-02-17

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

  12. MiR-630 Inhibits Endothelial-Mesenchymal Transition by Targeting Slug in Traumatic Heterotopic Ossification

    PubMed Central

    Sun, Yangbai; Cai, Jiangyu; Yu, Shiyang; Chen, Shuai; Li, Fengfeng; Fan, Cunyi

    2016-01-01

    Heterotopic ossification (HO) is the abnormal formation of mature bone in extraskeletal soft tissues that occurs as a result of inflammation caused by traumatic injury or associated with genetic mutation. Despite extensive research to identify the source of osteogenic progenitors, the cellular origins of HO are controversial and the underlying mechanisms, which are important for the early detection of HO, remain unclear. Here, we used in vitro and in vivo models of BMP4 and TGF-β2-induced HO to identify the cellular origin and the mechanisms mediating the formation of ectopic bone in traumatic HO. Our results suggest an endothelial origin of ectopic bone in early phase of traumatic HO and indicate that the inhibition of endothelial-mesenchymal transition by miR-630 targeting Slug plays a role in the formation of ectopic bone in HO. A matched case-control study showed that miR-630 is specifically downregulated during the early stages of HO and can be used to distinguish HO from other processes leading to bone formation. Our findings suggest a potential mechanism of post-traumatic ectopic bone formation and identify miR-630 as a potential early indicator of HO. PMID:26940839

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

    PubMed

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

    2014-01-01

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

  14. Endothelial nitric oxide synthase activation through obacunone-dependent arginase inhibition restored impaired endothelial function in ApoE-null mice.

    PubMed

    Yoon, Jeongyeon; Park, Minjin; Lee, Jeong hyung; Min, Byung Sun; Ryoo, Sungwoo

    2014-03-01

    Endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion and reduces nitric oxide bioavailability. During the screening course of arginase inhibitor, we found obacunone as an arginase inhibitor. We tested the hypothesis that obacunone regulates vascular endothelial NO production. Obacunone incubation inhibited arginase I and II activities in liver and kidney lysates, respectively, in dose-dependent manner. Obacunone reciprocally increased nitrite/nitrate (NOx) production in HUVECs. In isolated aortic rings, obacunone increased intracellular l-arginine concentration and enhanced eNOS coupling, leading to increased NO and decreased superoxide production, with no changes in protein expression. Vasoconstriction response to U46619 was attenuated in obacunone-treated aortic vessels compared to that in untreated vessels. Endothelium-dependent vasorelaxant response to acetylcholine was significantly increased in obacunone-treated vessels and was modulated by the NO-dependent signaling cascade. The dose-dependent vasorelaxant response to Ach was reduced in the aortic vessels of ApoE-/- mice fed a high-cholesterol diet. Obacunone incubation increased vasorelaxation to the level of a WT mouse, although the endothelium-independent response to sodium nitroprusside was identical among the groups. Therefore, obacunone may help treat cardiovascular diseases derived from endothelial dysfunction and may be useful for designing pharmaceutical compounds.

  15. Magnetic fluid hyperthermia inhibits the growth of breast carcinoma and downregulates vascular endothelial growth factor expression.

    PubMed

    Wang, Guihua; Xu, Derong; Chai, Qin; Tan, Xiaolang; Zhang, Yu; Gu, Ning; Tang, Jintian

    2014-05-01

    The application of magnetic fluid hyperthermia (MFH) with nanoparticles has been shown to inhibit tumor growth in several animal models. However, the feasibility of using MFH in vivo to treat breast cancer is uncertain, and the mechanism is unclear. In the present study, it was observed that the intratumoral administration of MFH induced hyperthermia significantly in rats with Walker-265 breast carcinomas. The hyperthermia treatment with magnetic nanoparticles inhibited tumor growth in vivo and promoted the survival of the tumor-bearing rats. Furthermore, it was found that MFH treatment downregulated the protein expression of vascular endothelial growth factor (VEGF) in the tumor tissue, as observed by immunohistochemistry. MFH treatment also decreased the gene expression of VEGF and its receptors, VEGF receptor 1 and 2, and inhibited angiogenesis in the tumor tissues. Taken together, these results indicate that the application of MFH with nanoparticles is feasible for the treatment of breast carcinoma. The MFH-induced downregulation of angiogenesis may also contribute to the induction of an anti-tumor effect.

  16. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    SciTech Connect

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-11-07

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-{alpha} (TNF-{alpha}). siRNA was designed and synthesized targeting tumor necrosis factor-{alpha} receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-{alpha} expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-{alpha} expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

  17. Soy isoflavones attenuate human monocyte adhesion to endothelial cell-specific CD54 by inhibiting monocyte CD11a.

    PubMed

    Nagarajan, Shanmugam; Stewart, Bradford W; Badger, Thomas M

    2006-09-01

    Soy-based diets have been shown to protect against the development of atherosclerosis; however, the underlying mechanism(s) remain unknown. Interaction between activated monocytes and inflamed endothelial cells is an early event in atherogenesis. Therefore, we examined whether treatment of monocytes with soy phytochemicals could inhibit their adhesion to the endothelial cell-specific protein, CD54, a key factor in monocyte adhesion. Female Sprague-Dawley rats were fed AIN-93G diets containing soy protein isolate or casein. Sera from soy-fed rats inhibited CD54-dependent monocyte adhesion, whereas sera from casein-fed rats did not. To determine whether isoflavones in the sera of soy-fed rats were involved in this inhibition, monocytes were preincubated with soy isoflavones. Isoflavone treatment inhibited monocyte adhesion to CD54 protein, as well as to endothelial cells expressing CD54. Monocyte expression of CD11a, the cognate receptor for CD54, was unaffected by isoflavones. However, binding of the activation epitope-specific antibody mAb24, which binds specifically to the active form of CD11a, was significantly lower in soy isoflavone-treated monocytes than in media-treated cells. These findings suggest that inhibition of CD54-dependent monocyte adhesion by soy isoflavones is mediated in part by affinity regulation of CD11a. Inhibition of monocyte adhesion to endothelial cells by isoflavones resulted in reduced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, these data suggest that the athero-protective effect of soy diets may be mediated by blocking monocyte-endothelial cell interaction.

  18. Increase in E-selectin expression in umbilical vein endothelial cells by anticancer drugs and inhibition by cimetidine.

    PubMed

    Kawase, Jin; Ozawa, Soji; Kobayashi, Kenichi; Imaeda, Yoshihiro; Umemoto, Shunji; Matsumoto, Sumio; Ueda, Masakazu

    2009-12-01

    E-selectin is expressed on the surfaces of stimulated vascular endothelial cells and is sometimes involved in cancer cell metastasis. The H2-receptor antagonist cimetidine inhibits the increase in E-selectin expression on vascular endothelial cells that is induced by interleukin-1beta (IL-1beta) and cimetidine. It also inhibits the adhesion of sialyl-Lewis-antigen-positive cancer cells to vascular endothelial cells, ultimately inhibiting hematogenous metastasis. Anticancer drugs are essential to cancer therapy, but whether they can alter the expression of E-selectin in vascular endothelial cells remains unclear. Whether cimetidine inhibits the expression of E-selectin in the same manner in the presence or absence of anticancer drugs also remains unknown. Human umbilical vein endothelial cells were cultured with 5-fluorouracil (5-FU), doxorubicin (DXR), cisplatin (CDDP), or IL-1beta and with or without cimetidine. The expression of E-selectin at the mRNA and protein levels was then determined using quantitative reverse transcription-polymerase chain reaction and immunohistochemical staining, respectively. The E-selectin mRNA level increased in cells exposed to 5-FU, DXR, or CDDP, but the addition of cimetidine had no effect on the E-selectin mRNA level. The expression of E-selectin protein was also significantly higher after the addition of 5-FU, DXR, or CDDP, compared with that of a negative control. However, when cimetidine was added prior to the addition of 5-FU, DXR, or CDDP, the expression of E-selectin was significantly suppressed. Thus, cimetidine significantly inhibited the expression of E-selectin at the protein level without affecting its expression at the mRNA level in cells treated with anticancer drugs. In conclusion, anticancer drugs increased the expression of E-selectin and this increase was inhibited by cimetidine. These findings suggest that the administration of cimetidine during treatment with anticancer drugs might be useful for preventing

  19. Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

    NASA Technical Reports Server (NTRS)

    Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

    2002-01-01

    During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

  20. Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway.

    PubMed

    Sha, Xiaojin; Meng, Shu; Li, Xinyuan; Xi, Hang; Maddaloni, Massimo; Pascual, David W; Shan, Huimin; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-feng

    2015-07-31

    Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.

  1. Inhibition of endocytosis exacerbates TNF-α-induced endothelial dysfunction via enhanced JNK and p38 activation.

    PubMed

    Choi, Hyehun; Nguyen, Hong N; Lamb, Fred S

    2014-04-15

    Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that causes endothelial dysfunction. Endocytosis of TNF-α receptors (TNFR) precedes endosomal reactive oxygen species (ROS) production, which is required for NF-κB activation in vascular smooth muscle cells. It is unknown how endocytosis of TNFRs impacts signaling in endothelial cells. We hypothesized that TNF-α-induced endothelial dysfunction is induced by both endosomal and cell surface events, including NF-κB and mitogen-activated protein kinases (MAPKs) activation, and endocytosis of the TNFR modifies signaling. Mesenteric artery segments from C57BL/6 mice were treated with TNF-α (10 ng/ml) for 22 h in tissue culture, with or without signaling inhibitors (dynasore for endocytosis, SP600125 for JNK, SB203580 for p38, U0126 for ERK), and vascular function was assessed. Endothelium-dependent relaxation to acetylcholine (ACh) was impaired by TNF-α, and dynasore exacerbated this, whereas JNK or p38 inhibition prevented these effects. In cultured endothelial cells from murine mesenteric arteries, dynasore potentiated JNK and p38 but not ERK phosphorylation and promoted cell death. NF-κB activation by TNF-α was decreased by dynasore. JNK inhibition dramatically increased both the magnitude and duration of TNF-α-induced NF-κB activation and potentiated intercellular adhesion molecule-1 (ICAM-1) activation. Dynasore still inhibited NF-κB activation in the presence of SP600125. Thus TNF-α-induced endothelial dysfunction is both JNK and p38 dependent. Endocytosis modulates the balance of NF-κB and MAPK signaling, and inhibition of NF-κB activation by JNK limits this pro-proliferative signal, which may contribute to endothelial cell death in response to TNF-α.

  2. A natural small molecule harmine inhibits angiogenesis and suppresses tumour growth through activation of p53 in endothelial cells.

    PubMed

    Dai, Fujun; Chen, Yihua; Song, Yajuan; Huang, Li; Zhai, Dong; Dong, Yanmin; Lai, Li; Zhang, Tao; Li, Dali; Pang, Xiufeng; Liu, Mingyao; Yi, Zhengfang

    2012-01-01

    Activation of p53 effectively inhibits tumor angiogenesis that is necessary for tumor growth and metastasis. Reactivation of the p53 by small molecules has emerged as a promising new strategy for cancer therapy. Several classes of small-molecules that activate the p53 pathway have been discovered using various approaches. Here, we identified harmine (β-carboline alkaloid) as a novel activator of p53 signaling involved in inhibition of angiogenesis and tumor growth. Harmine induced p53 phosphorylation and disrupted the p53-MDM2 interaction. Harmine also prevented p53 degradation in the presence of cycloheximide and activated nuclear accumulation of p53 followed by increasing its transcriptional activity in endothelial cells. Moreover, harmine not only induced endothelial cell cycle arrest and apoptosis, but also suppressed endothelial cell migration and tube formation as well as induction of neovascularity in a mouse corneal micropocket assay. Finally, harmine inhibited tumor growth by reducing tumor angiogenesis, as demonstrated by a xenograft tumor model. Our results suggested a novel mechanism and bioactivity of harmine, which inhibited tumor growth by activating the p53 signaling pathway and blocking angiogenesis in endothelial cells.

  3. Inhibition of major integrin αV β3 reduces Staphylococcus aureus attachment to sheared human endothelial cells.

    PubMed

    McDonnell, C J; Garciarena, C D; Watkin, R L; McHale, T M; McLoughlin, A; Claes, J; Verhamme, P; Cummins, P M; Kerrigan, S W

    2016-12-01

    Essentials Staphylococcus aureus (S. aureus) binds and impairs function of vascular endothelial cells (EC). We investigated the molecular signals triggered by S. aureus adhesion to EC. Inhibition of the EC integrin αVβ3 reduces S. aureus binding and rescues EC function. αVβ3 blockade represents an attractive target to treat S. aureus bloodborne infections.

  4. Inhibition of angiogenesis by vitamin D-binding protein: characterization of anti-endothelial activity of DBP-maf.

    PubMed

    Kalkunte, Satyan; Brard, Laurent; Granai, Cornelius O; Swamy, Narasimha

    2005-01-01

    Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis (IC(50) = 7.8 +/- 0.15 microg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.

  5. Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein.

    PubMed Central

    Murugesan, G; Fox, P L

    1996-01-01

    Endothelial cell (EC) movement is required for the development and repair of blood vessels. We have previously shown that LDL oxidized by transition metals almost completely suppressed the wound-healing migratory response of vascular EC in vitro. We now report that lysophosphatidylcholine (lysoPC), a lipid component of oxidized LDL, has an important role in the antimigratory activity of the lipoprotein. Purified 1-palmitoyl lysoPC inhibited movement with a half-maximal activity at 12-15 micrometers, and near complete inhibition at 20 micrometers; the inhibitory concentration of lysoPC was consistent with its abundance in oxidized LDL. The inhibition was not due to cytotoxicity since protein synthesis was unaffected and since EC movement was restored after removal of lysoPC. Lysophospholipid activity was dependent on lipid structure. LysoPC's containing 1-position C16 or C18 saturated fatty acids were antimigratory, but those containing C < or = 14 saturated fatty acids or polyunsaturated fatty acids were not. The activity of 1-palmitoyl lysolipids with various head groups was examined. Lysophosphatidylinositol was more antimigratory than lysophosphatidylglycerol and lysophosphatidylcholine, which were more potent than lysophosphatidylserine and lysophosphatidylethanolamine. Monoglyceride was inactive while lysophosphatidate had promigratory activity. These results are consistent with head group size rather than charge as a critical determinant of activity. To show that lysophospholipids within an intact lipoprotein were active, LDL was treated with bee venom phospholipase A2 (PLA2). The modified lipoprotein inhibited EC movement to the same extent as iron-oxidized LDL and antimigratory activity correlated with the amount of lysoPC formed. To determine antimigratory activity of lysoPC present in oxidized LDL, lipid extracts from oxidized LDL were fractionated by normal phase HPLC. The fraction comigrating with lysoPC had nearly the same activity as the total extract

  6. Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells.

    PubMed

    Ghoshal, Pushpankur; Rajendran, Mythilypriya; Odo, Nadine; Ikuta, Tohru

    2014-01-01

    Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.

  7. N-ethylmaleimide-sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells

    PubMed Central

    Zhou, Yong; Yang, Shui-Xiang; Yue, Yu-Nan; Wei, Xiao-Fei; Liu, Yan

    2016-01-01

    The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N-ethylmaleimide sensitive factor (NSF) and Weibel-Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N-terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription-quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF-shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF-shRNA. Therefore NSF-siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome. PMID:27277949

  8. Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye

    PubMed Central

    Michael, Iacovos P; Westenskow, Peter D; Hacibekiroglu, Sabiha; Greenwald, Alissa Cohen; Ballios, Brian G; Kurihara, Toshihide; Li, Zhijie; Warren, Carmen M; Zhang, Puzheng; Aguilar, Edith; Donaldson, Laura; Marchetti, Valentina; Baba, Takeshi; Hussein, Samer M; Sung, Hoon-Ki; Iruela-Arispe, M Luisa; Rini, James M; van der Kooy, Derek; Friedlander, Martin; Nagy, Andras

    2014-01-01

    Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF “Sticky-traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Sticky-traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity. PMID:24705878

  9. Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene

    NASA Astrophysics Data System (ADS)

    von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

    1995-02-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as β-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

  10. The vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 inhibits aromatase.

    PubMed

    Banerjee, Susana; Zvelebil, Marketa; Furet, Pascal; Mueller-Vieira, Ursula; Evans, Dean B; Dowsett, Mitch; Martin, Lesley-Ann

    2009-06-01

    Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

  11. Direct endothelial junction restoration results in significant tumor vascular normalization and metastasis inhibition in mice

    PubMed Central

    Agrawal, Vijayendra; Maharjan, Sony; Kim, Kyeojin; Kim, Nam-Jung; Son, Jimin; Lee, Keunho; Choi, Hyun-Jung; Rho, Seung-Sik; Ahn, Sunjoo; Won, Moo-Ho; Ha, Sang-Jun; Koh, Gou Young; Kim, Young-Myeong; Suh, Young-Ger; Kwon, Young-Guen

    2014-01-01

    Tumor blood vessels are leaky and immature, which causes inadequate blood supply to tumor tissues resulting in hypoxic microenvironment and promotes metastasis. Here we have explored tumor vessel modulating activity of Sac-1004, a recently developed molecule in our lab, which directly potentiates VE-cadherin-mediated endothelial cell junction. Sac-1004 could enhance vascular junction integrity in tumor vessels and thereby inhibit vascular leakage and enhance vascular perfusion. Improved perfusion enabled Sac-1004 to have synergistic anti-tumor effect on cisplatin-mediated apoptosis of tumor cells. Interestingly, characteristics of normalized blood vessels namely reduced hypoxia, improved pericyte coverage and decreased basement membrane thickness were readily observed in tumors treated with Sac-1004. Remarkably, Sac-1004 was also able to inhibit lung and lymph node metastasis in MMTV and B16BL6 tumor models. This was in correlation with a reduction in epithelial-to-mesenchymal transition of tumor cells with considerable diminution in expression of related transcription factors. Moreover, cancer stem cell population dropped substantially in Sac-1004 treated tumor tissues. Taken together, our results showed that direct restoration of vascular junction could be a significant strategy to induce normalization of tumor blood vessels and reduce metastasis. PMID:24811731

  12. Oral Administration of Blueberry Inhibits Angiogenic Tumor Growth and Enhances Survival of Mice with Endothelial Cell Neoplasm

    PubMed Central

    Fang, Huiqing; Khanna, Savita; Harper, Justin; Phillips, Gary; Sen, Chandan K.

    2009-01-01

    Abstract Endothelial cell neoplasms are the most common soft tissue tumor in infants. Subcutaneous injection of spontaneously transformed murine endothelial (EOMA) cells results in development of hemangioendothelioma (HE). We have previously shown that blueberry extract (BBE) treatment of EOMA cells in vitro prior to injection in vivo can significantly inhibit the incidence and size of developing HE. In this study, we sought to determine whether oral BBE could be effective in managing HE and to investigate the mechanisms through which BBE exerts its effects on endothelial cells. A dose-dependent decrease in HE tumor size was observed in mice receiving daily oral gavage feeds of BBE. Kaplan-Meier survival curve showed significantly enhanced survival for mice with HE tumors given BBE, compared to control. BBE treatment of EOMA cells inhibited both c-Jun N-terminal kinase (JNK) and NF-κB signaling pathways that culminate in monocyte chemoattractant protein-1 (MCP-1) expression required for HE development. Antiangiogenic effects of BBE on EOMA cells included decreased proliferation by BrdU assay, decreased sprouting on Matrigel, and decreased transwell migration. Thus, this work provides first evidence demonstrating that BBE can limit tumor formation through antiangiogenic effects and inhibition of JNK and NF-κB signaling pathways. Oral administration of BBE represents a potential therapeutic antiangiogenic strategy for treating endothelial cell neoplasms in children. Antioxid Redox Signal 11, 47–58. PMID:18817478

  13. Itraconazole Side Chain Analogues: Structure–Activity Relationship Studies for Inhibition of Endothelial Cell Proliferation, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, and Hedgehog Signaling

    PubMed Central

    Shi, Wei; Nacev, Benjamin A.; Aftab, Blake T.; Head, Sarah; Rudin, Charles M.; Liu, Jun O.

    2012-01-01

    Itraconazole is an antifungal drug that was recently found to possess potent antiangiogenic activity and anti-hedgehog (Hh) pathway activity. To search for analogues of itraconazole with greater potency and to understand the structure–activity relationship in both antiangiogenic and Hh targeting activity, 25 itraconazole side chain analogues were synthesized and assayed for inhibition of endothelial cell proliferation and Gli1 transcription in a medulloblastoma (MB) culture. Through this analysis, we have identified analogues with increased potency for inhibiting endothelial cell proliferation and the Hh pathway, as well as VEGFR2 glycosylation that was recently found to be inhibited by itraconazole. An SAR analysis of these activities revealed that potent activity of the analogues against VEGFR2 glycosylation was generally driven by side chains of at least four carbons in composition with branching at the α or β position. SAR trends for targeting the Hh pathway were divergent from those related to HUVEC proliferation or VEGFR2 glycosylation. These results also suggest that modification of the sec-butyl side chain can lead to enhancement of the biological activity of itraconazole. PMID:21936514

  14. (-)-Epigallocatechin gallate inhibits TNF-α-induced PAI-1 production in vascular endothelial cells.

    PubMed

    Cao, Yanli; Wang, Difei; Wang, Xiaoli; Zhang, Jin; Shan, Zhongyan; Teng, Weiping

    2013-11-01

    : (-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, reduces the incidence of cardiovascular diseases such as atherosclerosis. Plasminogen activator inhibitor-1 (PAI-1) accelerates thrombus formation upon ruptured atherosclerotic plaques. However, it is not known whether or not EGCG inhibits PAI-1 production induced by tumor necrosis factor-α (TNF-α) in endothelial cells. This study tested the hypothesis that EGCG might have an inhibitory effect on PAI-1 production induced by TNF-α. Human umbilical vein endothelial cells were cultured and incubated with TNF-α and/or EGCG. The expression of p-extracellular regulated protein kinases (p-ERK1/2) and tumor necrosis factor receptor (TNFR1) protein was quantified by Western blotting, and PAI-1 levels were measured by enzyme-linked immunosorbent assay. The results showed that TNF-α increased PAI-1 production in both a dose-dependent and time-dependent manner, and EGCG prevented TNF-α-mediated PAI-1 production and reduced phosphorylation of ERK1/2. The ERK1/2 inhibitor, PD98059 (20 μmol/L), downregulated TNF-α-induced PAI-1 expression 57.69 ± 2.46% (P < 0.01), but had no effect in cells pretreated with EGCG. TNF-α stimulation resulted in a significant decrease in TNFR1, an effect that was abolished by pretreatment with EGCG. These results suggest that EGCG could provide vascular benefits in inflammatory cardiovascular diseases such as decreased thrombus formation associated with ruptured atherosclerotic plaques.

  15. Pharmacological inhibition of S-nitrosoglutathione reductase improves endothelial vasodilatory function in rats in vivo

    PubMed Central

    Chen, Qiumei; Sievers, Richard E.; Varga, Monika; Kharait, Sourabh; Haddad, Daniel J.; Patton, Aaron K.; Delany, Christopher S.; Mutka, Sarah C.; Blonder, Joan P.; Dubé, Gregory P.; Rosenthal, Gary J.

    2013-01-01

    Nitric oxide (NO) exerts a wide range of cellular effects in the cardiovascular system. NO is short lived, but S-nitrosoglutathione (GSNO) functions as a stable intracellular bioavailable NO pool. Accordingly, increased levels can facilitate NO-mediated processes, and conversely, catabolism of GSNO by the regulatory enzyme GSNO reductase (GSNOR) can impair these processes. Because dysregulated GSNOR can interfere with processes relevant to cardiovascular health, it follows that inhibition of GSNOR may be beneficial. However, the effect of GSNOR inhibition on vascular activity is unknown. To study the effects of GSNOR inhibition on endothelial function, we treated rats with a small-molecule inhibitor of GSNOR (N6338) that has vasodilatory effects on isolated aortic rings and assessed effects on arterial flow-mediated dilation (FMD), an NO-dependent process. GSNOR inhibition with a single intravenous dose of N6338 preserved FMD (15.3 ± 5.4 vs. 14.2 ± 6.3%, P = nonsignificant) under partial NO synthase inhibition that normally reduces FMD by roughly 50% (14.1 ± 2.9 vs. 7.6 ± 4.4%, P < 0.05). In hypertensive rats, daily oral administration of N6338 for 14 days reduced blood pressure (170.0 ± 5.3/122.7 ± 6.4 vs. 203.8 ± 1.9/143.7 ± 7.5 mmHg for vehicle, P < 0.001) and vascular resistance index (1.5 ± 0.4 vs. 3.2 ± 1.0 mmHg·min·l−1 for vehicle, P < 0.001), and restored FMD from an initially impaired state (7.4 ± 1.7%, day 0) to a level (13.0 ± 3.1%, day 14, P < 0.001) similar to that observed in normotensive rats. N6338 also reversed the pathological kidney changes exhibited by the hypertensive rats. GSNOR inhibition preserves FMD under conditions of impaired NO production and protects against both microvascular and conduit artery dysfunction in a model of hypertension. PMID:23349456

  16. Dehydroepiandrosterone inhibits the TNF-alpha-induced inflammatory response in human umbilical vein endothelial cells.

    PubMed

    Gutiérrez, Gisela; Mendoza, Criselda; Zapata, Estrella; Montiel, Angélica; Reyes, Elba; Montaño, Luis Felipe; López-Marure, Rebeca

    2007-01-01

    Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis. We determined the effect of pharmacological doses of DHEA upon the adhesion of monocytic U937 cells to human umbilical vein endothelial cells (HUVEC), as well as the expression of adhesion and chemoattractant molecules, the translocation of NF-kappaB, the degradation of IkappaB-alpha and the production of reactive oxygen species (ROS) in HUVEC. Adhesion of U937 cells to DHEA-treated HUVEC was evaluated by co-culture experiments using [(3)H]-thymidine-labeled U937 cells. The expression of adhesion and chemoattractant molecules was evaluated by flow cytometry and RT-PCR, respectively; NF-kappaB translocation was determined by Electrophoretic Mobility Shift Assay (EMSA) and IkappaB-alpha degradation by Western blot. ROS production was determined by the reduction of fluorescent DCFDA. TNF-alpha was used to induce inflammatory responses in HUVEC. One hundred micromolar of DHEA-treatment inhibited the TNF-alpha-induced expression of ICAM-1, E-selectin, ROS production and U937 cells adhesion to HUVEC, and interfered with NF-kappaB translocation and IkappaB-alpha degradation. DHEA at the above mention concentration also inhibited the mRNA expression of MCP-1 and IL-8 in basal conditions but not in TNF-alpha-stimulated conditions. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process, therefore it could be used as an alternative in the treatment of chronic inflammatory diseases such as atherosclerosis.

  17. Developmental endothelial locus-1 inhibits MIF production through suppression of NF-κB in macrophages.

    PubMed

    Lee, Seung-Hwan; Kim, Dong-Young; Kang, Yoon-Young; Kim, Hyesoon; Jang, Jungin; Lee, Mi-Ni; Oh, Goo Taeg; Kang, Sang-Wook; Choi, Eun Young

    2014-04-01

    Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that regulates leukocyte recruitment, thereby playing a pivotal role in the regulation of innate and adaptive immunity and tumor progression. Elevated levels of MIF are associated with numerous inflammatory disorders and cancers. To determine whether developmental endothelial locus-1 (Del-1) regulated MIF, RAW264.7 macrophages were treated with Del-1 and assessed using ELISA. The results showed that MIF was downregulated in macrophages by Del-1, an endogenous anti-inflammatory protein that was previously shown to limit leukocyte adhesion and migration. Treatment of RAW264.7 macrophages with Del-1 inhibited constitutive and lipopolysaccharide (LPS)-induced MIF secretion. Recombinant Del-1 protein attenuated the phosphorylation of IκBα induced by a relatively low concentration of LPS in THP-1 monocytes, but did not inhibit IκBα phosphorylation in response to a relatively high concentration of LPS. Concomitantly, translocation of NF-κB to the nucleus was inhibited by Del-1 in LPS-activated macrophages. In addition, conditioned medium harvested from cells transfected with a Del-1 expression plasmid suppressed NF-κB activation in response to relatively low concentrations of TNF-α, albeit not the activation that was induced by a relatively high concentration of TNF-α. On the other hand, although Del-1 enhanced the macrophage expression of p53, a known negative regulator of MIF production, MIF production was not significantly affected by the level of p53 in mouse bone marrow-derived macrophages. These findings suggested that Del-1 controls NF-κB-activated MIF production in macrophages, and the potential application of Del-1 to therapeutic modalities for chronic inflammation-associated cancers.

  18. Asymmetric dimethylarginine does not inhibit arginase activity and is pro-proliferative in pulmonary endothelial cells.

    PubMed

    Chen, Bernadette; Strauch, Krista; Jin, Yi; Cui, Hongmei; Nelin, Leif D; Chicoine, Louis G

    2014-07-01

    Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. L-Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater L-arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (L-arginine, ADMA, N(G) -monomethyl-L-arginine (L-NMMA) and N(G) -nitro-L-arginine methyl ester (L-NAME)). Only L-arginine resulted in measurable urea production (Km = 6.9 ± 0.8 mmol/L; Vmax = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, L-NMMA and L-NAME in the presence of 1 mmol/L l-arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.

  19. Inhibition of cultured bovine aortic endothelial cell proliferation by sodium spirulan, a new sulfated polysaccharide isolated from Spirulina platensis.

    PubMed

    Kaji, Toshiyuki; Fujiwara, Yasuyuki; Hamada, Chieko; Yamamoto, Chika; Shimada, Satomi; Lee, Jung-Bum; Hayashi, Toshimitsu

    2002-06-01

    Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, which consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with sulfate groups, other minor saccharides and sodium ion. Vascular endothelial cells are present on the inner surface of blood vessels in a monolayer and have anticoagulant properties. To address the question whether Na-SP influences the maintenance of endothelial cell monolayers, we investigated the proliferation of cultured bovine aortic endothelial cells treated with Na-SP. It was found that Na-SP has an inhibitory activity on endothelial cell proliferation accompanied with suppression of whole protein synthesis but without non-specific cell damage. The inhibitory activity of Na-SP was the strongest when compared to that of heparan sulfate, heparin, dextran sulfate, dermatan sulfate, chondroitin sulfate A/C and hyaluronan. Furthermore, it was shown that the inhibitory activity of Na-SP disappeared by either desulfation or depolymerization. The present data suggest that Na-SP is a unique sulfated polysaccharide that strongly inhibits vascular endothelial cell proliferation, and the inhibitory activity requires polymerization of sulfated O-rhamnosyl-acofriose repeating units.

  20. Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

    SciTech Connect

    Pan, Christopher C.; Bloodworth, Jeffrey C.; Mythreye, Karthikeyan; Lee, Nam Y.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.

  1. The Traditional Japanese Formula Keishibukuryogan Inhibits the Production of Inflammatory Cytokines by Dermal Endothelial Cells

    PubMed Central

    Yoshihisa, Yoko; Furuichi, Megumi; Ur Rehman, Mati; Ueda, Chieko; Makino, Teruhiko; Shimizu, Tadamichi

    2010-01-01

    Keishibukuryogan (KBG) is one of the traditional herbal formulations widely administered to patients with blood stagnation for improving blood circulation; currently, it is the most frequently prescribed medicine in Japan. KBG has been reported to improve conjunctional microcirculation. The aim of this study was to evaluate the role of KBG and paeoniflorin, a bioactive compound of KBG, in inhibiting the production of inflammatory cytokines using human dermal microvessel endothelial cells (HDMECs). The authors observed that lipopolysaccharide (LPS; 1 μg/mL) stimulated the secretion of proinflammatory cytokines in HDMECs. KBG treatment (10 mg/mL) significantly suppressed the mRNA levels of migration inhibitory factor (MIF), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in LPS-stimulated cultured HDMECs. Similarly, paeoniflorin significantly suppressed the mRNA levels of these cytokines in LPS-stimulated cultured HDMECs. ELISA showed that KBG and paeoniflorin suppressed the production of MIF, IL-6, IL-8, and TNF-α in LPS-stimulated HDMECs. Moreover, KBG and paeoniflorin decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) in these cells. These results suggest that KBG may be useful for improving microvascular inflammation in patients with skin diseases. PMID:21253500

  2. The Biflavonoid Amentoflavone Inhibits Neovascularization Preventing the Activity of Proangiogenic Vascular Endothelial Growth Factors*

    PubMed Central

    Tarallo, Valeria; Lepore, Laura; Marcellini, Marcella; Dal Piaz, Fabrizio; Tudisco, Laura; Ponticelli, Salvatore; Lund, Frederik Wendelboe; Roepstorff, Peter; Orlandi, Augusto; Pisano, Claudio; De Tommasi, Nunziatina; De Falco, Sandro

    2011-01-01

    The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low μm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules. PMID:21471210

  3. Effects of vascular endothelial growth factor signaling inhibition on human erythropoiesis.

    PubMed

    Bhatta, Sumita S; Wroblewski, Kristen E; Agarwal, Kelly L; Sit, Laura; Cohen, Ezra E W; Seiwert, Tanguy Y; Karrison, Theodore; Bakris, George L; Ratain, Mark J; Vokes, Everett E; Maitland, Michael L

    2013-01-01

    Inhibition of vascular endothelial growth factor (VEGF) signaling increases red blood cell (RBC) counts, and erythropoiesis markers have been proposed to guide antiangiogenic therapy in humans. We analyzed RBC measurements in patients enrolled in three studies: a phase II trial of axitinib in thyroid cancer; a study of sorafenib in advanced solid tumors; and a randomized trial of fluorouracil, hydroxyurea, and radiation with and without bevacizumab for head and neck cancer. In the sorafenib trial, plasma erythropoietin concentrations were measured at baseline, day 8, and day 35. Over the first 84 days of treatment, RBC counts increased for each day on sorafenib (2.7 M/μL [95% confidence interval (CI), 1.5-3.9]) and axitinib (4.3 M/μL [95% CI, 2.2-6.5]). RBCs declined over the first 68 days of cytotoxic chemoradiotherapy alone (-12.8 M/μL per day [95% CI, -15.7 to -9.8]) but less so with added bevacizumab (-7.2 M/μL per day [95% CI, -9.5 to -4.9]). Erythropoietin levels increased, on average, by 9.5 mIU/mL between day 8 and day 35 of sorafenib exposure. No significant relationships between elevations in RBCs and changes in volume status or blood pressure or between elevations in erythropoietin and smoking status were found. VEGF signaling inhibition is associated with increased RBC and erythropoietin production in humans. The effects of these changes are subtle at physiologic doses and are unlikely to be clinically useful biomarkers for guiding the administration of or predicting treatment responses to VEGF pathway inhibitors.

  4. Flavonoids inhibit hypoxia-induced vascular endothelial growth factor expression by a HIF-1 independent mechanism.

    PubMed

    Ansó, Elena; Zuazo, Alicia; Irigoyen, Marta; Urdaci, María C; Rouzaut, Ana; Martínez-Irujo, Juan J

    2010-06-01

    Flavonoids are a group of polyphenolic dietary compounds that have been proposed to possess chemopreventive properties against lung cancer. In this work we analyzed the effect of a group of 20 structurally related flavonoids, including flavones, flavonols and isoflavones, on the production of vascular endothelial growth factor (VEGF) induced by hypoxia in NCI-H157 cells. VEGF is the main regulator of physiological and pathological angiogenesis and is highly stimulated by hypoxia-inducible factor 1 (HIF-1). We found that apigenin, luteolin, fisetin and quercetin inhibited hypoxia-induced VEGF expression in the low micromolar range. Structure-activity relationships demonstrated that flavone derivatives were the most active compounds and that hydroxylation of the A ring at the positions 5 and 7 and of the B ring at the 4' position were important for this activity. Interestingly, only a group of VEGF inhibitors, including apigenin, flavone and 4',7-dihydroxiflavone, reduced the expression of HIF-1alpha under these conditions, whereas others, such as fisetin, luteolin, galangin or quercetin, induced HIF-1alpha expression while reducing those of VEGF. When cells were exposed to hypoxia in the presence of these flavonoids, HIF-1alpha translocated to the nucleus and interacted with p300/CBP, but this complex was transcriptionally inactive. Taken together these findings indicate that flavonoids impair VEGF transcription by an alternative mechanism that did not depend on nuclear HIF levels. We also found that flavonoids suppressed hypoxia-induced STAT3 tyrosine phosphorylation and that this activity correlated with their potency as VEGF inhibitors, suggesting that inhibition of STAT3 function may play a role in this process.

  5. BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells.

    PubMed

    Choi, Hye-Eun; Yoo, Min-Sang; Choi, Jung-Hye; Lee, Jae Yeol; Kim, Je Hak; Kim, Ji Han; Lee, Joon Kwang; Kim, Gyu Il; Park, Yong; Chi, Yong Ha; Paik, Soo Heui; Lee, Joo Han; Lee, Kyung-Tae

    2011-11-01

    Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.

  6. Widdrol, a sesquiterpene isolated from Juniperus chinensis, inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2 signaling.

    PubMed

    Jin, Soojung; Yun, Hee Jung; Jeong, Hyun Young; Oh, You Na; Park, Hyun-Jin; Yun, Seung-Geun; Kim, Byung Woo; Kwon, Hyun Ju

    2015-09-01

    Widdrol is an odorous compound derived from Juniperus chinensis that is widely used in traditional medicine to treat fever, inflammation and cancer. It was previously reported that widdrol has antitumor activity by apoptosis induction in cancer cells in vitro. However, its anti-angiogenic activity remains elusive. In the present study, we investigated the anti‑angiogenic activity of widdrol and the molecular mechanisms involved. Widdrol inhibited cell proliferation via G1 phase arrest induction in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Additionally, it was associated with a decreased expression of cyclin-dependent kinase 2 (CDK2) and an increased expression of p21, a CDK inhibitor. Widdrol significantly inhibited the cell migration and tube formation of HUVECs using an in vitro angiogenesis assay. The results showed that widdrol suppressed phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream proteins, such as AKT, focal adhesion kinase (FAK) and endothelial nitric oxide synthase (eNOS). Moreover, widdrol effectively reduced tumor growth and blood vessel formation in colon tumor xenograft mice. Collectively, these results suggested that widdrol may act as a potential anti-angiogenic agent by inhibiting vessel sprouting and growth, which may have implications for angioprevention.

  7. HDAC6 inhibition prevents TNF-α-induced caspase 3 activation in lung endothelial cell and maintains cell-cell junctions

    PubMed Central

    Yu, Jinyan; Ma, Mengshi; Ma, Zhongsen; Fu, Jian

    2016-01-01

    Pro-inflammatory mediators such as TNF-α induce caspase activation in endothelial cells, which leads to degradation of cellular proteins, induction of apoptotic signaling, and endothelial cell dysfunction. New therapeutic agents that can inhibit caspase activation may provide protection against inflammatory injury to endothelial cells. In the present study, we examined the effects of selective histone deacetylase 6 (HDAC6) inhibition on TNF-α induced caspase 3 activation and cell-cell junction dysfunction in lung endothelial cells. We also assessed the protective effects of HDAC6 inhibition against lung inflammatory injury in a mouse model of endotoxemia. We demonstrated that selective HDAC6 inhibition or knockdown of HDAC6 expression was able to prevent caspase 3 activation in lung endothelial cells and maintain lung endothelial cell-cell junctions. Mice pre-treated with HDAC6 inhibitors exhibited decreased endotoxin-induced caspase 3 activation and reduced lung vascular injury as indicated by the retention of cell-cell junction protein VE-Cadherin level and alleviated lung edema. Collectively, our data suggest that HDAC6 inhibition is a potent therapeutic strategy against inflammatory injury to endothelial cells. PMID:27419634

  8. A BMP7 Variant Inhibits Tumor Angiogenesis In Vitro and In Vivo through Direct Modulation of Endothelial Cell Biology

    PubMed Central

    Pallini, Roberto; Vakana, Eliza; Wyss, Lisa; Blosser, Wayne; Ricci-Vitiani, Lucia; D’Alessandris, Quintino Giorgio; Morgante, Liliana; Giannetti, Stefano; Maria Larocca, Luigi; Todaro, Matilde; Benfante, Antonina; Colorito, Maria Luisa; Stassi, Giorgio; De Maria, Ruggero; Rowlinson, Scott; Stancato, Louis

    2015-01-01

    Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis. PMID:25919028

  9. Curcumin inhibits advanced glycation end product-induced oxidative stress and inflammatory responses in endothelial cell damage via trapping methylglyoxal.

    PubMed

    Sun, Yan Ping; Gu, Jun Fei; Tan, Xiao Bin; Wang, Chun Fei; Jia, Xiao Bin; Feng, Liang; Liu, Ji Ping

    2016-02-01

    Methylglyoxal (MGO)-induced carbonyl stress and pro-inflammatory responses have been suggested to contribute to endothelial dysfunction. Curcumin (Cur), a polyphenolic compound from Curcuma longa L., may protect endothelial cells against carbonyl stress-induced damage by trapping dicarbonyl compounds such as MGO. However, Cur-MGO adducts have not been studied in depth to date and it remains to be known whether Cur-MGO adducts are able to attenuate endothelial damage by trapping MGO. In the present study, 1,2-diaminobenzene was reacted with MGO to ensure the reliability of the reaction system. Cur was demonstrated to trap MGO at a 1:1 ratio to form adducts 1, 2 and 3 within 720 min. The structures of these adducts were identified by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. The kinetic curves of Cur (10(-7), 10(-6) and 10(-5) M) were measured from 0-168 h by fluorescent intensity. Cur significantly inhibited the formation of advanced glycation end products (AGEs). The differences in oxidative damage and the levels of pro-inflammatory cytokines following MGO + HSA or Cur-MGO treatment were investigated in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to the Cur-MGO reaction adducts significantly reduced the intracellular ROS levels and improved cell viability compared with MGO alone. Furthermore, there was a significant reduction in the expression levels of transforming growth factor-β1 and intercellular adhesion molecule(-1) following treatment with Cur-MGO adducts compared with MGO alone. These results provide further evidence that the trapping of MGO by Cur inhibits the formation of AGEs. The current study indicates that the protective effect of Cur on carbonyl stress and pro-inflammatory responses in endothelial damage occurs via the trapping of MGO.

  10. Role of transforming growth factor-[beta]1 in inhibiting endothelial cell proliferation in experimental alcoholic liver disease.

    PubMed Central

    Nanji, A. A.; Tahan, S. R.; Golding, M.; Khwaja, S.; Rahemtulla, A.; Lalani, E. N.

    1996-01-01

    We used the intragastric feeding rat model for alcoholic liver disease to investigate the relationship between transforming growth factor (TGF)-beta 1 and inhibition of endothelial cell proliferation. Twelve groups of male Wistar rats (four to five rats per group) were fed ethanol or dextrose with either corn oil or saturated fat for 1-, 2-, and 4-week periods. All control animals were pair fed the same diets as ethanol-fed rats except that ethanol was isocalorically replaced by dextrose. In the ethanol-fed groups, nonparenchymal cells were isolated and TGF-beta 1 was measured in the nonparenchymal cell supernatant. Liver pathology and endothelial cell proliferation with an antibody to proliferating cell nuclear antigen were studied in all groups. Plasma TGF-beta 1 was measured in all rats. Pathological changes (fatty liver, necrosis, and inflammation) were observed only in the corn oil/ethanol-fed rats at 4 weeks. Significantly higher levels of TGF-beta 1 were seen in both plasma and nonparenchymal cell supernatant in rats fed corn oil and ethanol; plasma levels of TGF-beta 1 were not significantly different between the dextrose-fed controls and saturated fat/ethanol-fed rats. A significant inverse correlation (r = -0.89, P < 0.01) was seen between plasma TGF-beta 1 and the number of endothelial cells arrested at G1/S. Immunohistochemistry revealed the presence of TGF-beta 1 staining in interstitial macrophages only in rats fed corn oil and ethanol. The present study provides evidence for a role for TGF-beta 1 in inhibiting endothelial cell proliferation in experimental alcoholic liver disease. Arrest of endothelial cells may lead to their differentiation and/or to produce mediators that could stimulate other cells such as Ito cells. Sustained TGF-beta 1 may also lead to Ito cell production of extracellular matrix. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8774130

  11. Hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor VEGF, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability.

    PubMed

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

    2008-06-01

    Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism

  12. Renal endothelial function is associated with the anti-proteinuric effect of ACE inhibition in 5/6 nephrectomized rats.

    PubMed

    Vettoretti, Simone; Vavrinec, Peter; Ochodnicky, Peter; Deelman, Leo E; De Zeeuw, Dick; Henning, Rob H; Buikema, Hendrik

    2016-05-01

    In healthy rats, the physiological variation of baseline endothelial function of intrarenal arteries correlates with the severity of renal damage in response to a subsequent specific renal injury. However, whether such a variation in endothelial function may also condition or predict the variable response to angiotensin-converting enzyme-inhibiting treatment in these individuals has not been addressed before. To study this, 5/6 nephrectomy was performed to induce renal injury and chronic kidney disease in a group of healthy Wistar rats. At the time of nephrectomy, interlobar arteries were obtained from the extirpated right kidney and studied in vitro for endothelium-dependent relaxation to acetylcholine. Six weeks thereafter, treatment with lisinopril was started (n = 11) and continued for 9 wk. Proteinuria (metabolic cages) and systolic blood pressure (SBP; tail cuff) were evaluated weekly, and these were analyzed in relation to renal endothelial function at baseline. 5/6 Nephrectomy induced an increase in SBP and progressive proteinuria. Treatment with lisinopril reduced SBP and slowed proteinuria, albeit to a variable degree among individuals. The acetylcholine-induced renal artery dilation at baseline negatively correlated with lisinopril-induced reduction of proteinuria (r(2) = 0.648, P = 0.003) and with the decrease in SBP (r(2) = 0.592, P = 0.006). Our data suggest that angiotensin-converting enzyme-inhibitor attenuates the progression of renal damage the most in those individuals with decreased basal renal endothelial-mediated vasodilation.

  13. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  14. MicroRNA-221/222 regulate ox-LDL-induced endothelial apoptosis via Ets-1/p21 inhibition.

    PubMed

    Qin, Bing; Cao, Yuze; Yang, Huan; Xiao, Bo; Lu, Zhengqi

    2015-07-01

    Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play an essential role in atherosclerosis. MicroRNAs (miRNAs) are a class of short non-coding RNAs, acting as posttranscriptional regulators of protein-coding genes involved in vascular cell biology. MiRNA-221 and miRNA-222 (miR-221/222) are known to be involved in the regulation of endothelial inflammation and angiogenesis. However, the function of miR-221/222 in ox-LDL-induced ECs apoptosis and atherosclerosis is still unknown. Here, we showed that miR-221/222 expression was markedly down-regulated in ox-LDL-induced apoptotic human umbilical cord vein endothelial cells. MiR-221/222 inhibition enhanced apoptosis in ECs, whereas over-expression of miR-221/222 could partly alleviate apoptotic cell death mediated by ox-LDL through suppression of Ets-1 and its downstream target p21. These findings suggest that manipulation of the miR-221/222-Ets-1-p21 pathway may offer a novel strategy for treatment of endothelial apoptosis and atherosclerosis.

  15. Repair of wounded monolayers of cultured bovine aortic endothelial cells is inhibited by calcium spirulan, a novel sulfated polysaccharide isolated from Spirulina platensis.

    PubMed

    Kaji, Toshiyuki; Fujiwara, Yasuyuki; Inomata, Yuki; Hamada, Chieko; Yamamoto, Chika; Shimada, Satomi; Lee, Jung-Bum; Hayashi, Toshimitsu

    2002-03-08

    Calcium spirulan (Ca-SP) is a novel sulfated polysaccharide isolated from a blue-green alga Spirulina platensis. Ca-SP inhibits thrombin by activation of heparin cofactor II. Therefore, it could serve as an origin of anti-atherogenic medicines. Since maintenance of vascular endothelial cell monolayers is important for prevention of vascular lesions such as atherosclerosis, the effect of Ca-SP at 20 microg/ml or less on the repair of wounded bovine aortic endothelial cell monolayers in culture was investigated in the present study. When the monolayers were wounded and cultured in the presence of Ca-SP, the polysaccharide inhibited the appearance of the cells in the wounded area. The inhibition was also observed even when the repair was promoted by excess basic fibroblast growth factor, which is one of the autocrine growth factors that are involved in the endothelial cell monolayer maintenance. On the other hand, Ca-SP inhibited the cell growth and the incorporation of [3H]thymidine into the acid-insoluble fraction of proliferating endothelial cells, suggesting that Ca-SP inhibits endothelial cell proliferation. From these results, it is concluded that Ca-SP may retard the repair process of damaged vascular endothelium through inhibition of vascular endothelial cell proliferation by induction of a lower ability to respond to stimulation by endogenous basic fibroblast growth factor.

  16. Hydroxytyrosol targets extracellular matrix remodeling by endothelial cells and inhibits both ex vivo and in vivo angiogenesis.

    PubMed

    García-Vilas, Javier A; Quesada, Ana R; Medina, Miguel Ángel

    2017-04-15

    The health benefits of olive oil are attributed to their bioactive compounds, such as hydroxytyrosol. Previously, we demonstrated that hydroxytyrosol inhibits angiogenesis in vitro. The present study aimed to: i) get further insight into the effects of hydroxytyrosol on extracellular matrix remodeling; and ii) test whether hydroxytyrosol is able to inhibit angiogenesis ex vivo and in vivo. Hydroxytyrosol induced a shift toward inhibition of proteolysis in endothelial cells, with decreased expression of extracellular matrix remodeling-enzyme coding genes and increased levels of some of their inhibitors. Furthermore, this work demonstrated that hydroxytyrosol, at concentrations within the range of its content in virgin olive oil that can be absorbed from moderate and sustained virgin olive oil consumption, is a strong inhibitor of angiogenesis ex vivo and in vivo. These results suggest the need for translational studies to evaluate the potential use of hydroxytyrosol for angio-prevention and angiogenesis inhibition in clinical setting.

  17. Current status of vascular endothelial growth factor inhibition in age-related macular degeneration.

    PubMed

    Mousa, Shaker A; Mousa, Shaymaa S

    2010-06-01

    Angiogenesis, the process by which new vessels are created from pre-existing vasculature, has become the subject of intense research in recent years. Increased rates of angiogenesis are associated with several disease states, including cancer, age-related macular degeneration (AMD), psoriasis, rheumatoid arthritis, and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is an important modulator of angiogenesis, and has been implicated in the pathology of a number of conditions, including AMD, diabetic retinopathy, and cancer. AMD is a progressive disease of the macula and the third major cause of blindness worldwide. If not treated appropriately, AMD can progress to involve both eyes. Until recently, the treatment options for AMD have been limited, with photodynamic therapy (PDT) the mainstay of treatment. Although PDT is effective at slowing disease progression, it rarely results in improved vision. Several therapies have been or are now being developed for neovascular AMD, with the goal of inhibiting VEGF. These VEGF inhibitors include the RNA aptamer pegaptanib, partial and full-length antibodies ranibizumab and bevacizumab, the VEGF receptor decoy aflibercept, small interfering RNA-based therapies bevasiranib and AGN 211745, sirolimus, and tyrosine kinase inhibitors, including vatalanib, pazopanib, TG 100801, TG 101095, AG 013958, and AL 39324. At present, established therapies have met with great success in reducing the vision loss associated with neovascular AMD, whereas those still under investigation offer the potential for further advances. In AMD patients, these therapies slow the rate of vision loss and in some cases increase visual acuity. Although VEGF-inhibitor therapies are a milestone in the treatment of these disease states, several concerns need to be addressed before their impact can be fully realized.

  18. Dihydroartemisinin inhibits vascular endothelial growth factor-induced endothelial cell migration by a p38 mitogen-activated protein kinase-independent pathway.

    PubMed

    Guo, Ling; Dong, Fengyun; Hou, Yinglong; Cai, Weidong; Zhou, Xia; Huang, Ai-Ling; Yang, Min; Allen, Thaddeus D; Liu, Ju

    2014-12-01

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has been demonstrated to possess a strong antiangiogenic activity. However, the molecular mechanisms underlying this effect remain unclear. Endothelial cell (EC) migration is an essential component of angiogenesis, and the p38 mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in the regulation of migration induced by vascular endothelial growth factor (VEGF). The aim of the present study was to investigate the effects of DHA on EC migration and the p38 MAPK signaling pathway. Human umbilical vein ECs (HUVECs) were treated with DHA and VEGF-induced migration was analyzed. The activation of p38 MAPK was detected by western blot analysis, and the migration assays were performed with a p38-specific inhibitor, SB203850. It was revealed that 20 μM DHA significantly reduced EC migration in the transwell migration assay, wound healing assay and electrical cell-substrate impedance sensing real-time analysis. However, DHA did not affect p38 MAPK phosphorylation or expression. In the absence or presence of SB203850, DHA induced a similar proportional reduction of EC migration in the three migration assays. Therefore, the present study demonstrated that DHA inhibits VEGF-induced EC migration via a p38 MAPK-independent pathway.

  19. Maprotiline inhibits LPS-induced expression of adhesion molecules (ICAM-1 and VCAM-1) in human endothelial cells

    PubMed Central

    Rafiee, Laleh; Hajhashemi, Valiollah; Javanmard, Shaghayegh Haghjooy

    2016-01-01

    Regardless of the known anti-inflammatory potential of heterocyclic antidepressants, the mechanisms concerning their modulating effects are not completely known. In our earlier work, maprotiline, a heterocyclic antidepressants, considerably inhibited infiltration of polymorphonuclear cell leucocytes into the inflamed paw. To understand the mechanism involved, we evaluated the effect of vascular cell adhesion molecule (VCAM-1), intracellular adhesion molecule (ICAM-1) expression in stimulated endothelial cells. Endothelial cells were stimulated with lipopolysaccharide (LPS) in the presence and absence of maprotiline (10-8 to 10-6 M) and ICAM-1 and VCAM-1 expression were measured using real-time quantitative reverse transcription polymerase chain reaction. Maprotiline significantly decreased the LPS-induced expression of VCAM-1 at all applied concentrations. The expression of ICAM-1 decreased in the presence of maprotiline at 10-6 M concentration (P<0.05). Since maprotiline inhibits the expression of adhesion molecules, ICAM-1 and VCAM-1 in LPS-stimulated human endothelial cells, it can be a possible way through which maprotiline exerts its anti-inflammatory properties. PMID:27168753

  20. Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

    PubMed

    Bentur, Ohad S; Schwartz, Doron; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Chernin, Gil; Schwartz, Idit F

    2015-08-15

    Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.

  1. Cyclosporin A inhibits flow-mediated activation of endothelial nitric-oxide synthase by altering cholesterol content in caveolae.

    PubMed

    Lungu, Andreea O; Jin, Zheng-Gen; Yamawaki, Hideyuki; Tanimoto, Tatsuo; Wong, Chelsea; Berk, Bradford C

    2004-11-19

    Fluid shear stress generated by blood flowing over the endothelium is a major determinant of arterial tone, vascular remodeling, and atherogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an essential role in regulation of vascular function and structure by blood flow. Although cyclosporin A (CsA), an inhibitory ligand of cyclophilin A, is a widely used immunosuppressive drug, it causes arterial hypertension in part by impairing eNOS-dependent vasodilation. Here we show that CsA inhibits fluid shear stress-mediated eNOS activation in endothelial cells via decreasing cholesterol content in caveolae. Exposure of cultured bovine aortic endothelial cells to 1 mum CsA for 1 h significantly inhibited NO production and eNOS phosphorylation at Ser-1179 induced by flow (shear stress=dynes/cm2). The effect of CsA was not related to inhibition of two known eNOS kinases, protein kinase B (Akt) and protein kinase A, because CsA did not affect Akt or protein kinase A activation. In rabbit aorta perfused ex vivo, CsA also significantly inhibited flow-induced eNOS phosphorylation at Ser-1179 but had no effect on Akt measured by phosphorylation at Ser-473. However, CsA treatment decreased cholesterol content in caveolae and displaced eNOS from caveolae, which may be caused by CsA disrupting the association of caveolin-1 and cyclophilin A. The magnitude of the cholesterol depleting effect was similar to that of beta-cyclodextrin, a cholesterol-binding molecule, and beta-cyclodextrin had a similar inhibitory effect on flow-mediated eNOS activation. Treating bovine aortic endothelial cells for 24 h with 30 mug/ml cholesterol blocked the CsA effect and restored eNOS phosphorylation in response to flow. These data suggest that decreasing cholesterol content in caveolae by CsA is a potentially important pathogenic mechanism for CsA-induced endothelial dysfunction and hypertension.

  2. 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.

  3. Omentin inhibits TNF-α-induced expression of adhesion molecules in endothelial cells via ERK/NF-κB pathway.

    PubMed

    Zhong, Xia; Li, Xiaonan; Liu, Fuli; Tan, Hui; Shang, Deya

    2012-08-24

    In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-α-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-α-activated signal pathway of nuclear factor-κB (NF-κB) by preventing NF-κB inhibitory protein (IκBα) degradation and NF-κB/DNA binding activity. Omentin pretreatment significantly inhibited TNF-α-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-α-induced NF-κB activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α. These results suggest that omentin may inhibit TNF-α-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-κB pathway.

  4. Prevention of VEGF-induced growth and tube formation in human retinal endothelial cell by aldose reductase inhibition

    PubMed Central

    Yadav, Umesh CS; Srivastava, SK; Ramana, KV

    2012-01-01

    Objective Since diabetes-induced vascular endothelial growth factor (VEGF) is implicated in retinal angiogenesis, we aimed to examine the role of aldose reductase (AR) in VEGF–induced human retinal endothelial cell (HREC) growth and tube formation. Materials and Methods HREC were stimulated with VEGF and cell-growth was determined by MTT assay. AR inhibitor, fidarestat, to block the enzyme activity and AR siRNA to ablate AR gene expression in HREC were used to investigate the role of AR in neovascularization using cell-migration and tube formation assays. Various signaling intermediates and angiogenesis markers were assessed by Western blot analysis. Immuno-histochemical analysis of diabetic rat eyes was performed to examine VEGF expression in the retinal layer. Results Stimulation of primary HREC with VEGF caused increased cell growth and migration, and AR inhibition with fidarestat or ablation with siRNA significantly prevented it. VEGF-induced tube formation in HREC was also significantly prevented by fidarestat. Treatment of HREC with VEGF also increased the expression of VCAM, AR, and phosphorylation and activation of Akt and p38-MAP kinase, which were prevented by fidarestat. VEGF-induced expression of VEGFRII in HREC was also prevented by AR inhibition or ablation. Conclusions Our results indicate that inhibition of AR in HREC prevents tube formation by inhibiting the VEGF-induced activation of the Akt and p38-MAPK pathway and suggest a mediatory role of AR in ocular neovascularization generally implicated in retinopathy and AMD. PMID:22658411

  5. Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish

    PubMed Central

    Zhang, Changwen; Ellis, Jillian L.

    2016-01-01

    ABSTRACT Alcoholic liver disease (ALD) results from alcohol overconsumption and is among the leading causes of liver-related morbidity and mortality worldwide. Elevated expression of vascular endothelial growth factor (VEGF) and its receptors has been observed in ALD, but how it contributes to ALD pathophysiology is unclear. Here, we investigated the impact of VEGF signaling inhibition on an established zebrafish model of acute alcoholic liver injury. Kdrl activity was blocked by chemical inhibitor treatment or by genetic mutation. Exposing 4-day-old zebrafish larvae to 2% ethanol for 24 h induced hepatic steatosis, angiogenesis and fibrogenesis. The liver started self-repair once ethanol was removed. Although inhibiting Kdrl did not block the initial activation of hepatic stellate cells during ethanol treatment, it suppressed their proliferation, extracellular matrix protein deposition and fibrogenic gene expression after ethanol exposure, thus enhancing the liver repair. It also ameliorated hepatic steatosis and attenuated hepatic angiogenesis that accelerated after the ethanol treatment. qPCR showed that hepatic stellate cells are the first liver cell type to increase the expression of VEGF ligand and receptor genes in response to ethanol exposure. Both hepatic stellate cells and endothelial cells, but not hepatic parenchymal cells, expressed kdrl upon ethanol exposure and were likely the direct targets of Kdrl inhibition. Ethanol-induced steatosis and fibrogenesis still occurred in cloche mutants that have hepatic stellate cells but lack hepatic endothelial cells, and Kdrl inhibition suppressed both phenotypes in the mutants. These results suggest that VEGF signaling mediates interactions between activated hepatic stellate cells and hepatocytes that lead to steatosis. Our study demonstrates the involvement of VEGF signaling in regulating sustained liver injuries after acute alcohol exposure. It also provides a proof of principle of using the zebrafish model

  6. H2S inhibits pulmonary arterial endothelial cell inflammation in rats with monocrotaline-induced pulmonary hypertension.

    PubMed

    Feng, Shasha; Chen, Siyao; Yu, Wen; Zhang, Da; Zhang, Chunyu; Tang, Chaoshu; Du, Junbao; Jin, Hongfang

    2017-03-01

    This study aimed to determine whether hydrogen sulfide (H2S) inhibits pulmonary arterial endothelial inflammation in rats with monocrotaline (MCT)-induced pulmonary hypertension and its possible mechanisms. Twenty-four male Wistar rats were divided randomly into control, MCT, and MCT+H2S treatment groups. Human pulmonary arterial endothelial cells (HPAEC) were cultured and divided into four groups: control, MCT, MCT+H2S, and H2S. Pulmonary artery pressure was determined using a right cardiac catheterization procedure 3 weeks after MCT administration. Pulmonary vascular morphological changes and inflammatory infiltration were measured. Endogenous H2S levels, cystathionine-γ-lyase (CSE) expression, and inflammatory cytokines were determined both in vivo and in vitro. In addition, phosphorylation of NF-κB p65 and IκBα was detected by western blotting, and NF-κB p65 nuclear translocation, as well as its DNA-binding activity, was determined. Pulmonary hypertension and vascular remolding developed 3 wks after MCT administration, with elevated lung tissue inflammatory infiltration and cytokine level associated with activation of the NF-κB pathway, both in vivo and in vitro. However, the endogenous H2S/CSE pathway was downregulated in MCT rats. By contrast, an H2S donor markedly reduced pulmonary artery pressure, pulmonary vascular structural remolding, and increased lung inflammatory infiltration and cytokine levels of MCT-treated rats. Meanwhile, H2S reversed the activation of the NF-κB pathway successfully. The downregulated pulmonary arterial endothelial H2S/CSE pathway is involved in the pulmonary inflammatory response in MCT-treated pulmonary hypertensive rats. H2S attenuated endothelial inflammation by inhibiting the NF-κB pathway.

  7. Morin, a Flavonoid from Moraceae, Inhibits Cancer Cell Adhesion to Endothelial Cells and EMT by Downregulating VCAM1 and Ncadherin.

    PubMed

    Lee, JeongHee; Jin, Hana; Lee, Won Sup; Nagappan, Arulkumar; Choi, Yung Hyun; Kim, Gon Sup; Jung, JinMyung; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Kim, Hye Jung

    2016-01-01

    Morin, a flavonoid found in figs and other Moraceae species, displays a variety of biological actions, exerting antioxidant, antiinflammatory and anticarcinogenic effects. Here, we investigated the anticancer activity of morin focusing on antiadhesive influence. We performed experiments with MDAMB231 human breast cancer cells. Morin inhibited TNFinduced cancer cell adhesion to human umbilical vein endothelial cells (HUVECs) without showing any toxicity. It further inhibited the expression of VCAM1 on MDAMB231 cells as well as HUVECs. Morin also decreased the expression of Ncadherin on MDAMB231 cells. In addition, there was apparent antimetastatic activity in vivo. In conclusion, this study suggested that morin inhibits cancer cell adhesion to HUVECs by reducing VCAM1, and EMT by targeting Ncadherin, and that it features antimetastatic activity in vivo. Further investigation of possible antimetastatic activity of morin against human breast cancer cells is warranted.

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

    SciTech Connect

    Zhong, Xia; Li, Xiaonan; Liu, Fuli; Tan, Hui; Shang, Deya

    2012-08-24

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

  9. Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells

    PubMed Central

    Du, Xueliang; Matsumura, Takeshi; Edelstein, Diane; Rossetti, Luciano; Zsengellér, Zsuzsanna; Szabó, Csaba; Brownlee, Michael

    2003-01-01

    In this report, we show that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain activates the three major pathways of hyperglycemic damage found in aortic endothelial cells by inhibiting GAPDH activity. In bovine aortic endothelial cells, GAPDH antisense oligonucleotides activated each of the pathways of hyperglycemic vascular damage in cells cultured in 5 mM glucose to the same extent as that induced by culturing cells in 30 mM glucose. Hyperglycemia-induced GAPDH inhibition was found to be a consequence of poly(ADP-ribosyl)ation of GAPDH by poly(ADP-ribose) polymerase (PARP), which was activated by DNA strand breaks produced by mitochondrial superoxide overproduction. Both the hyperglycemia-induced decrease in activity of GAPDH and its poly(ADP-ribosyl)ation were prevented by overexpression of either uncoupling protein–1 (UCP-1) or manganese superoxide dismutase (MnSOD), which decrease hyperglycemia-induced superoxide. Overexpression of UCP-1 or MnSOD also prevented hyperglycemia-induced DNA strand breaks and activation of PARP. Hyperglycemia-induced activation of each of the pathways of vascular damage was abolished by blocking PARP activity with the competitive PARP inhibitors PJ34 or INO-1001. Elevated glucose increased poly(ADP-ribosyl)ation of GAPDH in WT aortae, but not in the aortae from PARP-1–deficient mice. Thus, inhibition of PARP blocks hyperglycemia-induced activation of multiple pathways of vascular damage. PMID:14523042

  10. Phloroglucinol Inhibits the Bioactivities of Endothelial Progenitor Cells and Suppresses Tumor Angiogenesis in LLC-Tumor-Bearing Mice

    PubMed Central

    Kwon, Yi-Hong; Jung, Seok-Yun; Kim, Jae-Won; Lee, Sang-Hun; Lee, Jun-Hee; Lee, Boo-Yong; Kwon, Sang-Mo

    2012-01-01

    Background There is increasing evidence that phloroglucinol, a compound from Ecklonia cava, induces the apoptosis of cancer cells, eventually suppressing tumor angiogenesis. Methodology/Principal Findings This is the first report on phloroglucinol's ability to potentially inhibit the functional bioactivities of endothelial progenitor cells (EPCs) and thereby attenuate tumor growth and angiogenesis in the Lewis lung carcinoma (LLC)-tumor-bearing mouse model. Although Phloroglucinol did not affect their cell toxicity, it specifically inhibited vascular endothelial growth factor (VEGF) dependent migration and capillary-like tube formation of EPCs. Our matrigel plug assay clearly indicated that orally injected phloroglucinol effectively disrupts VEGF-induced neovessel formation. Moreover, we demonstrated that when phloroglucinol is orally administered, it significantly inhibits tumor growth and angiogenesis as well as CD45−/CD34+ progenitor mobilization into peripheral blood in vivo in the LLC-tumor-bearing mouse model. Conclusions/Significance These results suggest a novel role for phloroglucinol: Phloroglucinol might be a modulator of circulating EPC bioactivities, eventually suppressing tumorigenesis. Therefore, phloroglucinol might be a candidate compound for biosafe drugs that target tumor angiogenesis. PMID:22496756

  11. H2 inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells.

    PubMed

    Song, Guohua; Tian, Hua; Liu, Jia; Zhang, Hongle; Sun, Xuejun; Qin, Shucun

    2011-09-01

    H(2) is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H(2) in endothelial cells. H(2) significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-L-cysteine. H(2) inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H(2) inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H(2) probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.

  12. Colominic acid inhibits the proliferation of cultured bovine aortic endothelial cells and injures their monolayers: cell density-dependent effects prevented by sulfation.

    PubMed

    Yamamoto, Chika; Morita, Yuki; Yamaguchi, Shinya; Hayashi, Toshimitsu; Kaji, Toshiyuki

    2006-01-18

    Colominic acid (CA), produced by Escherichia coli K1, is a polymer of sialic acid linked through alpha (2-->8) glycosidic linkages. Although there are several studies on the biological activities of chemically sulfated CA, the activity of CA has been incompletely understood. In the present study, we investigated the effects of CA, prepared as an alpha2,8-linked homopolymer of N-acetylneuraminic acid, on the proliferation and monolayer maintenance of bovine aortic endothelial cells in culture. The results indicate that CA potently inhibits the proliferation of sparse endothelial cells without nonspecific cell damage. The inhibitory effect of CA was markedly stronger than those of sodium spirulan and calcium spirulan, known polysaccharides that inhibit endothelial cell proliferation. On the other hand, in dense endothelial cells, CA induced nonspecific cell damage and markedly injured the monolayer. These results indicate that CA has two distinct effects on vascular endothelial cells: one is the inhibition of proliferation when the cell density is low, and the other is the nonspecific cytotoxicity when the cell density is high. Interestingly, these cell density-dependent effects of CA could be prevented by sulfation of the CA chains. Therefore, it is concluded that CA not only inhibits the proliferation of sparse endothelial cells without nonspecific cell damage but also injures dense cells in a monolayer by nonspecific cytotoxicity, which can be prevented by sulfation of the polysaccharide.

  13. Stat3 orchestrates interaction between endothelial and tumor cells and inhibition of Stat3 suppresses brain metastasis of breast cancer cells.

    PubMed

    Lee, Hsueh-Te; Xue, Jianfei; Chou, Ping-Chieh; Zhou, Aidong; Yang, Phillip; Conrad, Charles A; Aldape, Kenneth D; Priebe, Waldemar; Patterson, Cam; Sawaya, Raymond; Xie, Keping; Huang, Suyun

    2015-04-30

    Brain metastasis is a major cause of morbidity and mortality in patients with breast cancer. Our previous studies indicated that Stat3 plays an important role in brain metastasis. Here, we present evidence that Stat3 functions at the level of the microenvironment of brain metastases. Stat3 controlled constitutive and inducible VEGFR2 expression in tumor-associated brain endothelial cells. Furthermore, inhibition of Stat3 by WP1066 decreased the incidence of brain metastases and increased survival in a preclinical model of breast cancer brain metastasis. WP1066 inhibited Stat3 activation in tumor-associated endothelial cells, reducing their infiltration and angiogenesis. WP1066 also inhibited breast cancer cell invasion. Our results indicate that WP1066 can inhibit tumor angiogenesis and brain metastasis mediated by Stat3 in endothelial and tumor cells.

  14. Diacerein inhibits the pro-atherogenic & pro-inflammatory effects of IL-1 on human keratinocytes & endothelial cells

    PubMed Central

    Bao, Lei; Many, Benjamin; Chan, Lawrence S.

    2017-01-01

    We investigated IL-1-induced regulation of genes related to inflammation and atherogenesis in human keratinocytes and endothelial cells, and if ‘diacerein’, an oral IL-1 inhibiting drug currently approved for use in osteoarthritis, would reverse IL-1’s effects on these cells. Primary human keratinocytes and coronary artery endothelial cells were treated with either IL-1α or IL-1β, with and without diacerein. Using PCR-array, we assessed differential gene-expression regulated by IL-1 and diacerein. We identified 34 pro-atherogenic genes in endothelial cells and 68 pro-inflammatory genes in keratinocytes significantly (p<0.05) regulated at least 2-fold by IL-1, in comparison to control. Diacerein completely or partially reversed this regulation on almost all genes. Using ELISA, we confirmed diacerein’s ability to reverse IL-1-driven gene-regulation of 11 selected factors, at the protein level. The results support a novel idea that diacerein acts as an inhibitor of the pro-atherogenic and pro-inflammatory effects of IL-1. Diacerein may have therapeutic applications to diminish IL-1-induced skin inflammation in psoriasis and attenuate IL-1-induced development of atherosclerosis. Further investigation into diacerein’s effect on skin inflammation, atherogenesis and cardiovascular risk in animal models or humans is warranted. PMID:28323859

  15. Rocuronium Bromide Inhibits Inflammation and Pain by Suppressing Nitric Oxide Production and Enhancing Prostaglandin E2 Synthesis in Endothelial Cells

    PubMed Central

    2016-01-01

    Purpose Rocuronium bromide is a nondepolarizing neuromuscular blocking drug and has been used as an adjunct for relaxation or paralysis of the skeletal muscles, facilitation of endotracheal intubation, and improving surgical conditions during general anesthesia. However, intravenous injection of rocuronium bromide induces injection pain or withdrawal movement. The exact mechanism of rocuronium bromide-induced injection pain or withdrawal movement is not yet understood. We investigated whether rocuronium bromide treatment is involved in the induction of inflammation and pain in vascular endothelial cells. Methods For this study, calf pulmonary artery endothelial (CPAE) cells were used, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, nitric oxide detection, and prostaglandin E2 immunoassay were conducted. Results Rocuronium bromide treatment inhibited endothelial nitric oxide synthase and suppressed nitric oxide production in CPAE cells. Rocuronium bromide activated cyclooxygenase-2, inducible nitric oxide synthase and increased prostaglandin E2 synthesis in CPAE cells. Conclusions Rocuronium bromide induced inflammation and pain in CPAE cells. Suppressing nitric oxide production and enhancing prostaglandin E2 synthesis might be associated with rocuronium bromide-induced injection pain or withdrawal movement. PMID:28043117

  16. Inhibitive Effects of Mulberry Leaf-Related Extracts on Cell Adhesion and Inflammatory Response in Human Aortic Endothelial Cells

    PubMed Central

    Chao, P.-Y.; Lin, K.-H.; Chiu, C.-C.; Yang, Y.-Y.; Huang, M.-Y.; Yang, C.-M.

    2013-01-01

    Effects of mulberry leaf-related extracts (MLREs) on hydrogen peroxide-induced DNA damage in human lymphocytes and on inflammatory signaling pathways in human aortic endothelial cells (HAECs) were studied. The tested MLREs were rich in flavonols, especially bombyx faces tea (BT) in quercetin and kaempferol. Polyphenols, flavonoids, and anthocyanidin also abounded in BT. The best trolox equivalent antioxidant capacity (TEAC) was generated from the acidic methanolic extracts of BT. Acidic methanolic and water extracts of mulberry leaf tea (MT), mulberry leaf (M), and BT significantly inhibited DNA oxidative damage to lymphocytes based on the comet assay as compared to the H2O2-treated group. TNF-α-induced monocyte-endothelial cell adhesion was significantly suppressed by MLREs. Additionally, nuclear factor kappa B (NF-κB) expression was significantly reduced by BT and MT. Significant reductions were also observed in both NF-κB and activator protein (AP)-1 DNA binding by MLREs. Significant increases in peroxisome proliferator-activated receptor (PPAR) α and γ DNA binding by MLREs were also detected in M and MT extracts, but no evidence for PPAR α DNA binding in 50 μg/mL MT extract was found. Apparently, MLREs can provide distinct cytoprotective mechanisms that may contribute to its putative beneficial effects on suppressing endothelial responses to cytokines during inflammation. PMID:24371453

  17. Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression.

    PubMed

    Chen, Xi-Lin; Dodd, Geraldine; Thomas, Suzanne; Zhang, Xiaolan; Wasserman, Martin A; Rovin, Brad H; Kunsch, Charles

    2006-05-01

    The antioxidant response element (ARE) is a transcriptional control element that mediates expression of a set of antioxidant proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that activates ARE-containing genes. In endothelial cells, the ARE-mediated genes are upregulated by atheroprotective laminar flow through a Nrf2-dependent mechanism. We tested the hypothesis that activation of ARE-regulated genes via adenovirus-mediated expression of Nrf2 may suppress redox-sensitive inflammatory gene expression. Expression of Nrf2 in human aortic endothelial cells (HAECs) resulted in a marked increase in ARE-driven transcriptional activity and protected HAECs from H2O2-mediated cytotoxicity. Nrf2 suppressed TNF-alpha-induced monocyte chemoattractant protein (MCP)-1 and VCAM-1 mRNA and protein expression in a dose-dependent manner and inhibited TNF-alpha-induced monocytic U937 cell adhesion to HAECs. Nrf2 also inhibited IL-1beta-induced MCP-1 gene expression in human mesangial cells. Expression of Nrf2 inhibited TNF-alpha-induced activation of p38 MAP kinase. Furthermore, expression of a constitutively active form of MKK6 (an upstream kinase for p38 MAP kinase) partially reversed Nrf2-mediated inhibition of VCAM-1 expression, suggesting that p38 MAP kinase, at least in part, mediates Nrf2's anti-inflammatory action. In contrast, Nrf2 did not inhibit TNF-alpha-induced NF-kappaB activation. These data identify the Nrf2/ARE pathway as an endogenous atheroprotective system for antioxidant protection and suppression of redox-sensitive inflammatory genes, suggesting that targeting the Nrf2/ARE pathway may represent a novel therapeutic approach for the treatment of inflammatory diseases such as atherosclerosis.

  18. Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells.

    PubMed

    Liao, Chun-Hou; Lin, Feng-Yen; Wu, Yi-No; Chiang, Han-Sun

    2012-06-01

    Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens-testosterone and dihydrotestosterone-and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10-50 nM), dihydrotestosterone (5-50 nM), and estradiol (0.1-0.4 nM) on the adhesion of tumor necrosis factor-α (TNF-α)-stimulated HCAECs was determined at different time points (12-96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-α-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism.

  19. Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

    SciTech Connect

    Zhang, Wenjie; Zhang, Xiaomei; Lu, Hong; Matsukura, Makoto; Zhao, Jien; Shinohara, Makoto

    2013-05-10

    Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cell HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.

  20. Soy diet inhibits expression of inflammation-induced vascular cell adhesion molecules in endothelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently we reported that dietary soy attenuated atherogenesis in apolipoprotein E knockout (apoE-/-) mice. However, the molecular mechanisms contributing to the atheroprotective effect of soy-based diets is not clear. Since interactions between endothelial cells and monocytes play a pivotal role ...

  1. Chronic Inhibition of PPAR-γ Signaling Induces Endothelial Dysfunction In The Juvenile Lamb

    PubMed Central

    Sharma, Shruti; Barton, Jubilee; Rafikov, Ruslan; Aggarwal, Saurabh; Kuo, Hsuan-Chang; Oishi, Peter E.; Datar, Sanjeev A.; Fineman, Jeffrey R; Black, Stephen M.

    2013-01-01

    We have recently shown that the development of endothelial dysfunction in lambs with increased pulmonary blood flow (PBF) correlates with a decrease in peroxisome proliferator activated receptor-γ (PPAR-γ) signaling. Thus, in this study we determined if the loss of PPAR-γ signaling is necessary and sufficient to induce endothelial dysfunction by exposing lambs with normal PBF to the PPAR-γ antagonist, GW9662. Two-weeks of exposure to GW9662 significantly decreased both PPAR-γ protein and activity. In addition, although eNOS protein and nitric oxide metabolites (NOx) were significantly increased, endothelial dependent pulmonary vasodilation in response to acetylcholine was attenuated, indicative of endothelial dysfunction. To elucidate whether downstream mediators of vasodilation were impaired we examined soluble guanylate cyclase (sGC)- α and β subunit protein, cGMP levels, and phosphodiesterase 5 (PDE5) protein and activity, but we found no significant changes. However, we found that peroxynitrite levels were significantly increased in GW9662-treated lambs and this correlated with a significant increase in protein kinase G-1α (PKG-1α) nitration and a reduction in PKG activity. Peroxynitrite is formed by the interaction of NO with superoxide and we found that there was a significant increase in superoxide generation in GW9662-treated lambs. Further, we identified dysfunctional mitochondria as the primary source of the increased superoxide. Finally, we found that the mitochondrial dysfunction was due to a disruption in carnitine metabolism. We conclude that loss of PPAR-γ signaling is sufficient to induce endothelial dysfunction confirming its important role in maintaining a healthy vasculature. PMID:23257346

  2. Cryptotanshinone inhibits TNF-α-induced LOX-1 expression by suppressing reactive oxygen species (ROS) formation in endothelial cells.

    PubMed

    Ran, Xiaoli; Zhao, Wenwen; Li, Wenping; Shi, Jingshan; Chen, Xiuping

    2016-07-01

    Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha (TNF-α) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of TNF-α on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe CM-DCFH2-DA. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. TNF-α induced LOX-1 expression in a dose- and time-dependent manner in endothelial cells. TNF-α induced ROS formation, phosphorylation of NF-κB p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. NF-κB inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited TNF-α-induced LOX-1 expression. CPT and NAC suppressed TNF-α-induced LOX-1 expression and phosphorylation of NF-κB p65 and ERK in rat aorta. These data suggested that TNF-α induced LOX-1 expression via ROS activated NF-κB/ERK pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT.

  3. Cryptotanshinone inhibits TNF-α-induced LOX-1 expression by suppressing reactive oxygen species (ROS) formation in endothelial cells

    PubMed Central

    Ran, Xiaoli; Zhao, Wenwen; Li, Wenping

    2016-01-01

    Cryptotanshinone (CPT) is a natural compound isolated from traditional Chinese medicine Salvia miltiorrhiza Bunge. In the present study, the regulatory effect and potential mechanisms of CPT on tumor necrosis factor alpha (TNF-α) induced lectin-like receptor for oxidized low density lipoprotein (LOX-1) were investigated. Human umbilical vein endothelial cells (HUVECs) were cultured and the effect of TNF-α on LOX-1 expression at mRNA and protein levels was determined by Real-time PCR and Western blotting respectively. The formation of intracellular ROS was determined with fluorescence probe CM-DCFH2-DA. The endothelial ox-LDL uptake was evaluated with DiI-ox-LDL. The effect of CPT on LOX-1 expression was also evaluated with SD rats. TNF-α induced LOX-1 expression in a dose- and time-dependent manner in endothelial cells. TNF-α induced ROS formation, phosphorylation of NF-κB p65 and ERK, and LOX-1 expression, which were suppressed by rotenone, DPI, NAC, and CPT. NF-κB inhibitor BAY11-7082 and ERK inhibitor PD98059 inhibited TNF-α-induced LOX-1 expression. CPT and NAC suppressed TNF-α-induced LOX-1 expression and phosphorylation of NF-κB p65 and ERK in rat aorta. These data suggested that TNF-α induced LOX-1 expression via ROS activated NF-κB/ERK pathway, which could be inhibited by CPT. This study provides new insights for the anti-atherosclerotic effect of CPT. PMID:27382351

  4. IGFBP-3 inhibits TNF-α production and TNFR-2 signaling to protect against retinal endothelial cell apoptosis.

    PubMed

    Zhang, Qiuhua; Steinle, Jena J

    2014-09-01

    In models of diabetic retinopathy, insulin-like growth factor binding protein-3 (IGFBP-3) protects against tumor necrosis factors-alpha (TNF-α)-mediated apoptosis of retinal microvascular endothelial cells (REC), but the underlying mechanisms are unclear. Our current findings suggest that at least two discrete but complimentary pathways contribute to the protective effects of IGFBP-3; 1) IGFBP-3 directly activates the c-Jun kinase/tissue inhibitor of metalloproteinase-3/TNF-α converting enzyme (c-Jun/TIMP-3/TACE), pathway, which in turn inhibits TNF-α production; 2) IGFBP-3 acts through the IGFBP-3 receptor, low-density lipoprotein receptor-related protein 1 (LRP1), to inhibit signaling of TNF-α receptor 2 (TNFR2). Combined, these two IGFBP-3 pathways substantially reduce REC apoptosis and offer potential targets for the treatment of diabetic retinopathy.

  5. Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells

    PubMed Central

    Luzak, Boguslawa; Golanski, Jacek; Rozalski, Marek; Krajewska, Urszula; Olas, Beata

    2010-01-01

    Introduction Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. Material and methods This study encompasses two models of monitoring platelet reactivity: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. Results We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. Conclusions It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases. PMID:22371737

  6. Aspalathin and nothofagin from rooibos (Aspalathus linearis) inhibit endothelial protein C receptor shedding in vitro and in vivo.

    PubMed

    Kwak, Soyoung; Han, Min-Su; Bae, Jong-Sup

    2015-01-01

    Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos, which have been reported for their anti-oxidant activity. Increasing evidence has demonstrated that beyond its role in the activation of protein C, endothelial cell protein C receptor (EPCR) is also involved in vascular inflammation. EPCR activity is markedly changed by ectodomain cleavage and its release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of Asp and Not on EPCR shedding. Our results demonstrated that Asp and Not induced potent inhibition of phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α-, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-induced EPCR shedding. Asp and Not also inhibited the expression and activity of PMA-induced TACE in endothelial cells. Asp and Not also suppressed CLP-induced protein C decrease in mice and thrombin generation in HUVECs. In addition, treatment with Asp and Not resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of Asp and Not as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

  7. Rhein lysinate inhibits monocyte adhesion to human umbilical vein endothelial cells by blocking p38 signaling pathway.

    PubMed

    Lin, Yajun; Zhen, Yongzhan; Liu, Jiang; Wei, Jie; Tu, Ping; Hu, Gang

    2013-11-01

    The objective of this study was to investigate the effect of rhein lysinate (RHL) on monocyte adhesion and its mechanism. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth inhibition by drugs. The monocyte chemoattractant protein (MCP)-1 levels were assayed using MCP-1 ELISA. The expression of proteins was detected by Western blotting analysis. The results indicated that RHL inhibited monocyte adhesion in a dose- and time-dependent manner. RHL (<20 μmol/L) and lipopolysaccharide (LPS) had no effect on viability of human umbilical vein endothelial cells. Therefore, 20 μmol/L RHL was selected for this study. RHL inhibited secretion of MCP-1 induced by LPS and expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. In the meantime, both RHL and p38 inhibitor (SB203580) inhibited phosphorylation of p38 and mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) and transcription and expression of ICAM-1 and VCAM-1. In conclusion, RHL inhibits the transcription and expression of ICAM-1 and VCAM-1 by the p38/MAPKAPK-2 signaling pathway, and the effect of RHL on transcription and expression of ICAM-1 and VCAM-1 is similar to p38 inhibitor. RHL could be a prophylactic drug for atherosclerosis.

  8. Bevacizumab radiosensitizes non-small cell lung cancer xenografts by inhibiting DNA double-strand break repair in endothelial cells.

    PubMed

    Gao, Hui; Xue, Jianxin; Zhou, Lin; Lan, Jie; He, Jiazhuo; Na, Feifei; Yang, Lifei; Deng, Lei; Lu, You

    2015-08-28

    The aims of this study were to evaluate the effects of biweekly bevacizumab administration on a tumor microenvironment and to investigate the mechanisms of radiosensitization that were induced by it. Briefly, bevacizumab was administered intravenously to Balb/c nude mice bearing non-small cell lung cancer (NSCLC) H1975 xenografts; in addition, bevacizumab was added to NSCLC or endothelial cells (ECs) in vitro, followed by irradiation (IR). The anti-tumor efficacy, anti-angiogenic efficacy and repair of DNA double-strand breaks (DSBs) were evaluated. The activation of signaling pathways was determined using immunoprecipitation (IP) and WB analyses. Finally, biweekly bevacizumab administration inhibited the growth of H1975 xenografts and induced vascular normalization periodically. Bevacizumab more significantly increased cellular DSB and EC apoptosis when administered 1 h prior to 12 Gy/1f IR than when administered 5 days prior to IR, thereby inhibiting tumor angiogenesis and growth. In vitro, bevacizumab more effectively increased DSBs and apoptosis prior to IR and inhibited the clonogenic survival of ECs but not NSCLC cells. Using IP and WB analyses, we confirmed that bevacizumab can directly inhibit the phosphorylation of components of the VEGR2/PI3K/Akt/DNA-PKcs signaling pathway that are induced by IR in ECs. In conclusion, bevacizumab radiosensitizes NSCLC xenografts mainly by inhibiting DSB repair in ECs rather than by inducing vascular normalization.

  9. Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor-induced angiogenesis.

    PubMed

    Singleton, Patrick A; Garcia, Joe G N; Moss, Jonathan

    2008-06-01

    Many patients with cancer receive combinations of drug treatments that include 5-fluorouracil (5-FU) and bevacizumab. Therapeutic doses of 5-FU are often associated with unwanted side effects, and bevacizumab is costly. Therefore, we explored potential agents that can reduce the therapeutic concentration of these drugs. Our data indicate that methylnaltrexone (MNTX), a peripheral antagonist of the mu-opioid receptor, exerts a synergistic effect with 5-FU and bevacizumab on inhibition of vascular endothelial growth factor (VEGF)-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration, two key components in cancer-associated angiogenesis. MNTX inhibited EC proliferation with an IC(50) of approximately 100 nmol/L. Adding 100 nmol/L MNTX to EC shifted the IC(50) of 5-FU from approximately 5 micromol/L to approximately 7 nmol/L. Further, adding 50 ng/mL MNTX shifted the IC(50) of bevacizumab on inhibition of EC migration from approximately 25 to approximately 6 ng/mL. These synergistic effects were not observed with naltrexone, a tertiary mu-opioid receptor antagonist. On a mechanistic level, we observed that treatment of human EC with MNTX, but not naltrexone, increased receptor protein tyrosine phosphatase mu activity, which was independent of mu-opioid receptor expression. Silencing receptor protein tyrosine phosphatase mu expression (small interfering RNA) in human EC inhibited both synergy between MNTX and bevacizumab or 5-FU and increased VEGF-induced tyrosine phosphorylation of Src and p190 RhoGAP with enhanced activation of Akt and the actin cytoskeletal regulatory protein, RhoA, whereas silencing Src, Akt, or RhoA blocked VEGF-induced angiogenic events. Therefore, addition of MNTX could potentially lower the therapeutic doses of 5-FU and bevacizumab, which could improve index.

  10. GX1-mediated anionic liposomes carrying adenoviral vectors for enhanced inhibition of gastric cancer vascular endothelial cells.

    PubMed

    Xiong, Dan; Liu, Zhongbing; Bian, Tierong; Li, Juan; Huang, Wenjun; Jing, Pei; Liu, Li; Wang, Yunlong; Zhong, Zhirong

    2015-12-30

    Gastric cancer is a highly lethal malignancy and its 5-year survival rate remains depressed in spite of multiple treatment options. Targeting drug delivery to tumor vasculature may be a promising strategy for gastric cancer therapy, for it can block the nutrition source of tumor and inhibit the metastasis and invasion in a certain extent. In present study, we have prepared the drug-targeting delivery system of peptide GX1-mediated anionic liposomes carrying adenoviral vectors (GX1-Ad5-AL), in which the tumor suppressor gene of PTEN was integrated into DNA of Ad5 and the GX1 peptide could play targeting role to vascular of gastric cancer. The inhibition ability of GX1-Ad5-AL to human gastric cancer cell lines (SGC-7901) and human umbilical vein endothelial cells (HUVEC) was evaluated by MTT assay. Further, the cell migration assay was carried out in transwell inserts and the cells uptaking of GX1-Ad5-AL was detected by confocal laser scanning microscopy. The experimental results indicated that the average cell proliferation inhibition rates resulted from the drug delivery system of GX1-Ad5-AL in SGC-7901 and HUVEC were 68.36% and 64.13%, respectively which were higher than that resulted from GX1 or Ad5-AL. Meanwhile, results of cell migration experiment demonstrated that GX1-Ad5-AL could significantly suppress the migration of gastric cancer cell of SGC-7901. Moreover, both the imaging from confocal laser scanning microscopy and the quantitative analysis of fluorescence intensity showed that, GX1-Ad5-AL was more easily uptaken by SGC-7901 cells, as compared to Ad5-AL. Therefore, the formulation of GX1-Ad5-AL was effective for enhancing the inhibition effect and suppressing the migration of gastric cancer vascular endothelial cells.

  11. C-reactive protein inhibits insulin activation of endothelial nitric oxide synthase via the immunoreceptor tyrosine-based inhibition motif of FcgammaRIIB and SHIP-1.

    PubMed

    Tanigaki, Keiji; Mineo, Chieko; Yuhanna, Ivan S; Chambliss, Ken L; Quon, Michael J; Bonvini, Ezio; Shaul, Philip W

    2009-06-05

    Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcgammaRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcgammaRIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain-containing inositol 5'-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcgammaRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.

  12. Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors.

    PubMed

    Boyer, Laurent; Doye, Anne; Rolando, Monica; Flatau, Gilles; Munro, Patrick; Gounon, Pierre; Clément, René; Pulcini, Céline; Popoff, Michel R; Mettouchi, Amel; Landraud, Luce; Dussurget, Olivier; Lemichez, Emmanuel

    2006-06-05

    The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.

  13. Hop derived flavonoid xanthohumol inhibits endothelial cell functions via AMPK activation.

    PubMed

    Gallo, Cristina; Dallaglio, Katiuscia; Bassani, Barbara; Rossi, Teresa; Rossello, Armando; Noonan, Douglas M; D'Uva, Gabriele; Bruno, Antonino; Albini, Adriana

    2016-09-13

    Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Recently, increased attention has been addressed to the ability of flavonoids to prevent cancer by suppressing angiogenesis, strategy that we named "angioprevention". Several natural compounds exert their anti-tumor properties by activating 5' adenosine monophosphate-activated protein kinase (AMPK), a key regulator of metabolism in cancer cells. Drugs with angiopreventive activities, in particular metformin, regulate AMPK in endothelial cells. Here we investigated the involvement of AMPK in the anti-angiogenic effects of xanthohumol (XN), the major prenylated flavonoid of the hop plant, and mechanisms of action. The anti-angiogenic activity of XN was more potent than epigallocatechin-3-gallate (EGCG). Treatment of endothelial cells with XN led to increased AMPK phosphorylation and activity. Functional studies using biochemical approaches confirmed that AMPK mediates XN anti-angiogenic activity. AMPK activation by XN was mediated by CAMMKβ, but not LKB1. Analysis of the downstream mechanisms showed that XN-induced AMPK activation reduced nitric oxide (NO) levels in endothelial cells by decreasing eNOS phosphorylation. Finally, AKT pathway was inactivated by XN as part of its anti-angiogenic activity, but independently from AMPK, suggesting that these two signaling pathways proceed autonomously. Our study dissects the molecular mechanism by which XN exerts its potent anti-angiogenic activity, pointing out AMPK as a crucial signal transducer.

  14. Hop derived flavonoid xanthohumol inhibits endothelial cell functions via AMPK activation

    PubMed Central

    Gallo, Cristina; Dallaglio, Katiuscia; Bassani, Barbara; Rossi, Teresa; Rossello, Armando; Noonan, Douglas M.; D'Uva, Gabriele; Bruno, Antonino; Albini, Adriana

    2016-01-01

    Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Recently, increased attention has been addressed to the ability of flavonoids to prevent cancer by suppressing angiogenesis, strategy that we named “angioprevention”. Several natural compounds exert their anti-tumor properties by activating 5′ adenosine monophosphate-activated protein kinase (AMPK), a key regulator of metabolism in cancer cells. Drugs with angiopreventive activities, in particular metformin, regulate AMPK in endothelial cells. Here we investigated the involvement of AMPK in the anti-angiogenic effects of xanthohumol (XN), the major prenylated flavonoid of the hop plant, and mechanisms of action. The anti-angiogenic activity of XN was more potent than epigallocatechin-3-gallate (EGCG). Treatment of endothelial cells with XN led to increased AMPK phosphorylation and activity. Functional studies using biochemical approaches confirmed that AMPK mediates XN anti-angiogenic activity. AMPK activation by XN was mediated by CAMMKβ, but not LKB1. Analysis of the downstream mechanisms showed that XN-induced AMPK activation reduced nitric oxide (NO) levels in endothelial cells by decreasing eNOS phosphorylation. Finally, AKT pathway was inactivated by XN as part of its anti-angiogenic activity, but independently from AMPK, suggesting that these two signaling pathways proceed autonomously. Our study dissects the molecular mechanism by which XN exerts its potent anti-angiogenic activity, pointing out AMPK as a crucial signal transducer. PMID:27494895

  15. 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.

  16. Inhibition of human endothelial cell proliferation by ShIF, a vacuolar H(+)-ATPase-like protein.

    PubMed

    Tulin, Edgardo E; Onoda, Nobuhisa; Hasegawa, Masakazu; Nomura, Hitoshi; Kitamura, Toshio

    2002-01-24

    ShIF is a bone marrow stroma cell-derived factor originally identified to support proliferation of bone marrow cells in vitro. This protein shares high sequence homology to the yeast vacuolar H(+)-ATPase subunit, Vph1p, and the 116 kDa proton pump of the rat and bovine synaptic vesicle, Vpp1. We examined the function of ShIF in the proliferation of human umbilical vein endothelial cells (HUVEC). ShIF inhibited HUVEC proliferation in a dose-dependent manner. Recombinant ShIF added at 10 and 20 ng/ml inhibited HUVEC proliferation by 21.6 and 44.3%, respectively and increasing the concentration of ShIF to 100 ng/ml inhibited proliferation by as much as 55.5%. When HUVEC cells were cultured at various concentrations of ShIF in the presence of anti-ShIF antibody, the inhibitory effects of ShIF to HUVEC proliferation were abrogated by 89-91% indicating that the activity of ShIF to HUVEC was specific. HUVEC cultured in the presence of ShIF and bafilomycin, a specific inhibitor of ATPase, resulted to a 90% growth inhibition. Thus, ShIF may act as an antagonist to the ATPase complex by disrupting the production of cellular ATP thereby decreasing the ability of HUVEC to proliferate.

  17. Targeted inhibition of {alpha}v{beta}3 integrin with an RNA aptamer impairs endothelial cell growth and survival

    SciTech Connect

    Mi Jing; Zhang Xiuwu; Giangrande, Paloma H.; McNamara, James O.; Nimjee, Shahid M.; Sarraf-Yazdi, Shiva; Sullenger, Bruce A.; Clary, Bryan M. . E-mail: mi001@duke.edu

    2005-12-16

    {alpha}v{beta}3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. {alpha}v{beta}3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of {alpha}v{beta}3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-{alpha}v{beta}3 that binds recombinant {alpha}v{beta}3 integrin, for its ability to bind endogenous {alpha}v{beta}3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-{alpha}v{beta}3 binds {alpha}v{beta}3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-{alpha}v{beta}3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-{alpha}v{beta}3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-{alpha}v{beta}3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.

  18. Trichosanatine alleviates oxidized low-density lipoprotein induced endothelial cells injury via inhibiting the LOX-1/p38 MAPK pathway.

    PubMed

    Zhang, Lei; Jia, Yu-Hua; Zhao, Xiao-Shan; Zhou, Feng-Hua; Pan, Yun-Yun; Wan, Qiang; Cui, Xiao-Bing; Sun, Xue-Gang; Chen, Yu-Yao; Zhang, Yu; Cheng, Sai-Bo

    2016-01-01

    The LOX-1/p38 mitogen-activated protein kinase (MAPK) pathway has been proved to participate in the endothelial dysfunction in atherosclerosis. Trichosanatineis is an active compound isolated from the peel of Trichosanthes kirilowii. This study aims to determine whether trichosanatine prevents the oxidized low-density lipoprotein (ox-LDL)-induced insult through inhibition of the LOX-1/p38 MAPK pathway in HUVECs. HUVECs were treated with 150 mg/ml ox-LDL for 24 h to establish an ox-LDL-induced endothelial injury model. Cell viability, mitochondrial membrane potential (MMP), apoptosis, reactive oxygen species (ROS) level, LOX-1 and p38 MAPK expression level were measured. The results indicated that HUVECs were pretreated with either 100 mM trichosanatine or LOX-1 shRNA prior to exposure to ox-LDL for 24 h. Exposure of HUVECs to 150 mg/ml ox-LDL for 24 h significantly up-regulated the expression levels of LOX-1. The increased expression levels of LOX-1 were markedly attenuated by pretreatment with 100 mM trichosanatine. In addition, the ox-LDL-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with LOX-1 shRNA. Pretreatment of HUVECs with either trichosanatine or LOX-1 shRNA before exposure to ox-LDL significantly inhibited the ox-LDL-induced injuries, as evidenced by an increase in cell viability, a decrease in apoptotic cells, a ROS generation and a loss of MMP. In conclusion, we have demonstrated for the first time that the LOX-1/p38 MAPK pathway contributes to the ox-LDL-induced injury in HUVECs. Meanwhile, the trichosanatine protects the HUVECs against ox-LDL-induced injury at least in part by inhibiting the activated of LOX-1/p38 MAPK pathway.

  19. Trichosanatine alleviates oxidized low-density lipoprotein induced endothelial cells injury via inhibiting the LOX-1/p38 MAPK pathway

    PubMed Central

    Zhang, Lei; Jia, Yu-Hua; Zhao, Xiao-Shan; Zhou, Feng-Hua; Pan, Yun-Yun; Wan, Qiang; Cui, Xiao-Bing; Sun, Xue-Gang; Chen, Yu-Yao; Zhang, Yu; Cheng, Sai-Bo

    2016-01-01

    The LOX-1/p38 mitogen-activated protein kinase (MAPK) pathway has been proved to participate in the endothelial dysfunction in atherosclerosis. Trichosanatineis is an active compound isolated from the peel of Trichosanthes kirilowii. This study aims to determine whether trichosanatine prevents the oxidized low-density lipoprotein (ox-LDL)-induced insult through inhibition of the LOX-1/p38 MAPK pathway in HUVECs. HUVECs were treated with 150 mg/ml ox-LDL for 24 h to establish an ox-LDL-induced endothelial injury model. Cell viability, mitochondrial membrane potential (MMP), apoptosis, reactive oxygen species (ROS) level, LOX-1 and p38 MAPK expression level were measured. The results indicated that HUVECs were pretreated with either 100 mM trichosanatine or LOX-1 shRNA prior to exposure to ox-LDL for 24 h. Exposure of HUVECs to 150 mg/ml ox-LDL for 24 h significantly up-regulated the expression levels of LOX-1. The increased expression levels of LOX-1 were markedly attenuated by pretreatment with 100 mM trichosanatine. In addition, the ox-LDL-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with LOX-1 shRNA. Pretreatment of HUVECs with either trichosanatine or LOX-1 shRNA before exposure to ox-LDL significantly inhibited the ox-LDL-induced injuries, as evidenced by an increase in cell viability, a decrease in apoptotic cells, a ROS generation and a loss of MMP. In conclusion, we have demonstrated for the first time that the LOX-1/p38 MAPK pathway contributes to the ox-LDL-induced injury in HUVECs. Meanwhile, the trichosanatine protects the HUVECs against ox-LDL-induced injury at least in part by inhibiting the activated of LOX-1/p38 MAPK pathway. PMID:28078016

  20. Far-infrared radiation inhibits proliferation, migration, and angiogenesis of human umbilical vein endothelial cells by suppressing secretory clusterin levels.

    PubMed

    Hwang, Soojin; Lee, Dong-Hoon; Lee, In-Kyu; Park, Young Mi; Jo, Inho

    2014-04-28

    Far-infrared (FIR) radiation is known to lessen the risk of angiogenesis-related diseases including cancer. Because deficiency of secretory clusterin (sCLU) has been reported to inhibit angiogenesis of endothelial cells (EC), we investigated using human umbilical vein EC (HUVEC) whether sCLU mediates the inhibitory effects of FIR radiation. Although FIR radiation ranging 3-25μm wavelength at room temperature for 60min did not alter EC viability, further incubation in the culture incubator (at 37°C under 5% CO2) after radiation significantly inhibited EC proliferation, in vitro migration, and tube formation in a time-dependent manner. Under these conditions, we found decreased sCLU mRNA and protein expression in HUVEC and decreased sCLU protein secreted in culture medium. Expectedly, the replacement of control culture medium with the FIR-irradiated conditioned medium significantly decreased wound closure and tube formation of HUVEC, and vice versa. Furthermore, neutralization of sCLU with anti-sCLU antibody also mimicked all observed inhibitory effects of FIR radiation. Moreover, treatment with recombinant human sCLU protein completely reversed the inhibitory effects of FIR radiation on EC migration and angiogenesis. Lastly, vascular endothelial growth factor also increased sCLU secretion in the culture medium, and wound closure and tube formation of HUVEC, which were significantly reduced by FIR radiation. Our results demonstrate a novel mechanism by which FIR radiation inhibits the proliferation, migration, and angiogenesis of HUVEC, via decreasing sCLU.

  1. Foeniculum vulgare Mill. increases cytosolic Ca(2+) concentration and inhibits store-operated Ca(2+) entry in vascular endothelial cells.

    PubMed

    Han, A Young; Lee, Hui Su; Seol, Geun Hee

    2016-12-01

    This study assessed the effects of essential oil of Foeniculum vulgare Mill. (fennel oil) and of trans-anethole, the main component of fennel oil, on extracellular Ca(2+)-induced store-operated Ca(2+) entry (SOCE) into vascular endothelial (EA) cells and their mechanisms of action. Components of fennel oil were analyzed by gas chromatography-mass spectrometry. Cytosolic Ca(2+) concentration ([Ca(2+)]c) in EA cells was determined using Fura-2 fluorescence. In the presence of extracellular Ca(2+), fennel oil significantly increased [Ca(2+)]c in EA cells; this increase was significantly inhibited by the Ca(2+) channel blockers La(3+) and nifedipine. In contrast, fennel oil induced [Ca(2+)]c was significantly lower in Ca(2+)-free solution, suggesting that fennel oil increases [Ca(2+)]c mainly by enhancing Ca(2+) influx into EA cells. [Ca(2+)]c mobilization by trans-anethole was similar to that of fennel oil. Moreover, SOCE was suppressed by fennel oil and trans-anethole. SOCE was also attenuated by lanthanum (La(3+)), a non-selective cation channel (NSC) blocker; 2-aminoethoxydiphenyl borane (2-APB), an inositol 1,4,5-triphosphate (IP3) receptor inhibitor and SOCE blocker; and U73122, an inhibitor of phospholipase C (PLC). Further, SOCE was more strongly inhibited by La(3+) plus fennel oil or trans-anethole than by La(3+) alone. These findings suggest that fennel oil and trans-anethole significantly inhibit SOCE-induced [Ca(2+)]c increase in vascular endothelial cells and that these reactions may be mediated by NSC, IP3-dependent Ca(2+) mobilization, and PLC activation.

  2. Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen.

    PubMed

    Kanasaki, Keizo; Shi, Sen; Kanasaki, Megumi; He, Jianhua; Nagai, Takako; Nakamura, Yuka; Ishigaki, Yasuhito; Kitada, Munehiro; Srivastava, Swayam Prakash; Koya, Daisuke

    2014-06-01

    Kidney fibrosis is the final common pathway of all progressive chronic kidney diseases, of which diabetic nephropathy is the leading cause. Endothelial-to-mesenchymal transition (EndMT) has emerged as one of the most important origins of matrix-producing fibroblasts. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been introduced into the market as antidiabetes drugs. Here, we found that the DPP-4 inhibitor linagliptin ameliorated kidney fibrosis in diabetic mice without altering the blood glucose levels associated with the inhibition of EndMT and the restoration of microRNA 29s. Streptozotocin-induced diabetic CD-1 mice exhibited kidney fibrosis and strong immunoreactivity for DPP-4 by 24 weeks after the onset of diabetes. At 20 weeks after the onset of diabetes, mice were treated with linagliptin for 4 weeks. Linagliptin-treated diabetic mice exhibited a suppression of DPP-4 activity/protein expression and an amelioration of kidney fibrosis associated with the inhibition of EndMT. The therapeutic effects of linagliptin on diabetic kidneys were associated with the suppression of profibrotic programs, as assessed by mRNA microarray analysis. We found that the induction of DPP-4 observed in diabetic kidneys may be associated with suppressed levels of microRNA 29s in diabetic mice; linagliptin restored microRNA 29s and suppressed DPP-4 protein levels. Using cultured endothelial cells, we found that linagliptin inhibited TGF-β2-induced EndMT, and such anti-EndMT effects of linagliptin were mediated through microRNA 29 induction. These results indicate the possible novel pleiotropic action of linagliptin to restore normal kidney function in diabetic patients with renal impairment.

  3. Sirt 1 activator inhibits the AGE-induced apoptosis and p53 acetylation in human vascular endothelial cells.

    PubMed

    Li, Peng; Zhang, Lina; Zhou, Changyong; Lin, Nan; Liu, Aiguo

    2015-01-01

    Advanced glycation end products (AGEs) by nonenzymatic glycation reactions are extremely accumulated in the diabetic vascular cells, neurons, and glia, and are confirmed to play important role in the pathogenesis of diabetes mellitus -induced cardiovascular complications. Sirt 1, known as mammalian sirtuin, has been recognized to regulate insulin secretion and protect cells against oxidative stress, which is promoted by the accumulated AGEs in cardiovascular cells. In the present study, we treated human endothelial Eahy926 cells with AGEs, and determined the apoptosis induction, caspase activation, the Sirt 1 activity, the expression and acetylation of p53. Then we manipulated Sirt 1 activity with a Sirt 1 activator, Resveratrol (RSV), and a Sirt 1 inhibitor, sirtinol, in the AGE-BSA-treated Eahy926 cells, and then re-evaluated the apoptosis induction, caspase activation, the expression and acetylation of p53. Results demonstrated that AGEs induced apoptosis in the human endothelial Eahy926 cells, by promoting the cytochrome c release, activation of caspase 9/3. Also, the AGE-BSA treatment promoted the total p53 level and acetylated (Ac) p53, but reduced the Sirt 1 level and activity. On the other hand, the Sirt 1 inhibitor/activator not only deteriorated/ameliorated the promotion to p53 level and Ac p53, but also aggravated/inhibited the AGE-induced apoptosis and the promotion to apoptosis-associated signaling molecules. In conclusion, the present study confirmed the apoptosis promotion by AGEs in endothelial Eahy926 cells, by regulating the Sirt 1 activity and p53 signaling, it also implies the protective role of Sirt 1 activator against the AGE-induced apoptosis.

  4. Secreted APE1/Ref-1 inhibits TNF-α-stimulated endothelial inflammation via thiol-disulfide exchange in TNF receptor.

    PubMed

    Park, Myoung Soo; Choi, Sunga; Lee, Yu Ran; Joo, Hee Kyoung; Kang, Gun; Kim, Cuk-Seong; Kim, Soo Jin; Lee, Sang Do; Jeon, Byeong Hwa

    2016-03-11

    Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a multifunctional protein with redox activity and is proved to be secreted from stimulated cells. The aim of this study was to evaluate the functions of extracellular APE1/Ref-1 with respect to leading anti-inflammatory signaling in TNF-α-stimulated endothelial cells in response to acetylation. Treatment of TNF-α-stimulated endothelial cells with an inhibitor of deacetylase that causes intracellular acetylation, considerably suppressed vascular cell adhesion molecule-1 (VCAM-1). During TSA-mediated acetylation in culture, a time-dependent increase in secreted APE1/Ref-1 was confirmed. The acetyl moiety of acetylated-APE1/Ref-1 was rapidly removed based on the removal kinetics. Additionally, recombinant human (rh) APE1/Ref-1 with reducing activity induced a conformational change in rh TNF-α receptor 1 (TNFR1) by thiol-disulfide exchange. Following treatment with the neutralizing anti-APE1/Ref-1 antibody, inflammatory signals via the binding of TNF-α to TNFR1 were remarkably recovered, leading to up-regulation of reactive oxygen species generation and VCAM-1, in accordance with the activation of p66(shc) and p38 MAPK. These results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits TNF-α binding to TNFR1 by reductive conformational change, with suggestion as an endogenous inhibitor of vascular inflammation.

  5. ClC-3 deficiency prevents apoptosis induced by angiotensin II in endothelial progenitor cells via inhibition of NADPH oxidase.

    PubMed

    Liu, Jing; Zhang, Fei-Fei; Li, Lei; Yang, Jing; Liu, Jie; Guan, Yong-Yuan; Du, Yan-Hua

    2013-10-01

    Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization and re-endothelialization in response to tissue ischemia and endothelial injury. It is reported that the circulating EPCs number is decreased during hypertension. However, the detailed mechanism is still unclear. Our previous studies have shown that ClC-3 chloride channel is up-regulated with the development of hypertension. This study aims to test whether ClC-3 participates in EPC apoptosis under the condition of increased oxidative stress in angiotensin II (Ang II)-induced hypertension. The results showed that stimulation with 10(-6)mol/L Ang II significantly up-regulated the endogenous ClC-3 expression and increased intracellular reactive oxygen species (ROS) generation in EPCs of wild type mice, accompanied by an enhanced NADPH oxidase activity and the expression of gp91(phox) (NOX-2), a key catalytic subunit of NADPH oxidase. However, these effects of Ang II were significantly reduced in EPCs of ClC-3(-/-) mice. Compared with control, treatment with Ang II induced EPCs apoptosis in wild type mice, concomitantly with declined Bcl-2/Bax ratio, depressed mitochondrial membrane potential and activation of poly(ADP-ribose) polymerase, which was remarkably prevented by both ClC-3 knockout and NADPH oxidase inhibitor apocynin. In addition, the role of ClC-3 deficiency in protecting EPCs against Ang II-induced oxidative stress and apoptosis was further confirmed in Ang II-infused hypertensive mice in vivo. In conclusion, ClC-3 deficiency inhibited Ang II-induced EPC apoptosis via suppressing ROS generation derived from NADPH oxidase.

  6. Influence of GSH synthesis inhibition on temporal distribution of NAD+/NADH during vascular endothelial cells proliferation.

    PubMed

    Busu, C; Atanasiu, V; Caldito, G; Aw, T Y

    2014-01-01

    Pathological conditions states such as stroke, diabetes mellitus, hypertension, dyslipidemia are associated with increased levels of free radicals that alter normal function of the vascular endothelium and perturb vascular homeostasis. The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance. Any decrease in tissue or systemic GSH levels under the aforementioned pathologies would enhance oxidative damage to the vascular endothelium. Beside their role as coenzyme that participate in cellular metabolism, pyridine nucleotides serve also as substrate for enzymes involved in DNA repair and longevity. There is scant data on NAD+/NADH kinetics and distribution during human cells proliferation. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) NAD+ and nuclear NADH kinetics (6 h interval) over 72 h of endothelial cell proliferation. The IHEC cell line was used as a surrogate for human brain micro vascular endothelial cells. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase. When BSO was removed the pattern of nuclear NAD+ resembled that of control group, but nuclear NADH concentrations remained elevated, as in GSH deficient cells (p<0.01). The coincidence of high nuclear NADH and lower nuclear NAD+ with S-phase prolongation are suggestive of CtBP and NAD+-dependent DNA repair enzyme activation under conditions of decreased cellular GSH. These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.

  7. Ethyl-p-methoxycinnamate isolated from kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-α, and angiogenesis by blocking endothelial functions

    PubMed Central

    Umar, Muhammad Ihtisham; Asmawi, Mohd Zaini; Sadikun, Amirin; Majid, Amin Malik Shah Abdul; Al-Suede, Fouad Saleih R.; Hassan, Loiy Elsir Ahmed; Altaf, Rabia; Ahamed, Mohamed B. Khadeer

    2014-01-01

    OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and

  8. Advanced glycation inhibition and protection against endothelial dysfunction induced by coumarins and procyanidins from Mammea neurophylla.

    PubMed

    Dang, Bach Tai; Gény, Charlotte; Blanchard, Patricia; Rouger, Caroline; Tonnerre, Pierre; Charreau, Béatrice; Rakolomalala, Gilbertine; Randriamboavonjy, Joseph Iharinjaka; Loirand, Gervaise; Pacaud, Pierre; Litaudon, Marc; Richomme, Pascal; Séraphin, Denis; Derbré, Séverine

    2014-07-01

    Advanced glycation end-products (AGEs) are associated with many pathogenic disorders such as pathogenesis of diabetes or endothelial dysfunction leading to cardiovascular events. Therefore, the identification of new anti-AGE molecules or extracts aims at preventing such pathologies. Many Clusiaceae and Calophyllaceae species are used in traditional medicines to treat arterial hypertension as well as diabetes. Focusing on these plant families, an anti-AGE plant screening allowed us to select Mammea neurophylla for further phytochemical and biological studies. Indeed, both DCM and MeOH stem bark extracts demonstrated in vitro their ability to prevent inflammation in endothelial cells and to reduce vasoconstriction. A bioguided fractionation of these extracts allowed us to point out 4-phenyl- and 4-(1-acetoxypropyl)coumarins and procyanidins as potent inhibitors of AGE formation, potentially preventing endothelial dysfunction. The fractionation steps also led to the isolation of two new compounds, namely neurophyllols A and B from the DCM bark extract together with thirteen known mammea A and E coumarins (mammea A/AA, mammea A/AB, mammea A/BA, mammea A/BB, mammea A/AA cycloD, mammea A/AB cycloD, disparinol B, mammea A/AB cycloE, ochrocarpin A, mammea A/AA cycloF, mammea A/AB cycloF, mammea E/BA, mammea E/BB) as well as δ-tocotrienol, xanthones (1-hydroxy-7-methoxyxanthone, 2-hydroxyxanthone) and triterpenes (friedelin and betulinic acid). During this study, R,S-asperphenamate, previously described from fungal origin was also purified.

  9. Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells.

    PubMed

    Thangjam, Gagan S; Birmpas, Charalampos; Barabutis, Nektarios; Gregory, Betsy W; Clemens, Mary Ann; Newton, Joseph R; Fulton, David; Catravas, John D

    2016-05-15

    The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation.

  10. MicroRNA-16 inhibits feto-maternal angiogenesis and causes recurrent spontaneous abortion by targeting vascular endothelial growth factor

    PubMed Central

    Zhu, Yongsheng; Lu, Hong; Huo, Zhenghao; Ma, Zhanbin; Dang, Jie; Dang, Wei; Pan, Lin; Chen, Jing; Zhong, Huijun

    2016-01-01

    Recurrent spontaneous abortion (RSA) is a common health problem that affects women of reproductive age. Recent studies have indicated that microRNAs are important factors in miscarriage. This study investigated the role of miR-16 in regulating vascular endothelial growth factor (VEGF) expression and the pathogenesis of RSA. In this report, clinical samples revealed that miR-16 expression was significantly elevated in the villi and decidua of RSA patients. In vitro, miR-16 upregulation inhibited human umbilical vein endothelial cell proliferation, migration and tube formation. Conversely, the downregulation of miR-16 reversed these effects. In vivo, we demonstrated that abnormal miR-16 levels affect the weights of the placenta and embryo and the number of progeny and microvascular density, as well as cause recurrent abortions by controlling VEGF expression in pregnant mice. VEGF, a potential target gene of miR-16, was inversely correlated with miR-16 expression in the decidua of clinical samples. Furthermore, the luciferase reporter system demonstrated that miR-16 was found to directly downregulate the expression of VEGF by binding a specific sequence of its 3′-untranslated region (3′UTR). Collectively, these data strongly suggest that miR-16 regulates placental angiogenesis and development by targeting VEGF expression and is involved in the pathogenesis of RSA. PMID:27748453

  11. Curcumin supplementation could improve diabetes-induced endothelial dysfunction associated with decreased vascular superoxide production and PKC inhibition

    PubMed Central

    2010-01-01

    Background Curcumin, an Asian spice and food-coloring agent, is known for its anti-oxidant properties. We propose that curcumin can improve diabetes-induced endothelial dysfunction through superoxide reduction. Methods Diabetes (DM) was induced in rats by streptozotocin (STZ). Daily curcumin oral feeding was started six weeks after the STZ injection. Twelve weeks after STZ injection, mesenteric arteriolar responses were recorded in real time using intravital fluorescence videomicroscopy. Superoxide and vascular protein kinase C (PKC-βII) were examined by hydroethidine and immunofluorescence, respectively. Results The dilatory response to acetylcholine (ACh) significantly decreased in DM arterioles as compared to control arterioles. There was no difference among groups when sodium nitroprusside (SNP) was used. ACh responses were significantly improved by both low and high doses (30 and 300 mg/kg, respectively) of curcumin supplementation. An oxygen radical-sensitive fluorescent probe, hydroethidine, was used to detect intracellular superoxide anion (O2●-) production. O2●- production was markedly increased in DM arterioles, but it was significantly reduced by supplementation of either low or high doses of curcumin. In addition, with a high dose of curcumin, diabetes-induced vascular PKC-βII expression was diminished. Conclusion Therefore, it is suggested that curcumin supplementation could improve diabetes-induced endothelial dysfunction significantly in relation to its potential to decrease superoxide production and PKC inhibition. PMID:20946622

  12. Chronic Embolic Pulmonary Hypertension Caused by Pulmonary Embolism and Vascular Endothelial Growth Factor Inhibition.

    PubMed

    Neto-Neves, Evandro M; Brown, Mary B; Zaretskaia, Maria V; Rezania, Samin; Goodwill, Adam G; McCarthy, Brian P; Persohn, Scott A; Territo, Paul R; Kline, Jeffrey A

    2017-04-01

    Our understanding of the pathophysiological basis of chronic thromboembolic pulmonary hypertension (CTEPH) will be accelerated by an animal model that replicates the phenotype of human CTEPH. Sprague-Dawley rats were administered a combination of a single dose each of plastic microspheres and vascular endothelial growth factor receptor antagonist in polystyrene microspheres (PE) + tyrosine kinase inhibitor SU5416 (SU) group. Shams received volume-matched saline; PE and SU groups received only microspheres or SU5416, respectively. PE + SU rats exhibited sustained pulmonary hypertension (62 ± 13 and 53 ± 14 mmHg at 3 and 6 weeks, respectively) with reduction of the ventriculoarterial coupling in vivo coincident with a large decrement in peak rate of oxygen consumption during aerobic exercise, respectively. PE + SU produced right ventricular hypokinesis, dilation, and hypertrophy observed on echocardiography, and 40% reduction in right ventricular contractile function in isolated perfused hearts. High-resolution computed tomographic pulmonary angiography and Ki-67 immunohistochemistry revealed abundant lung neovascularization and cellular proliferation in PE that was distinctly absent in the PE + SU group. We present a novel rodent model to reproduce much of the known phenotype of CTEPH, including the pivotal pathophysiological role of impaired vascular endothelial growth factor-dependent vascular remodeling. This model may reveal a better pathophysiological understanding of how PE transitions to CTEPH in human treatments.

  13. Fucoidan inhibits lymphangiogenesis by downregulating the expression of VEGFR3 and PROX1 in human lymphatic endothelial cells.

    PubMed

    Yang, Yazong; Gao, Zixiang; Ma, Yanhong; Teng, Hongming; Liu, Zundong; Wei, Hengyun; Lu, Yanbing; Cheng, Xiaofang; Hou, Lin; Zou, Xiangyang

    2016-06-21

    Lymphangiogenesis is one of the promoters of tumor lymphatic metastasis. Fucoidan which is a fucose-enriched sulfated polysaccharide has effect on various pharmacological activities including anti-metastasis activity. However, the inhibitory effect of fucoidan on lymphangiogenesis remains unclear. Here, fucoidan extracted from U. pinnatifida sporophylls suppressed HLECs proliferation, migration and tube-like structure formation, and had inhibitory effect of tumor-induced lymphangiogenesis in vitro. Additionally, we found that fucoidan had a dose-dependent depressive effect on the expressions of PROX1, vascular endothelial growth factor receptor 3 (VEGFR3), NF-κB, phospho-PI3K and phospho-Akt in HLECs. Moreover, anti-lymphangiogenesis effect of fucoidan was assessed by using mouse tumor model. In summary, fucoidan inhibit tumor lymphangiogenesis and lymphatic metastasis by suppressing the NF-κB/PI3K/Akt signaling pathway through reduced levels of PROX1 and VEGFR3.

  14. Fucoidan inhibits lymphangiogenesis by downregulating the expression of VEGFR3 and PROX1 in human lymphatic endothelial cells

    PubMed Central

    Yang, Yazong; Gao, Zixiang; Ma, Yanhong; Teng, Hongming; Liu, Zundong; Wei, Hengyun; Lu, Yanbing; Cheng, Xiaofang; Hou, Lin; Zou, Xiangyang

    2016-01-01

    Lymphangiogenesis is one of the promoters of tumor lymphatic metastasis. Fucoidan which is a fucose-enriched sulfated polysaccharide has effect on various pharmacological activities including anti-metastasis activity. However, the inhibitory effect of fucoidan on lymphangiogenesis remains unclear. Here, fucoidan extracted from U. pinnatifida sporophylls suppressed HLECs proliferation, migration and tube-like structure formation, and had inhibitory effect of tumor-induced lymphangiogenesis in vitro. Additionally, we found that fucoidan had a dose-dependent depressive effect on the expressions of PROX1, vascular endothelial growth factor receptor 3 (VEGFR3), NF-κB, phospho-PI3K and phospho-Akt in HLECs. Moreover, anti-lymphangiogenesis effect of fucoidan was assessed by using mouse tumor model. In summary, fucoidan inhibit tumor lymphangiogenesis and lymphatic metastasis by suppressing the NF-κB/PI3K/Akt signaling pathway through reduced levels of PROX1 and VEGFR3. PMID:27203545

  15. A standardized bamboo leaf extract inhibits monocyte adhesion to endothelial cells by modulating vascular cell adhesion protein-1

    PubMed Central

    Choi, Sunga; Park, Myoung Soo; Lee, Yu Ran; Lee, Young Chul; Kim, Tae Woo; Do, Seon-Gil; Kim, Dong Seon

    2013-01-01

    Bamboo leaves (Phyllostachys pubescens Mazel ex J. Houz (Poacea)) have a long history of food and medical applications in Asia, including Japan and Korea. They have been used as a traditional medicine for centuries. We investigated the mechanism of anti-inflammatory activity of a bamboo leaf extract (BLE) on tumor necrosis factor-alpha (TNF-α)-induced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to BLE did not inhibit cell viability or cause morphological changes at concentrations ranging from 1 µg/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-α-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-α-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-α-activated HUVECs. BLE (0.05 mg/ml) also caused about 50% inhibition of interleukin-6 secretion from lipopolysaccharide-stimulated monocyte. The results indicate that BLE may be clinically useful as an anti-inflammatory or anti-oxidant for human cardiovascular disease including atherosclerosis. PMID:23422838

  16. 5'-nitro-indirubinoxime inhibits inflammatory response in TNF-alpha stimulated human umbilical vein endothelial cells.

    PubMed

    Kim, Eun-Jung; Park, Won-Hwan; Ahn, Sang-Gun; Yoon, Jung-Hoon; Kim, Si-Wouk; Kim, Soo-A

    2010-07-01

    Inflammation plays a critical role in the development of atherosclerosis and TNF-alpha, a major inflammatory cytokine, induces inflammatory responses by enhancing the expression of adhesion molecules and the secretion of inflammatory mediators. Indirubin is an active compound of Polygonum tinctorium Lour (P. tinctorium) that has the ability to suppress inflammation. Previously, we described the novel indirubin derivative, 5'-nitro-indirubinoxime (5'-NIO), and demonstrated that it has potent anti-proliferative activity against various human cancer cells. In this study, we examined the effect of 5'-NIO on the TNF-alpha induced inflammatory conditions of human umbilical vein endothelial cells (HUVECs). We found that 5'-NIO inhibited TNF-alpha induced MCP-1 and IL-8 expression at the RNA and protein levels in HUVECs. Specifically, 5'-NIO significantly inhibited the TNF-alpha stimulated release of MCP-1 and IL-8, with levels that were only 19.8% and 30.9% of those of untreated control cells, respectively. Furthermore, 5'-NIO largely inhibited the adhesion of U937 cells to HUVECs by decreasing the expression level of ICAM-1 and VCAM-1. Overall, these observations suggest that 5'-NIO has the potential for use as an anti-atherosclerotic agent.

  17. The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.

    PubMed Central

    Yoshizumi, M; Hsieh, C M; Zhou, F; Tsai, J C; Patterson, C; Perrella, M A; Lee, M E

    1995-01-01

    Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition. PMID:7760822

  18. Heme oxygenase-1 inhibits the expression of adhesion molecules associated with endothelial cell activation via inhibition of NF-kappaB RelA phosphorylation at serine 276.

    PubMed

    Seldon, Mark P; Silva, Gabriela; Pejanovic, Nadja; Larsen, Rasmus; Gregoire, Isabel Pombo; Filipe, Josina; Anrather, Josef; Soares, Miguel P

    2007-12-01

    Heme oxygenase-1 (HO-1; encoded by the Hmox1 gene) catalyzes the degradation of free heme into biliverdin, via a reaction that releases iron (Fe) and carbon monoxide. We report that HO-1 down-regulates the proinflammatory phenotype associated with endothelial cell (EC) activation by reducing intracellular nonprotein-bound Fe (labile Fe). EC isolated from Hmox1(-/-) mice have higher levels of intracellular labile Fe and reactive oxygen species (ROS) as compared with EC isolated from Hmox1(+/+) mice. Basal and TNF-induced expression of VCAM-1, ICAM-1, and E-selectin were increased in Hmox1(-/-) vs Hmox1(+/+) EC, an effect reversed by Fe chelation using deferoxamine mesylate (DFO). Fe chelation inhibits TNF-driven transcription of Vcam-1, Icam-1, and E-selectin, as assessed using luciferase reporter assays. This effect is associated with inhibition of the transcription factor NF-kappaB via a mechanism that is not associated with the inhibition of IkappaBalpha phosphorylation/degradation or NF-kappaB (i.e., RelA) nuclear translocation, although it affects very modestly NF-kappaB binding to DNA kappaB consensus sequences in the Vcam-1 and E-selectin promoters. HO-1 inhibits NF-kappaB (i.e., RelA) phosphorylation at Ser(276), a phosphoacceptor that is critical to sustain TNF-driven NF-kappaB activity in EC. This effect was mimicked by Fe chelation as well as by antioxidants (N-acetylcysteine). In conclusion, we demonstrate a novel mechanism via which HO-1 down-modulates the proinflammatory phenotype of activated EC, i.e., the inhibition of RelA phosphorylation at Ser(276).

  19. Cimetidine inhibits the adhesion of gastric cancer cells expressing high levels of sialyl Lewis x in human vascular endothelial cells by blocking E-selectin expression.

    PubMed

    Liu, Fu-Rong; Jiang, Cheng-Gang; Li, Yan-Shu; Li, Jia-Bin; Li, Feng

    2011-04-01

    Cimetidine has been shown to have anti-metastatic activity and improves the survival of patients with colorectal cancer. One hypothesis is its modulation of the expression of the cell adhesion molecule by target organ endothelial cells. Because of the inconclusive results in clinical trials of gastric cancer, we investigated the effects of cimetidine on the adhesion of gastric cancer cells to activated endothelial cells and on the expression of some cell adhesion molecules. Human endothelial cells were pre-incubated with cimetidine for 6 h, incubated with the cytokine tumor necrosis factor for 4 h, and the endothelial surface expression of E-selectin was evaluated by flow cytometry, immunostaining and ELISA. Further, we investigated E-selectin mRNA expression by RT-PCR. Three gastric cancer cell lines (SGC-7901, MGC-803, BGC-823) and a normal gastric epithelial cell line, GES-1, were studied for the surface expression of sialyl Lewis x by flow cytometry and immunostaining. Adherence of CFSE-labeled gastric cancer cells and GES-1 cells to endothelial cell monolayers was determined. Cimetidine significantly reduced E-selectin expression of activated endothelial cells, but did not influence E-selectin expression at the mRNA level. Three gastric cancer cell lines expressed high levels of sialyl Lewis x, whereas GES-1 did not. Cimetidine also significantly decreased gastric cancer cell adherence to stimulated endothelial cells. The inhibition of E-selectin expression corresponded to the reduction of tumor cell adherence. The effects of cimetidine on tumor adhesion were almost nullified by pre-incubation with E-selectin and sialyl Lewis x antibody. Furthermore, there was no significant change of GES-1 adherence to endothelial cells by TNF-α, cimetidine, E-selectin and sialyl Lewis x antibody. The inhibiton of gastric cancer cell adherence to cytokine-stimulated endothelial cells treated with cimetidine appears to result from blocking endothelial E-selectin expression

  20. Arginase-2 is cooperatively up-regulated by nitric oxide and histone deacetylase inhibition in human umbilical artery endothelial cells.

    PubMed

    Krause, Bernardo J; Hernandez, Cherie; Caniuguir, Andres; Vasquez-Devaud, Paola; Carrasco-Wong, Ivo; Uauy, Ricardo; Casanello, Paola

    2016-01-01

    Arginase-2 counteracts endothelial nitric oxide synthase (eNOS) activity in human endothelium, and its expression is negatively controlled by histone deacetylase (HDAC2). Conversely NO inhibits HDAC and previous studies suggest that arginase-2 is up-regulated by NO. We studied whether NO regulates arginase-2 expression in umbilical artery endothelial cells (HUAEC) increasing ARG2 promoter accessibility. HUAEC exposed to NOC-18 (NO donor, 1-100 μM, 0-24 h) showed an increase in arginase-2 but a decrease in eNOS mRNA levels in a time-dependent manner, with a maximal effect at 100 μM (24 h). Conversely NOS inhibition with L-NAME (100 μM) reduced arginase-2 mRNA and protein levels, an effect reverted by co-incubation with NOC-18. Treatment with TSA paralleled the effects of NO on arginase-2 and eNOS at mRNA and protein levels, with maximal effect at 10 μM. Co-incubation of NOC-18 (100 μM) with a sub-maximal concentration of TSA (1 μM) potentiated the increase in arginase-2 mRNA levels, whilst L-NAME prevented TSA-dependent arginase-2 induction. The effects on arginase-2 mRNA were paralleled by changes in chromatin accessibility, as well as increased levels of H3K9 and H4K12 acetylation, at ARG2 proximal (-579 to -367 and -280 to -73 bp from TSS) and core (-121 to +126 bp from TSS) promoter. Finally NO-dependent arginase-2 induction was prevented by pre-incubation for 10 min with the cysteine blocker MMTS (10 mM). These data showed for the first time that NO up-regulates arginase-2 expression in primary cultured human endothelial cells by an epigenetic-mediated mechanism increasing ARG2 promoter accessibility suggesting a negative regulatory loop for eNOS activity.

  1. Activation of protein kinase C inhibits potassium currents in cultured endothelial cells.

    PubMed

    Zhang, H; Weir, B; Daniel, E E

    1995-04-01

    The effect of protein kinase C on potassium channels in cultured endothelial cells was investigated by using whole-cell patch-clamp techniques. Activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not phorbol 12-monomyristate (PMM), an inactive analogue of phorbol esters, depressed an outward calcium-dependent potassium current. The inhibitory actions of PMA and PDBu could be reversed by the kinase inhibitor H-7. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum calcium pump, and LP-805, a novel vasodilator which also releases endothelium-derived relaxing factors, activated the outward calcium-dependent potassium conductance. PMA and PDBu, but not PMM, reduced the outward conductance induced by cyclopiazonic acid and LP-805. These effects of PMA and PDBu on potassium currents may be mediated either by phosphorylation of ion channels, or by decreasing intracellular calcium concentration.

  2. Estradiol inhibits vascular endothelial cells pro-inflammatory activation induced by C-reactive protein.

    PubMed

    Cossette, Émilie; Cloutier, Isabelle; Tardif, Kim; DonPierre, Geneviève; Tanguay, Jean-François

    2013-01-01

    In addition of being an important inflammatory biomarker and a risk factor for cardiovascular disease, much evidence indicates that the C-reactive protein (CRP) contributes to the atherosclerosis development process. This plasmatic protein synthesized by hepatocytes in response to inflammation and tissue injury induces pro-inflammatory molecules' expression by endothelial cells (ECs). Previous studies showed that the 17β-estradiol (E2) has beneficial effects on vascular cells by reducing in vitro pro-inflammatory molecules expressions in EC. Therefore, we hypothesize that E2 blocks or reduces CRP-mediated inflammatory responses by modulating endogenous production of CRP in EC and/or activation mechanisms. Using human aortic ECs (HAECs), we first evaluated CRP production by vascular EC and second demonstrated its self-induction. Indeed, recombinant human CRP stimulation induces a fivefold increase of CRP expression. A 1-h pre-treatment of E2 at a physiologic dose (10(-9 )M) leads to an important decrease of CRP production suggesting a partial blockage of its amplification loop mechanism. Furthermore, in HAEC, E2 reduces the secretion of the most potent agonist of CRP induction, the IL-6, by 21 %. E2 pre-treatment also decreased the expression of pro-inflammatory molecules IL-8, VCAM-1, and ICAM-1 induced by CRP and involved in leukocytes recruitment. In addition, we demonstrated that E2 could restore vascular endothelial growth factor-mediated EC migration response impaired by CRP suggesting another pro-angiogenic property of this hormone. These findings suggest that E2 can interfere with CRP pro-inflammatory effects via activation signals using its rapid, non-genomic pathway that may provide a new mechanism to improve vascular repair.

  3. eNOS-dependent S-nitrosylation of β-catenin prevents its association with TCF4 and inhibits proliferation of endothelial cells by Wnt3a.

    PubMed

    Zhang, Ying; Chidiac, Rony; Delisle, Chantal; Gratton, Jean-Philippe

    2017-03-20

    Nitric oxide (NO) produced by endothelial NO synthase (eNOS) modulates many functions in endothelial cells. S-nitrosylation (SNO) of cysteine residues on β-catenin by eNOS-derived NO has been shown to influence intercellular contacts between endothelial cells. However, the implication of SNO in the regulation of β-catenin transcriptional activity is ill-defined. Here we report that NO inhibits the transcriptional activity of β-catenin and endothelial cell proliferation induced by activation of Wnt/β-catenin signaling. Interestingly, induction by Wnt3a of β-catenin target genes, such as Axin2, is repressed in an eNOS-dependent manner by VEGF. We identify Cys466 of β-catenin as a target for SNO by eNOS-derived NO and as the critical residue for the repressive effects of NO on β-catenin transcriptional activity. Furthermore, we observed that Cys466 of β-catenin, located at the binding interface of the β-catenin/TCF4 transcriptional complex, is essential for disruption of this complex by NO. Importantly, Cys466 of β-catenin is necessary for the inhibitory effects of NO on Wnt3a-stimulated proliferation of endothelial cells. Thus our data define the mechanism responsible for the repressive effects of NO on the transcriptional activity of β-catenin and link eNOS-derived NO to the modulation by VEGF of Wnt/β-catenin-induced endothelial cell proliferation.

  4. Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (-)-Epigallocatechin Gallate.

    PubMed

    Tian, Rong; Ding, Yun; Peng, Yi-Yuan; Lu, Naihao

    2017-04-10

    Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate an inverse correlation between plant polyphenol consumption and the incidence of cardiovascular diseases. Here we showed that (-)-epigallocatechin gallate (EGCG), the main flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 μM EGCG versus 100 ± 5.6% for control, P < 0.01). UV-vis spectral and docking studies indicated that EGCG bound to the active site (heme) of MPO and resulted in the accumulation of compound II, which was unable to produce HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl formation: 65.0 ± 5.6% for 20 μM EGCG versus 100 ± 6.2% for control, P < 0.01) without influencing MPO and Nox2 release and superoxide formation, suggesting that EGCG specifically inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced injury (P < 0.05, all cases), although it did not induce cytotoxicity to HUVEC (P > 0.05, all cases). Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity and may participate in the regulation of immune responses at inflammatory sites.

  5. Calcium Dobesilate Inhibits the Alterations in Tight Junction Proteins and Leukocyte Adhesion to Retinal Endothelial Cells Induced by Diabetes

    PubMed Central

    Leal, Ermelindo C.; Martins, João; Voabil, Paula; Liberal, Joana; Chiavaroli, Carlo; Bauer, Jacques; Cunha-Vaz, José; Ambrósio, António F.

    2010-01-01

    OBJECTIVE Calcium dobesilate (CaD) has been used in the treatment of diabetic retinopathy in the last decades, but its mechanisms of action are not elucidated. CaD is able to correct the excessive vascular permeability in the retina of diabetic patients and in experimental diabetes. We investigated the molecular and cellular mechanisms underlying the protective effects of CaD against the increase in blood–retinal barrier (BRB) permeability induced by diabetes. RESEARCH DESIGN AND METHODS Wistar rats were divided into three groups: controls, streptozotocin-induced diabetic rats, and diabetic rats treated with CaD. The BRB breakdown was evaluated using Evans blue. The content or distribution of tight junction proteins (occludin, claudin-5, and zonula occluden-1 [ZO-1]), intercellular adhesion molecule-1 (ICAM-1), and p38 mitogen-activated protein kinase (p38 MAPK) was evaluated by Western blotting and immunohistochemistry. Leukocyte adhesion was evaluated in retinal vessels and in vitro. Oxidative stress was evaluated by the detection of oxidized carbonyls and tyrosine nitration. NF-κB activation was measured by enzyme-linked immunosorbent assay. RESULTS Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased occludin and claudin-5 levels and altered the distribution of ZO-1 and occludin in retinal vessels. These changes were inhibited by CaD treatment. CaD also inhibited the increase in leukocyte adhesion to retinal vessels or endothelial cells and in ICAM-1 levels, induced by diabetes or elevated glucose. Moreover, CaD decreased oxidative stress and p38 MAPK and NF-κB activation caused by diabetes. CONCLUSIONS CaD prevents the BRB breakdown induced by diabetes, by restoring tight junction protein levels and organization and decreasing leukocyte adhesion to retinal vessels. The protective effects of CaD are likely to involve the inhibition of p38 MAPK and NF-κB activation, possibly through the inhibition of oxidative

  6. The aryl hydrocarbon receptor (AhR) inhibits vanadate-induced vascular endothelial growth factor (VEGF) production in TRAMP prostates

    PubMed Central

    Fritz, Wayne A.; Lin, Tien-Min; Peterson, Richard E.

    2008-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) are basic helix-loop-helix/per-arnt-sim (PAS) family transcription factors. During angiogenesis and tumor growth, HIF-1α dimerizes with ARNT, inducing expression of many genes, including vascular endothelial growth factor (VEGF). ARNT also dimerizes with the aryl hydrocarbon receptor (AhR). AhR-null (Ahr−/−) transgenic adenocarcinoma of the mouse prostate (TRAMP) mice develop prostate tumors with greater frequency than AhR wild-type (Ahr+/+) TRAMP mice, even though prevalence of prostate epithelial hyperplasia is not inhibited. This suggests that Ahr inhibits prostate carcinogenesis. In TRAMP mice, prostatic epithelial hyperplasia results in stabilized HIF-1α, inducing expression of VEGF, a prerequisite for tumor growth and angiogenesis. Since ARNT is a common dimerization partner of AhR and HIF-1α, we hypothesized that the AhR inhibits prostate tumor formation by competing with HIF-1α for ARNT, thereby limiting VEGF production. Prostates from Ahr+/+, Ahr+/− and Ahr−/− C57BL/6J TRAMP mice were cultured in the presence of graded concentrations of vanadate, an inducer of VEGF through the HIF-1α–ARNT pathway. Vanadate induced VEGF protein in a dose-dependent fashion in Ahr+/− and Ahr−/− TRAMP cultures, but not in Ahr+/+ cultures. However, vanadate induced upstream proteins in the phosphatidylinositol 3-kinase-signaling cascade to a similar extent in TRAMPs of each Ahr genotype, evidenced by v-akt murine thymoma viral oncogene homolog (Akt) phosphorylation. These findings suggest that AhR sequesters ARNT, decreasing interaction with HIF-1α reducing VEGF production. Since VEGF is required for tumor vascularization and growth, these studies further suggest that reduction in VEGF correlates with inhibited prostate carcinogenesis in Ahr+/+ TRAMP mice. PMID:18359762

  7. Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells

    PubMed Central

    Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

    2017-01-01

    Background Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. Material/Methods We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). Results We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. Conclusions Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer. PMID:28087861

  8. Lung endothelial barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced mitochondrial oxidative damage via an Nrf2-dependent mechanism.

    PubMed

    Dong, Wen-Wen; Liu, Yu-Jian; Lv, Zhou; Mao, Yan-Fei; Wang, Ying-Wei; Zhu, Xiao-Yan; Jiang, Lai

    2015-11-01

    High-mobility group box 1 (HMGB1) contributes to lung vascular hyperpermeability during ventilator-induced lung injury. We aimed to determine whether the natural antioxidant resveratrol protected against HMGB1-induced endothelial hyperpermeability both in vitro and in vivo. We found that HMGB1 decreased vascular endothelial (VE)-cadherin expression and increased endothelial permeability, leading to mitochondrial oxidative damage in primary cultured mouse lung vascular endothelial cells (MLVECs). Both the mitochondrial superoxide dismutase 2 mimetic MnTBAP and resveratrol blocked HMGB1-induced mitochondrial oxidative damage, VE-cadherin downregulation, and endothelial hyperpermeability. In in vivo studies, anesthetized male ICR mice were ventilated for 4h using low tidal volume (6 ml/kg) or high tidal volume (HVT; 30 ml/kg) ventilation. The mice were injected intraperitoneally with resveratrol immediately before the onset of ventilation. We found that resveratrol attenuated HVT-associated lung vascular hyperpermeability and HMGB1 production. HVT caused a significant increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and Nrf2 target gene expression in lung tissues, which was further enhanced by resveratrol treatment. HMGB1 had no effect on Nrf2 activation, whereas resveratrol treatment activated the Nrf2 signaling pathway in HMGB1-treated MLVECs. Moreover, Nrf2 knockdown reversed the inhibitory effects of resveratrol on HMGB1-induced mitochondrial oxidative damage and endothelial hyperpermeability. The inhibitory effect of resveratrol on cyclic stretch-induced HMGB1 mRNA expression in primary cultured MLVECs was also abolished by Nrf2 knockdown. In summary, this study demonstrates that resveratrol protects against lung endothelial barrier dysfunction initiated by HVT. Lung endothelial barrier protection by resveratrol involves inhibition of mechanical stretch-induced HMGB1 release and HMGB1-induced mitochondrial oxidative damage

  9. Thiazolidinediones enhance vascular endothelial growth factor expression and induce cell growth inhibition in non-small-cell lung cancer cells

    PubMed Central

    2010-01-01

    Background It is known that thiazolidinediones are involved in regulating the expression of various genes, including the vascular endothelial growth factor (VEGF) gene via peroxisome proliferator-activated receptor γ (PPARγ); VEGF is a prognostic biomarker for non-small-cell lung cancer (NSCLC). Methods In this study, we investigated the effects of troglitazone and ciglitazone on the mRNA expression of VEGF and its receptors in human NSCLC cell lines, RERF-LC-AI, SK-MES-1, PC-14, and A549. These mRNA expressions were evaluated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. We also studied the effect of Je-11, a VEGF inhibitor, on the growth of these cells. Results In NSCLC cells, thiazolidinediones increased the mRNA expression of VEGF and neuropilin-1, but not that of other receptors such as fms-like tyrosine kinase and kinase insert domain receptor-1. Furthermore, the PPARγ antagonist GW9662 completely reversed this thiazolidinedione-induced increase in VEGF expression. Furthermore, the addition of VEGF inhibitors into the culture medium resulted in the reversal of thiazolidinedione-induced growth inhibition. Conclusions Our results indicated that thiazolidinediones enhance VEGF and neuropilin-1 expression and induce the inhibition of cell growth. We propose the existence of a pathway for arresting cell growth that involves the interaction of thiazolidinedione-induced VEGF and neuropilin-1 in NSCLC. PMID:20214829

  10. Salmon-derived thrombin inhibits development of chronic pain through an endothelial barrier protective mechanism dependent on APC

    PubMed Central

    Smith, Jenell R; Galie, Peter A; Slochower, David R; Weisshaar, Christine L.; Janmey, Paul A; Winkelstein, Beth A

    2015-01-01

    Many neurological disorders are initiated by blood-brain barrier breakdown, which potentiates spinal neuroinflammation and neurodegeneration. Peripheral neuropathic injuries are known to disrupt the blood-spinal cord barrier (BSCB) and to potentiate inflammation. But, it is not known whether BSCB breakdown facilitates pain development. In this study, a neural compression model in the rat was used to evaluate relationships among BSCB permeability, inflammation and pain-related behaviors. BSCB permeability increases transiently only after injury that induces mechanical hyperalgesia, which correlates with serum concentrations of pro-inflammatory cytokines, IL-7, IL-12, IL-1α and TNF-α. Mammalian thrombin dually regulates vascular permeability through PAR1 and activated protein C (APC). Since thrombin protects vascular integrity through APC, directing its affinity towards protein C, while still promoting coagulation, might be an ideal treatment for BSCB-disrupting disorders. Salmon thrombin, which prevents the development of mechanical allodynia, also prevents BSCB breakdown after neural injury and actively inhibits TNF-α-induced endothelial permeability in vitro, which is not evident the case for human thrombin. Salmon thrombin’s production of APC faster than human thrombin is confirmed using a fluorogenic assay and APC is shown to inhibit BSCB breakdown and pain-related behaviors similar to salmon thrombin. Together, these studies highlight the impact of BSCB on pain and establish salmon thrombin as an effective blocker of BSCB, and resulting nociception, through its preferential affinity for protein C. PMID:26708087

  11. Induction of heme oxygenase 1 by arsenite inhibits cytokine-induced monocyte adhesion to human endothelial cells

    SciTech Connect

    Sun Xi; Pi Jingbo; Liu Wenlan; Hudson, Laurie G.; Liu Kejian; Feng Changjian

    2009-04-15

    Heme oxygenase-1 (HO-1) is an oxidative stress responsive gene upregulated by various physiological and exogenous stimuli. Arsenite, as an oxidative stressor, is a potent inducer of HO-1 in human and rodent cells. In this study, we investigated the mechanistic role of arsenite-induced HO-1 in modulating tumor necrosis factor {alpha} (TNF-{alpha}) induced monocyte adhesion to human umbilical vein endothelial cells (HUVEC). Arsenite pretreatment, which upregulated HO-1 in a time- and concentration-dependent manner, inhibited TNF-{alpha}-induced monocyte adhesion to HUVEC and intercellular adhesion molecule 1 protein expression by 50% and 40%, respectively. Importantly, knockdown of HO-1 by small interfering RNA abolished the arsenite-induced inhibitory effects. These results indicate that induction of HO-1 by arsenite inhibits the cytokine-induced monocyte adhesion to HUVEC by suppressing adhesion molecule expression. These findings established an important mechanistic link between the functional monocyte adhesion properties of HUVEC and the induction of HO-1 by arsenite.

  12. Inhibition of TNF-alpha induced cell death in human umbilical vein endothelial cells and Jurkat cells by protocatechuic acid.

    PubMed

    Zhou-Stache, J; Buettner, R; Artmann, G; Mittermayer, C; Bosserhoff, A K

    2002-11-01

    The Chinese herb radix Salviae miltiorrhizae (RSM) is used in traditional Chinese medicine as a treatment for cardiovascular and cerebrovascular diseases. Several components of the plant extract from salvia mitorrhiza bunge have been determined previously, one of which is protocatechuic acid (PAC). It has been found, in the study, that PAC inhibited TNF-alpha-induced cell death of human umbilical vein endothelial cells (HUVECs) and Jurkat cells in a concentration of 100 microM when applied 2 h prior to TNF-alpha exposure. Molecular studies revealed that PAC activated NF-kappaB with a maximum effect after 30 min of treatment. Inhibition of NF-kappaB action by MG132 and NF-kappaB inhibitory peptide suppressed the cell-protective effect of PAC. Further, degradation of IkBalpha occurred in response to PAC treatment. The results provide evidence that activation of NF-kappaB plays an important role in mediating the cell-protecting effect of PAC on HUVECs and Jurkat cells. Further studies are required to test whether PAC, a component of radix salviae miltiorrhizae, could be useful in preventing in vivo cell death resulting from cardiovascular or cerebrovascular diseases.

  13. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    SciTech Connect

    Pourgholami, Mohammad H.; Khachigian, Levon M.; Fahmy, Roger G.; Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson

    2010-07-09

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  14. Inhibition of Plasmodium falciparum Field Isolates-Mediated Endothelial Cell Apoptosis by Fasudil: Therapeutic Implications for Severe Malaria

    PubMed Central

    Zang-Edou, Estelle S.; Bisvigou, Ulrick; Taoufiq, Zacharie; Lékoulou, Faustin; Lékana-Douki, Jean Bernard; Traoré, Yves; Mazier, Dominique; Touré-Ndouo, Fousseyni S.

    2010-01-01

    Plasmodium falciparum infection can abruptly progress to severe malaria, a life-threatening complication resulting from sequestration of parasitized red blood cells (PRBC) in the microvasculature of various organs such as the brain and lungs. PRBC adhesion can induce endothelial cell (EC) activation and apoptosis, thereby disrupting the blood-brain barrier. Moreover, hemozoin, the malarial pigment, induces the erythroid precursor apoptosis. Despite the current efficiency of antimalarial drugs in killing parasites, severe malaria still causes up to one million deaths every year. A new strategy targeting both parasite elimination and EC protection is urgently needed in the field. Recently, a rho-kinase inhibitior Fasudil, a drug already in clinical use in humans for cardio- and neuro-vascular diseases, was successfully tested on laboratory strains of P. falciparum to protect and to reverse damages of the endothelium. We therefore assessed herein whether Fasudil would have a similar efficiency on P. falciparum taken directly from malaria patients using contact and non-contact experiments. Seven (23.3%) of 30 PRBC preparations from different patients were apoptogenic, four (13.3%) acting by cytoadherence and three (10%) via soluble factors. None of the apoptogenic PRBC preparations used both mechanisms indicating a possible mutual exclusion of signal transduction ligand. Three PRBC preparations (42.9%) induced EC apoptosis by cytoadherence after 4 h of coculture (“rapid transducers”), and four (57.1%) after a minimum of 24 h (“slow transducers”). The intensity of apoptosis increased with time. Interestingly, Fasudil inhibited EC apoptosis mediated both by cell-cell contact and by soluble factors but did not affect PRBC cytoadherence. Fasudil was found to be able to prevent endothelium apoptosis from all the P. falciparum isolates tested. Our data provide evidence of the strong anti-apoptogenic effect of Fasudil and show that endothelial cell-P. falciparum

  15. Radiosensitization of Human Vascular Endothelial Cells Through Hsp90 Inhibition With 17-N-Allilamino-17-Demethoxygeldanamycin

    SciTech Connect

    Kabakov, Alexander E. Makarova, Yulia M.; Malyutina, Yana V.

    2008-07-01

    Purpose: In addition to invasive tumor cells, endothelial cells (ECs) of the tumor vasculature are an important target for anticancer radiotherapy. The purpose of the present work is to investigate how 17-N-allilamino-17-demethoxygeldanamycin (17AAG), known as an anticancer drug inhibiting heat shock protein 90 (Hsp90), modifies radiation responses of human vascular ECs. Methods and Materials: The ECs cultured from human umbilical veins were exposed to {gamma}-irradiation, whereas some EC samples were pretreated with growth factors and/or 17AAG. Postirradiation cell death/survival and morphogenesis were assessed by means of terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick end labeling or annexin V staining and clonogenic and tube-formation assays. The 17AAG-affected expression and phosphorylation of radioresistance-related proteins were probed by means of immunoblotting. Dominant negative or constitutively activated Akt was transiently expressed in ECs to manipulate Akt activity. Results: It was found that nanomolar concentrations of 17AAG sensitize ECs to relatively low doses (2-6 Gy) of {gamma}-irradiation and abolish the radioprotective effects of vascular endothelial growth factor and basic fibroblast growth factor. The drug-induced radiosensitization of ECs seems to be caused by prevention of Hsp90-dependent phosphorylation (activation) of Akt that results in blocking the radioprotective phosphatidylinositol 3-kinase/Akt pathway. Conclusions: Clinically achievable concentrations of 17AAG can decrease the radioresistance intrinsic to vascular ECs and minimize the radioprotection conferred upon them by tumor-derived growth factors. These findings characterize 17AAG as a promising radiosensitizer for the tumor vasculature.

  16. EETs alleviate ox-LDL-induced inflammation by inhibiting LOX-1 receptor expression in rat pulmonary arterial endothelial cells.

    PubMed

    Jiang, Jun-xia; Zhang, Shui-juan; Liu, Ya-nan; Lin, Xi-xi; Sun, Yan-hong; Shen, Hui-juan; Yan, Xiao-feng; Xie, Qiang-min

    2014-03-15

    Oxidized low-density lipoprotein (Ox-LDL) is associated with atherosclerotic events through the modulation of arachidonic acid (AA) metabolism and activation of inflammatory signaling. Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) mitigate inflammation through nuclear factor-κB (NF-κB). In this study, we explored the effects and mechanisms of exogenous EETs on the ox-LDL-induced inflammation of pulmonary artery endothelial cells (PAECs), which were cultured from rat pulmonary arteries. We determined that pre-treatment with 11,12-EET or 14,15-EET attenuated the ox-LDL-induced expression and release of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1) in a concentration-dependent manner. In addition, the ox-LDL-induced expression of CYP2J4 was upregulated by 11,12-EET and 14,15-EET (1μM). Furthermore, the endothelial receptor of lectin-like oxidized low-density lipoprotein (LOX-1) was downregulated in PAECs treated with EETs. The inflammatory responses evoked by ox-LDL (100μg/mL) were blocked by pharmacological inhibitors of Erk1/2 mitogen-activated protein kinase (MAPK) (U0126), p38 MAPK (SB203580), and NF-κB (PDTC). In addition, we confirmed that 11,12-EET suppresses phosphorylation of p38, degradation of IκBα, and activation of NF-κB (p65), whereas 14,15-EET can significantly suppress the phosphorylation of p38 and Erk1/2. Our results indicate that EETs exert beneficial effects on ox-LDL-induced inflammation primarily through the inhibition of LOX-1 receptor upregulation, MAPK phosphorylation, and NF-κB activation and through the upregulation of CYP2J4 expression. This study helps focus the current understanding of the contribution of EETs to the regulation of the inflammation of pulmonary vascular endothelial cells. Furthermore, the therapeutic potential of targeting the EET pathway in pulmonary vascular disease will be highlighted.

  17. Vasopeptidase inhibition improves insulin sensitivity and endothelial function in the JCR:LA-cp rat.

    PubMed

    Russell, James C; Kelly, Sandra E; Schäfer, Stefan

    2004-08-01

    The insulin-resistant, hyperinsulinemic, normoglycemic, and obese JCR:LA-cp rat was used to study the effects of ramipril (an ACE inhibitor) and AVE7688 (a dual inhibitor of ACE and neutral endopeptidases) on insulin sensitivity and vascular function. Both compounds reduced the surge of plasma insulin in a meal tolerance test by approximately 50%. Ramipril had no effect on acetylcholine-induced relaxation but increased the sensitivity to sodium nitroprus-side at low concentrations. AVE7688 significantly reduced the EC50 for acetylcholine to relax phenylephrine-contracted aortic rings. None of the compounds affected the baseline coronary flow and reactive hyperemia. Coronary flow response to bradykinin in AVE7688- and ramipril-treated rat hearts showed a significantly lower EC50 than in control rats. Maximum flow rate was not different between groups. In summary, both ramipril and AVE7688 had significant hypoinsulinemic and insulin-sensitizing effects. Whereas ramipril had limited vascular effects, AVE7688 had more marked beneficial vascular effects, probably of endothelial origin and possibly related to lowered insulin levels.

  18. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    PubMed Central

    Wang, Lefeng; Mehta, Sanjay; Brock, Michael

    2017-01-01

    Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix]) of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction. PMID:28250575

  19. Temsirolimus Inhibits Proliferation and Migration in Retinal Pigment Epithelial and Endothelial Cells via mTOR Inhibition and Decreases VEGF and PDGF Expression

    PubMed Central

    Siedlecki, Jakob; Haritoglou, Christos; Kampik, Anselm; Kernt, Marcus

    2014-01-01

    Due to their high prevalence, retinal vascular diseases including age related macular degeneration (AMD), retinal vein occlusions (RVO), diabetic retinopathy (DR) and diabetic macular edema have been major therapeutic targets over the last years. The pathogenesis of these diseases is complex and yet not fully understood. However, increased proliferation, migration and angiogenesis are characteristic cellular features in almost every retinal vascular disease. The introduction of vascular endothelial growth factor (VEGF) binding intravitreal treatment strategies has led to great advances in the therapy of these diseases. While the predominant part of affected patients benefits from the specific binding of VEGF by administering an anti-VEGF antibody into the vitreous cavity, a small number of non-responders exist and alternative or additional therapeutic strategies should therefore be evaluated. The mammalian target of rapamycin (mTOR) is a central signaling pathway that eventually triggers up-regulation of cellular proliferation, migration and survival and has been identified to play a key role in angiogenesis. In the present study we were able to show that both retinal pigment epithelial (RPE) cells as wells as human umbilical vein endothelial cells (HUVEC) are inhibited in proliferating and migrating after treatment with temsirolimus in non-toxic concentrations. Previous studies suggest that the production of VEGF, platelet derived growth factor (PDGF) and other important cytokines is not only triggered by hypoxia but also by mTOR itself. Our results indicate that temsirolimus decreases VEGF and PDGF expression on RNA and protein levels significantly. We therefore believe that the mTOR inhibitor temsirolimus might be a promising drug in the future and it seems worthwhile to evaluate complementary therapeutic effects with anti-VEGF drugs for patients not profiting from mono anti-VEGF therapy alone. PMID:24586308

  20. Edaravone protected human brain microvascular endothelial cells from methylglyoxal-induced injury by inhibiting AGEs/RAGE/oxidative stress.

    PubMed

    Li, Wenlu; Xu, Hongjiao; Hu, Yangmin; He, Ping; Ni, Zhenzhen; Xu, Huimin; Zhang, Zhongmiao; Dai, Haibin

    2013-01-01

    Subjects with diabetes experience an increased risk of cerebrovascular disease and stroke compared with nondiabetic age-matched individuals. Increased formation of reactive physiological dicarbonyl compound methylglyoxal (MGO) seems to be implicated in the development of diabetic vascular complication due to its protein glycation and oxidative stress effect. Edaravone, a novel radical scavenger, has been reported to display the advantageous effects on ischemic stroke both in animals and clinical trials; however, little is known about whether edaravone has protective effects on diabetic cerebrovascular injury. Using cultured human brain microvascular endothelial cells (HBMEC), protective effects of edaravone on MGO and MGO enhancing oxygen-glucose deprivation (OGD) induced injury were investigated. Cell injury was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, cell account, lactate dehydrogenase (LDH) release and Rhodamine 123 staining. Advanced glycation end-products (AGEs) formation and receptor for advanced glycation end-products (RAGE) expression were measured by western blotting. Cellular oxidative stress was measured by reactive oxygen species (ROS) release. Treatment of MGO for 24 h significantly induced HBMEC injury, which was inhibited by pretreatment of edaravone from 10-100 µmol/l. What's more, treatment of MGO enhanced AGEs accumulation, RAGE expression and ROS release in the cultured HBMEC, which were inhibited by 100 µmol/l edaravone. Finally, treatment of MGO for 24 h and then followed by 3 h OGD insult significantly enhanced cell injury when compared with OGD insult only, which was also protected by 100 µmol/l edaravone. Thus, edaravone protected HBMEC from MGO and MGO enhancing OGD-induced injury by inhibiting AGEs/RAGE/oxidative stress.

  1. Losartan Attenuates Myocardial Endothelial-To-Mesenchymal Transition in Spontaneous Hypertensive Rats via Inhibiting TGF-β/Smad Signaling

    PubMed Central

    Wu, Miao; Peng, Zhenyu; Zu, Changhao; Ma, Jing; Lu, Shijuan; Zhong, Jianghua; Zhang, Saidan

    2016-01-01

    Background Losartan plays an important role in the inhibition of myocardial fibrosis. But the underlying mechanism is not entirely clear. Emerging evidences have indicated that endothelial-to-mesenchymal transition (EndMT) plays a crucial role in cardiac fibrosis. Here the present study aims to first investigated the effect of Losartan on EndMT in cardiac fibrosis of spontaneous hypertensive rats (SHRs). Methods Male SHRs were randomly divided into three groups and fed for 12 weeks, namely the SHR group (Group S), the Losartan-treated group (Group L) and the Prazosin-treated group (Group P). Wistar-Kyoto rats served as controls (Group W). The histological changes were evaluated by Masson’s trichrome. Co-expression of CD31 and fibroblast-specific protein 1 (FSP1) were used as the markers of EndMT through immunofluorescence. The expressions of FSP1, CD31, TGF-β, Smad were detected by Western blot analysis. Results It was identified that elevated blood pressure induced a significant increase in myocardial fibrosis and EndMT in SHRs, which was reversed by Losartan and Prazosin treatment. Furthermore, the activity of TGF-β/Smad signaling was detected in the four groups. TGF-β/Smad signaling was activated in SHRs and suppressed by Losartan or Prazosin treatment. Losartan exhibited more efficiently than Prazosin in inhibiting TGF-β/Smad signaling activation, EndMT and myocardial fibrosis. Conclusion These results showed that EndMT played an important role in promoting hypertensive cardiac fibrosis, and that losartan could suppress cardiac fibrosis through the inhibition of EndMT via classical TGF-β/Smad pathway. PMID:27176484

  2. Andrographolide, a Novel NF-κB Inhibitor, Inhibits Vascular Smooth Muscle Cell Proliferation and Cerebral Endothelial Cell Inflammation

    PubMed Central

    Chang, Chao-Chien; Duann, Yeh-Fang; Yen, Ting-Lin; Chen, Yu-Ying; Jayakumar, Thanasekaran; Ong, Eng-Thiam; Sheu, Joen-Rong

    2014-01-01

    Background Aberrant vascular smooth muscle cell (VSMC) proliferation and cerebral endothelial cell (CEC) dysfunction contribute significantly in the pathogenesis of cardiovascular diseases. Therefore, inhibition of these cellular events would be by candidate agents for treating these diseases. In the present study, the mechanism of anti-proliferative and anti-inflammatory effects of andrographolides, a novel nuclear factor-κB inhibitor, was investigated in VSMC and CEC cells. Methods VSMCs and CECs were isolated from rat artery and mouse brain, respectively, and cultured before experimentation. The effect of andro on platelet-derived growth factor-BB (PDGF-BB) induced VSMC cell proliferation was evaluated by cell number, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of extracellular signal regulated kinase 1/2 (ERK1/2), proliferating cell nuclear antigen (PCNA), and the effects on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and, cyclooxygenase-2 (COX2) were detected by Western blotting. Results Andro significantly inhibited PDGF-BB (10 ng/ml) induced cell proliferation in a concentration (20-100 μM) dependent manner, which may be due to reducing the expression of ERK1/2, and by inhibiting the expression of PCNA. Andro also remarkably diminished LPS-induced iNOS and COX2 expression. Conclusions The results of this study suggested that the effects of andro against VSMCs proliferation and CECs dysfunction may represent a promising approach for treatment of vascular diseases. PMID:27122804

  3. Inhibition of interleukin-1β-induced endothelial tissue factor expression by the synthetic cannabinoid WIN 55,212-2

    PubMed Central

    Scholl, Antje; Ivanov, Igor; Hinz, Burkhard

    2016-01-01

    The role of cannabinoids in thrombosis remains controversial. In view of the primary importance of tissue factor (TF) in blood coagulation and its involvement in the pathology of several cardiovascular, inflammatory and neoplastic diseases, a regulation of this initial procoagulant signal seems to be of particular interest. Using human umbilical vein endothelial cells (HUVEC) the present study investigated the impact of the synthetic cannabinoid WIN 55,212-2 on interleukin (IL)-1β-induced TF expression and activity. WIN 55,212-2 caused a time- and concentration-dependent suppression of IL-1β-induced TF protein accompanied by decreases in TF mRNA and activity. Inhibition of TF protein expression by WIN 55,212-2 was mimicked by its cannabinoid receptor-inactive enantiomer WIN 55,212-3 but not by structurally unrelated phyto-, endo- and synthetic cannabinoids. In addition, the inhibitory effect of WIN 55,212-2 was not reversed by antagonists to cannabinoid receptors (CB1, CB2) or transient receptor potential vanilloid 1. Mechanistic approaches revealed WIN 55,212-2 to suppress IL-1β-induced TF expression via inhibition of ceramide formation and via decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinases. Further inhibitor experiments demonstrated neutral sphingomyelinase (nSMase) to confer ceramide generation upon IL-1β treatment with the parallel IL-1β-mediated activation of MAPKs occurring via an nSMase-independent pathway. Finally, a receptor-independent inhibition of IL-1β-induced TF protein by WIN 55,212-2 was confirmed in human blood monocytes. Collectively, this data provide a hitherto unknown receptor-independent anticoagulatory action of the cannabinoid WIN 55,212-2. PMID:27556861

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

    PubMed

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

    2015-01-01

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

  5. Red Raspberry Phenols Inhibit Angiogenesis: A Morphological and Subcellular Analysis Upon Human Endothelial Cells.

    PubMed

    Sousa, M; Machado, V; Costa, R; Figueira, M E; Sepodes, B; Barata, P; Ribeiro, L; Soares, R

    2016-07-01

    Polyphenols are a class of natural compounds whose potential as antioxidant, anti-inflammatory, and anti-angiogenesis has been reported in many pathological conditions. Red raspberry extract, rich in polyphenols, has been reported to exert anti-inflammatory effects and prevent cell proliferation in distinct animal models. However, the signaling pathways involved remain unknown. Herein, we used human microvascular endothelial cells (HMVECs) to determine the influence of red raspberry phenolic compound extract concentrations, ranging from 10 to 250 µg gallic acid equivalents (GAE)/mL, on endothelium viability (MTS assay), proliferation (BrdU incorporation), migration (injury assay), and capillary-like structures formation (Matrigel assay). Protein expression in cell lysates was determined by Western blot analysis. We showed that red raspberry extracts reduced cell viability (GI50  = 87,64 ± 6,59 μg GAE/mL) and proliferation in a dose-dependent manner. A significant abrogation of cells ability to migrate to injured areas, even at low concentrations, was observed by injury assay. Cell assembly into capillary-like structures on Matrigel also decreased in a dose dependent-manner for higher extract concentrations, as well as the number of branching points per unit of area. Protein expression analysis showed a dose-dependent decrease in Phospho-VEGFR2 expression, implying abrogation of VEGF signaling activity. We also showed for the first time that red raspberry phenolic compounds induce the rearrangement of filamentous actin cytoskeleton, with an isotropy increase found for higher testing concentrations. Taken together, our findings corroborate the anti-angiogenic potential of red raspberry phenolic compounds and provide new insights into their mode of action upon endothelium. J. Cell. Biochem. 117: 1604-1612, 2016. © 2015 Wiley Periodicals, Inc.

  6. Dual NEP/ECE inhibition improves endothelial function in mesenteric resistance arteries of 32-week-old SHR.

    PubMed

    Lemkens, Pieter; Spijkers, Leon Ja; Meens, Merlijn J; Nelissen, Jelly; Janssen, Ben; Peters, Stephan Lm; Schiffers, Paul Mh; De Mey, Jo Gr

    2017-03-16

    Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 μM l-NAME), blockade of ETA- and ETB-receptors (10 μM bosentan) and stimulation of the endothelium with 0.001-10 μM acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. l-NAME (1) increased arterial contractile responses to K(+) (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 μM) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of l-NAME during contractions induced by either 10 μM PHE or 40 mM K(+) were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.Hypertension Research advance online publication, 16 March 2017; doi:10.1038/hr.2017.38.

  7. PJ-34 inhibits PARP-1 expression and ERK phosphorylation in glioma-conditioned brain microvascular endothelial cells.

    PubMed

    Motta, Carla; D'Angeli, Floriana; Scalia, Marina; Satriano, Cristina; Barbagallo, Davide; Naletova, Irina; Anfuso, Carmelina Daniela; Lupo, Gabriella; Spina-Purrello, Vittoria

    2015-08-15

    Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD(+), the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

  8. Resuscitation of traumatic hemorrhagic shock patients with hypertonic saline-without dextran-inhibits neutrophil and endothelial cell activation.

    PubMed

    Junger, Wolfgang G; Rhind, Shawn G; Rizoli, Sandro B; Cuschieri, Joseph; Shiu, Maria Y; Baker, Andrew J; Li, Linglin; Shek, Pang N; Hoyt, David B; Bulger, Eileen M

    2012-10-01

    Posttraumatic inflammation and excessive neutrophil activation cause multiple organ dysfunction syndrome (MODS), a major cause of death among hemorrhagic shock patients. Traditional resuscitation strategies may exacerbate inflammation; thus, novel fluid treatments are needed to reduce such posttraumatic complications. Hypertonic resuscitation fluids inhibit inflammation and reduce MODS in animal models. Here we studied the anti-inflammatory efficacy of hypertonic fluids in a controlled clinical trial. Trauma patients in hypovolemic shock were resuscitated in a prehospital setting with 250 mL of either 7.5% hypertonic saline (HS; n = 9), 7.5% hypertonic saline + 6% dextran 70 (HSD; n = 8), or 0.9% normal saline (NS; n = 17). Blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Multicolor flow cytometry was used to quantify neutrophil expression of cell-surface activation/adhesion (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers as well as oxidative burst activity. Circulating concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVACM-1), P- and E-selectins, myeloperoxidase (MPO), and matrix metalloproteinase 9 (MMP-9) were assessed by immunoassay. Multiple organ dysfunction syndrome, leukocytosis, and mortality were lower in the HS and HSD groups than in the NS group. However, these differences were not statistically significant. Hypertonic saline prevented priming and activation and neutrophil oxidative burst and CD11b and CD66b expression. Hypertonic saline also reduced circulating markers of neutrophil degranulation (MPO and MMP-9) and endothelial cell activation (sICAM-1, sVCAM-1, soluble E-selectin, and soluble P-selectin). Hypertonic saline + 6% dextran 70 was less capable than HS of suppressing the upregulation of most of these activation markers. This study demonstrates that initial resuscitation with HS, but neither NS nor HSD, can attenuate

  9. Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells

    PubMed Central

    Yue, Grace GL; Fan, Jun-Ting; Lee, Julia KM; Zeng, Guang-Zhi; Ho, Tina WF; Fung, Kwok-Pui; Leung, Ping-Chung; Tan, Ning-Hua; Lau, Clara BS

    2011-01-01

    BACKGROUND AND PURPOSE Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed. EXPERIMENTAL APPROACH Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules. KEY RESULTS RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC50 values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased. CONCLUSIONS AND IMPLICATIONS RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent. PMID:21518338

  10. MicroRNA-210 Modulates Endothelial Cell Response to Hypoxia and Inhibits the Receptor Tyrosine Kinase Ligand Ephrin-A3*S⃞

    PubMed Central

    Fasanaro, Pasquale; D'Alessandra, Yuri; Di Stefano, Valeria; Melchionna, Roberta; Romani, Sveva; Pompilio, Giulio; Capogrossi, Maurizio C.; Martelli, Fabio

    2008-01-01

    MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation. PMID:18417479

  11. Chemical Structures of 4-Oxo-Flavonoids in Relation to Inhibition of Oxidized Low-Density Lipoprotein (LDL)-Induced Vascular Endothelial Dysfunction

    PubMed Central

    Yi, Long; Jin, Xin; Chen, Chun-Ye; Fu, Yu-Jie; Zhang, Ting; Chang, Hui; Zhou, Yong; Zhu, Jun-Dong; Zhang, Qian-Yong; Mi, Man-Tian

    2011-01-01

    Vascular endothelial dysfunction induced by oxidative stress has been demonstrated to be the initiation step of atherosclerosis (AS), and flavonoids may play an important role in AS prevention and therapy. Twenty-three flavonoids categorized into flavones, flavonols, isoflavones, and flavanones, all with 4-oxo-pyronenucleus, were examined for what structural characteristics are required for the inhibitory effects on endothelial dysfunction induced by oxidized low-density lipoprotein (oxLDL). Human vascular endothelial cells EA.hy926 were pretreated with different 4-oxo-flavonoids for 2 hs, and then exposed to oxLDL for another 24 hs. Cell viability and the level of malondialdehyde (MDA), nitric oxide (NO) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured, respectively. Then, correlation analysis and paired comparison were used to analyze the structure–activity relationships. Significant correlations were observed between the number of −OH moieties in total or in B-ring and the inhibitory effectson endothelial dysfunction. Furthermore, 3′,4′-ortho-dihydroxyl on B-ring, 3-hydroxyl on C-ring and 2,3-double bondwere correlated closely to the inhibitory effects of flavonolson cell viability decrease and lipid peroxidation. 5,7-meta-dihydroxyl group on A-ring was crucial for the anti-inflammatory effects of flavones and isoflavones in endothelial cells. Moreover, the substituted position of B-ring on C3 rather than C2 was important for NO release. Additionally, hydroxylation at C6 position significantly attenuated the inhibitory effects of 4-oxo-flavonoids on endothelial dysfunction. Our findings indicated that the effective agents in inhibiting endothelial dysfunction include myricetin, quercetin, luteolin, apigenin, genistein and daidzein. Our work might provide some evidence for AS prevention and a strategy for the design of novel AS preventive agents. PMID:22016603

  12. Chemical structures of 4-oxo-flavonoids in relation to inhibition of oxidized low-density lipoprotein (LDL)-induced vascular endothelial dysfunction.

    PubMed

    Yi, Long; Jin, Xin; Chen, Chun-Ye; Fu, Yu-Jie; Zhang, Ting; Chang, Hui; Zhou, Yong; Zhu, Jun-Dong; Zhang, Qian-Yong; Mi, Man-Tian

    2011-01-01

    Vascular endothelial dysfunction induced by oxidative stress has been demonstrated to be the initiation step of atherosclerosis (AS), and flavonoids may play an important role in AS prevention and therapy. Twenty-three flavonoids categorized into flavones, flavonols, isoflavones, and flavanones, all with 4-oxo-pyronenucleus, were examined for what structural characteristics are required for the inhibitory effects on endothelial dysfunction induced by oxidized low-density lipoprotein (oxLDL). Human vascular endothelial cells EA.hy926 were pretreated with different 4-oxo-flavonoids for 2 hs, and then exposed to oxLDL for another 24 hs. Cell viability and the level of malondialdehyde (MDA), nitric oxide (NO) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured, respectively. Then, correlation analysis and paired comparison were used to analyze the structure-activity relationships. Significant correlations were observed between the number of -OH moieties in total or in B-ring and the inhibitory effectson endothelial dysfunction. Furthermore, 3',4'-ortho-dihydroxyl on B-ring, 3-hydroxyl on C-ring and 2,3-double bondwere correlated closely to the inhibitory effects of flavonolson cell viability decrease and lipid peroxidation. 5,7-meta-dihydroxyl group on A-ring was crucial for the anti-inflammatory effects of flavones and isoflavones in endothelial cells. Moreover, the substituted position of B-ring on C3 rather than C2 was important for NO release. Additionally, hydroxylation at C6 position significantly attenuated the inhibitory effects of 4-oxo-flavonoids on endothelial dysfunction. Our findings indicated that the effective agents in inhibiting endothelial dysfunction include myricetin, quercetin, luteolin, apigenin, genistein and daidzein. Our work might provide some evidence for AS prevention and a strategy for the design of novel AS preventive agents.

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

    SciTech Connect

    Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi; Shinya, Tomohiro; Sato, Keizo; Takahashi, Satoru

    2015-07-03

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

  14. Inhibition of THP-1 cell adhesion to endothelial cells by alpha-tocopherol and alpha-tocotrienol is dependent on intracellular concentration of the antioxidants.

    PubMed

    Noguchi, Noriko; Hanyu, Ryuhei; Nonaka, Aya; Okimoto, Yuko; Kodama, Tatsuhiko

    2003-06-15

    Vitamin E analogs such as alpha-tocopherol and alpha-tocotrienol have been shown to reduce endothelial expression of adhesion molecules. The reactivity of alpha-tocopherol and alpha-tocotrienol in inhibiting lipid peroxidation in vitro was essentially identical but the inhibition of adhesion of THP-1 cells, a monocytic-"like" cell line, to endothelial cells differs substantially. To determine the mechanism underlying this response, human umbilical vein endothelial cells (HUVECs) were assessed for their ability to accumulate vitamin E analogs. alpha-Tocotrienol accumulated in HUVECs to levels approximately 10-fold greater than that of alpha-tocopherol. The decrease in expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion of THP-1 cells to HUVECs by alpha-tocopherol and alpha-tocotrienol was also determined. Both alpha-tocopherol and alpha-tocotrienol suppressed VCAM-1 expression and adhesion of THP-1 cells to HUVECs in a concentration-dependent manner. The efficacy of tocotrienol for reduction of VCAM-1 expression and adhesion of THP-1 cells to HUVECs was also 10-fold higher than that of tocopherol. The inhibitory effects of vitamin E analogs on the adhesiveness of endothelial cells clearly correlated with their intracellular concentrations. The data demonstrated that, in assessing the biological responses of antioxidants, intracellular accumulation and metabolism were additional important factors that must be considered.

  15. Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells.

    PubMed

    Gu, Junzhong; Zhang, Huiyuan; Ji, Bingyang; Jiang, Hui; Zhao, Tao; Jiang, Rongcai; Zhang, Zhiren; Tan, Shengjiang; Ahmed, Asif; Gu, Yuchun

    2017-03-08

    Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis.

  16. Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells

    PubMed Central

    Gu, Junzhong; Zhang, Huiyuan; Ji, Bingyang; Jiang, Hui; Zhao, Tao; Jiang, Rongcai; Zhang, Zhiren; Tan, Shengjiang; Ahmed, Asif; Gu, Yuchun

    2017-01-01

    Serotonin or 5-hydroxytryptamine (5-HT) has been shown to be essential in lots of physiological and pathological processes. It is well known that 5-HT and 5-HT transporter (5-HTT) play important roles in the pulmonary artery in pulmonary hypertension. However, little is known about the function of 5-HTT in other arteries. In this study we found that the expression of 5-HTT was elevated in injured carotid arteries and over-expression of 5-HTT induced proliferation of smooth muscle cells (SMCs); however, this phenotype could be reversed by knocking-down of 5-HTT or endothelial cells conditional medium (EC-CM). A 5-HTT inhibitor, fluoxetine, treated animals also exhibited reduced restenosis after injury. We identified that miR-195 was packaged in the extracellular vesicles from EC-CM. We further confirmed that extracellular vesicles could transfer miR-195 from ECs to SMCs to inhibit the expression of 5-HTT in SMCs and the proliferation of SMCs. These results provide the first evidence that ECs communicate with SMCs via micro-RNA195 in the regulation of the proliferation of SMCs through 5-HTT, which will contribute to a better understanding of communications between ECs and SMCs via micro-RNA. Our findings suggest a potential target for the control of vessel restenosis. PMID:28272473

  17. MicroRNA Mediation of Endothelial Inflammatory Response to Smooth Muscle Cells and its Inhibition by Atheroprotective Shear Stress

    PubMed Central

    Chen, Li-Jing; Chuang, Li; Huang, Yi-Hsuan; Zhou, Jing; Lim, Seh Hong; Lee, Chih-I; Lin, Wei-Wen; Lin, Ting-Er; Wang, Wei-Li; Chen, Linyi; Chien, Shu; Chiu, Jeng-Jiann

    2015-01-01

    Rationale In atherosclerotic lesions, synthetic smooth muscle cells (sSMCs) induce aberrant microRNA (miR) profiles in endothelial cells (ECs) under flow stagnation. Increase in shear stress induces favorable miR modulation to mitigate sSMC-induced inflammation. Objective To address the role of miRs in sSMC-induced EC inflammation and its inhibition by shear stress. Methods and Results Co-culturing ECs with sSMCs under static condition causes initial increases of four anti-inflammatory miRs (146a/708/451/98) in ECs followed by decreases below basal levels at 7 days; the increases for miR-146a/708 peaked at 24 h and those for miR-451/98 lasted for only 6-12 h. Shear stress (12 dynes/cm2) to co-cultured ECs for 24 h augments these four miR expressions. In vivo, these four miRs are highly expressed in neointimal ECs in injured arteries under physiological levels of flow, but not expressed under flow stagnation. MiR-146a, -708, -451, and -98 target interleukin (IL)-1 receptor-associated kinase, inhibitor of nuclear factor-κB (NF-κB) kinase subunit-γ, IL-6 receptor, and conserved helix-loop-helix ubiquitous kinase, respectively, to inhibit NF-κB signaling, which exerts negative feedback control on the biogenesis of these miRs. NF-E2-related factor-2 (Nrf-2) is critical for shear-induction of miR-146a in co-cultured ECs. Silencing either Nrf-2 or miR-146a led to increased neointima formation of injured rat carotid artery under physiological levels of flow. Overexpressing miR-146a inhibits neointima formation of rat or mouse carotid artery induced by injury or flow cessation. Conclusions Nrf-2-mediated miR-146a expression is augmented by atheroprotective shear stress in ECs adjacent to sSMCs to inhibit neointima formation of injured arteries. PMID:25623956

  18. Docosahexaenoic acid inhibits vascular endothelial growth factor (VEGF)-induced cell migration via the GPR120/PP2A/ERK1/2/eNOS signaling pathway in human umbilical vein endothelial cells.

    PubMed

    Chao, Che-Yi; Lii, Chong-Kuei; Ye, Siou-Yu; Li, Chien-Chun; Lu, Chia-Yang; Lin, Ai-Hsuan; Liu, Kai-Li; Chen, Haw-Wen

    2014-05-07

    Cell migration plays an important role in angiogenesis and wound repair. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that is essential for endothelial cell survival, proliferation, and migration. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, shows both anti-inflammatory and antioxidant activities in vitro and in vivo. This study investigated the molecular mechanism by which DHA down-regulates VEGF-induced cell migration. HUVECs were used as the study model, and the MTT assay, Western blot, wound-healing assay, and phosphatase activity assay were used to explore the effects of DHA on cell migration. GPR120 is the putative receptor for DHA action. The results showed that DHA, PD98059 (an ERK1/2 inhibitor), and GW9508 (a GPR120 agonist) inhibited VEGF-induced cell migration. In contrast, pretreatment with okadaic acid (OA, a PP2A inhibitor) and S-nitroso-N-acetyl-DL-penicillamine (an NO donor) reversed the inhibition of cell migration by DHA. VEGF-induced cell migration was accompanied by phosphorylation of ERK1/2 and eNOS. Treatment of HUVECs with DHA increased PP2A enzyme activity and decreased VEGF-induced phosphorylation of ERK1/2 and eNOS. However, pretreatment with OA significantly decreased DHA-induced PP2A enzyme activity and reversed the DHA inhibition of VEGF-induced ERK1/2 and eNOS phosphorylation. These results suggest that stimulation of PP2A activity and inhibition of the VEGF-induced ERK1/2/eNOS signaling pathway may be involved in the DHA suppression of VEGF-induced cell migration. Thus, the effect of DHA on angiogenesis and wound repair is at least partly by virtue of its attenuation of cell migration.

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

    SciTech Connect

    Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.; Kuo, M.-L.; Ho, Y.-S.; Lee, W.-S.

    2008-05-15

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

  20. Vaccarin attenuates the human EA.hy926 endothelial cell oxidative stress injury through inhibition of Notch signaling.

    PubMed

    Xie, Fengshan; Cai, Weiwei; Liu, Yanling; Li, Yue; Du, Bin; Feng, Lei; Qiu, Liying

    2015-01-01

    Endothelial cell injury is an essential component of atherosclerosis and hypertension. Atherosclerosis and other macrovascular diseases are the most common complications of diabetes. Vaccarin is a major flavonoid glycoside in Vaccariae semen, and is expected to be useful in the treatment of vascular diseases. The aim of the present study was to evaluate the possible effects of vaccarin in human umbilical vein endothelial cells (EA.hy926) induced by hydrogen peroxide (H2O2) and its underlying mechanism in the prevention and treatment of H2O2 injury. In this study, the EA.hy926 cells were exposed to 250, 500 and 1000 µM H2O2 for 2 and 4 h in the absence or presence of vaccarin, and the cell injury induced by H2O2 was examined via SRB. Cell migratory ability, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) levels and decreasing superoxide dismutase (SOD) activity were evaluated by the wound healing assay and corresponding assay kits. Cell apoptosis was detected by flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit and Hoechst staining. Furthermore, western blot detected the protein expressions of Notch1, Hes1 and caspase-3. Following treatment with H2O2, it was found that H2O2 stimulated cell injury in a dose-dependent manner, including reducing cell viability and cell migratory ability, increasing LDH leakage and MDA levels, and decreasing SOD activity. H2O2 further accelerated cell apoptosis via activation of Notch1 and the downstream molecule Hes1. Preincubation with vaccarin was found to protect EA.hy926 cells from H2O2-induced cell oxidative stress injury, which promoted cell viability and cell migratory ability, inhibited the level of LDH and MDA, but enhanced the activity of SOD. In particular, in addition to downregulation Notch signaling, vaccarin treatments also downregulated caspase-3, a cell apoptotic pathway-related protein. These findings indicated that vaccarin may be able to selectively protect

  1. Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

    PubMed

    Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

    2017-03-01

    Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

  2. Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells.

    PubMed

    Zhang, Feng-Lei; Gao, Hai-Qing; Wu, Jian-Min; Ma, Ya-Bing; You, Bei-An; Li, Bao-Ying; Xuan, Jun-Hua

    2006-08-01

    The interaction of advanced glycation end products (AGE) with their cell surface receptors for AGEs (RAGE) has been causally implicated in the pathogenesis of diabetic vascular complications and has been shown to stimulate cell adhesion molecule expression in endothelial cells via induction of reactive oxygen species (ROS). Alternatively, grape seed proanthocyanidin extracts (GSPE), which are naturally occurring polyphenolic compounds, have been reported to possess potent radical scavenging and antioxidant properties and to display significant cardiovascular protective action. In this study, we investigated whether GSPE could inhibit AGE-induced cell adhesion molecule expression through interference with ROS generations in human umbilical vein endothelial cells. AGE-modified bovine serum albumin (AGE-BSA) was prepared by incubating BSA with a high concentration of glucose. Stimulation of cultured human umbilical vein endothelial cells with 200 microg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM-1), whereas both unmodified BSA and GSPE alone were without effect. However, preincubation of different concentrations of GSPE markedly downregulated AGE-BSA-induced VCAM-1 expression at the surface protein and mRNA level in a concentration-dependent manner, but the increased ICAM-1 expression was not affected by GSPE treatment. Meanwhile, the inhibition by GSPE of intracellular ROS generation was also observed at defined time periods. These results demonstrate that GSPE can inhibit the enhanced VCAM-1 expression but not ICAM-1 in AGE-exposed endothelial cells by suppressing ROS generation. Hence, GSPE may have therapeutic potential in the prevention and treatment of vascular complications in patients with diabetes.

  3. Diallyl trisulfide inhibits migration, invasion and angiogenesis of human colon cancer HT-29 cells and umbilical vein endothelial cells, and suppresses murine xenograft tumour growth

    PubMed Central

    Lai, Kuang-Chi; Hsu, Shu-Chun; Yang, Jai-Sing; Yu, Chien-Chih; Lein, Jin-Cherng; Chung, Jing-Gung

    2015-01-01

    Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. We examined the cytotoxic, anti-metastatic, anti-cancer and anti-angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases-2, -7 and -9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary-like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex-vivo angiogenesis. We investigated the anti-tumour effects of DATS against human colon cancer xenografts in BALB/cnu/nu mice and its anti-angiogenic activity in vivo. In this in-vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS. PMID:25403643

  4. Impact of DPP-4 inhibition on acute and chronic endothelial function in humans with type 2 diabetes on background metformin therapy.

    PubMed

    Widlansky, Michael E; Puppala, Venkata K; Suboc, Tisha M; Malik, Mobin; Branum, Amberly; Signorelli, Kara; Wang, Jingli; Ying, Rong; Tanner, Michael J; Tyagi, Sudhi

    2017-01-01

    Cell culture and animal work indicate that dipeptidyl peptidase-4 (DPP-4) inhibition may exert cardiovascular benefits through favorable effects on the vascular endothelium. Prior human studies evaluating DPP-4 inhibition have shown conflicting results that may in part be related to heterogeneity of background anti-diabetes therapies. No study has evaluated the acute response of the vasculature to DPP-4 inhibition in humans. We recruited 38 patients with type 2 diabetes on stable background metformin therapy for a randomized, double-blind, placebo-controlled crossover trial of DPP-4 inhibition with sitagliptin (100 mg/day). Each treatment period was 8 weeks long separated by 4 weeks of washout. Endothelial function and plasma markers of endothelial activation (intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)) were measured prior to and 2 hours following acute dosing of sitagliptin or placebo, as well as following 8 weeks of intervention with each pill. Thirty subjects completed the study and were included in analyses. Neither acute nor chronic sitagliptin therapy resulted in significant changes in vascular endothelial function. While post-acute sitagliptin ICAM-1 levels were lower than that post-chronic sitagliptin, the ICAM-1 concentration was not significantly different than pre-acute sitagliptin levels or levels measured in relationship to placebo. There were no significant changes in plasma VCAM-1 levels at any time point. Acute and chronic sitagliptin therapies have neutral effects on the vascular endothelium in the setting of metformin background therapy. In conclusion, our findings suggest DPP-4 inhibition has a neutral effect on cardiovascular risk in patients without a history of heart failure or renal insufficiency.

  5. Inhibition of vascular endothelial growth factor by small interfering RNA upregulates differentiation, maturation and function of dendritic cells

    PubMed Central

    WANG, HAIYAN; ZHANG, LUPING; ZHANG, SHAOYAN; LI, YANNIAN

    2015-01-01

    This study aimed to investigate the effects of vascular endothelial growth factor (VEGF) secreted by MCF-7 breast cancer cells on the differentiation, maturation and function of dendritic cells (DCs). Small interfering RNAs (siRNAs) directed against the VEGF gene were designed and transfected into MCF-7 breast cancer cells at an optimal concentration (100 nmol/l) using cationic liposome transfection reagent, whereas the control group was transfected with only transfection reagent. Western blot analysis and ELISA were used to determine VEGF protein expression and VEGF concentration, respectively. Mononuclear cells were cultured with the culture supernatants from primary MCF-7 cells (control group) and siRNA-treated MCF-7 cells (siRNA group). The DC phenotypes, including CD1a, CD80, CD83, CD86 and HLA-DR, were evaluated by flow cytometry. The MTT assay was used to assess the cytotoxicity of DC-mediated tumor-specific cytotoxic T lymphocytes (CTLs) against MCF-7 cells in the two different culture supernatants. The VEGF-targeted constructed siRNA inhibited VEGF expression in MCF-7 cells. Cultivation with the culture supernatants from MCF-7 cells treated with siRNA affected DC morphology. DCs in the siRNA group exhibited a significantly higher expression of CD86, CD80, CD83 and HLA-DR compared to the cells in the control group, whereas the expression of CD1a in the siRNA group was significantly lower compared to that in the control group. The cytotoxic activity of CTLs mediated by DCs was significantly altered by siRNA transfection. These results indicated that VEGF may play a significant role in tumor development, progression and immunosuppression. PMID:25452786

  6. Vaccarin attenuates high glucose-induced human EA•hy926 endothelial cell injury through inhibition of Notch signaling.

    PubMed

    Qiu, Yuyu; Du, Bin; Xie, Fengshan; Cai, Weiwei; Liu, Yanling; Li, Yue; Feng, Lei; Qiu, Liying

    2016-03-01

    Endothelial cell injury is a critical component of atherosclerosis and hypertension. Vaccarin is considered to be of potential benefit in the treatment of vascular diseases. The aim of the present study was to evaluate the possible effects of vaccarin in human EA·hy926 cells induced by high glucose, and to investigate its underlying mechanism in the prevention and treatment of high glucose‑induced injury. In the present study, EA·hy926 cells were exposed to 90, 180 and 270 mM high glucose for 24 h, and the induced cell injury was examined using a sulforhodamine B assay. Following treatment with high glucose, it was found that high glucose stimulated cell injury, resulting in reduced cell viability and migratory ability, increased lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) activity. High glucose further accelerated cell apoptosis via activating Notch1 and Hairy and enhancer of split 1. It was found that preincubation with vaccarin protected the EA·hy926 cells from high glucose‑induced cell injury, which promoted cell viability and migratory ability, inhibited the expression levels of LDH and MDA, and enhanced the activity of SOD. Cell migratory ability, LDH leakage, MDA levels and decreasing SOD activity were evaluated using a wound healing assay and corresponding assay kits. Cell apoptosis was detected by flow cytometry with an Annexin V‑fluorescein isothiocyanate/propidium iodide apoptosis detection kit and Hoechst staining. Furthermore, western blotting was used to detect the protein expression levels of Notch1, Hes1 and caspase‑3. In particular, in addition to inducing the downregulation of Notch signaling, vaccarin treatment downregulated the cell apoptotic pathway‑associated protein caspase 3. These findings suggested that vaccarin may be able to selectively protect the vascular endothelium from dysfunction induced by high glucose.

  7. Increased expression of microRNA-221 inhibits PAK1 in endothelial progenitor cells and impairs its function via c-Raf/MEK/ERK pathway

    SciTech Connect

    Zhang, Xiaoping; Mao, Haian; Chen, Jin-yuan; Wen, Shengjun; Li, Dan; Ye, Meng; Lv, Zhongwei

    2013-02-15

    Highlights: ► MicroRNA-221 is upregulated in the endothelial progenitor cells of atherosclerosis patients. ► PAK1 is a direct target of microRNA-221. ► MicroRNA-221 inhibits EPCs proliferation through c-Raf/MEK/ERK pathway. -- Abstract: Coronary artery disease (CAD) is associated with high mortality and occurs via endothelial injury. Endothelial progenitor cells (EPCs) restore the integrity of the endothelium and protect it from atherosclerosis. In this study, we compared the expression of microRNAs (miRNAs) in EPCs in atherosclerosis patients and normal controls. We found that miR-221 expression was significantly up-regulated in patients compared with controls. We predicted and identified p21/Cdc42/Rac1-activated kinase 1 (PAK1) as a novel target of miR-221 in EPCs. We also demonstrated that miR-221 targeted a putative binding site in the 3′UTR of PAK1, and absence of this site was inversely associated with miR-221 expression in EPCs. We confirmed this relationship using a luciferase reporter assay. Furthermore, overexpression of miR-221 in EPCs significantly decreased EPC proliferation, in accordance with the inhibitory effects induced by decreased PAK1. Overall, these findings demonstrate that miR-221 affects the MEK/ERK pathway by targeting PAK1 to inhibit the proliferation of EPCs.

  8. Inhibition of mTOR down-regulates scavenger receptor, class B, type I (SR-BI) expression, reduces endothelial cell migration and impairs nitric oxide production.

    PubMed

    Fruhwürth, Stefanie; Krieger, Sigurd; Winter, Katharina; Rosner, Margit; Mikula, Mario; Weichhart, Thomas; Bittman, Robert; Hengstschläger, Markus; Stangl, Herbert

    2014-07-01

    The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.

  9. Geniposide inhibited endothelial-mesenchymal transition via the mTOR signaling pathway in a bleomycin-induced scleroderma mouse model

    PubMed Central

    Qi, Qing; Mao, Yueping; Tian, Yongzhen; Zhu, Ke; Cha, Xushan; Wu, Minghua; Zhou, Xiaodong

    2017-01-01

    Aim: Geniposide is an iridoid glycoside isolated from the gardenia plant. It has multiple biological activities. The roles of geniposide in systemic sclerosis (SSc) and in endothelial-to-mesenchymal transition (EndMT) are unclear. We investigated the protective effects of geniposide in a bleomycin-induced SSc mouse model, and its potential mechanisms. Methods: The effects of geniposide were evaluated as follows: (1) histological and immunochemical changes in mouse skin tissue; (2) changes in cellular morphology of human umbilical vein endothelial cells (HUVECs); (3) expression of endothelial cell biomarkers (E-Cadherin, CD31, and CD34), mesenchymal cell markers (FSP1, Collagen, and α-SMA), and key factors of EndMT (Slug, Snail, and Twist) using real time PCR, Western blot, and immunofluorescence; (4) tube formation in HUVECs; (5) mTOR signaling pathway transcription factors using Western blot analysis. Results: Treatment with bleomycin induced up-regulation of mesenchymal cell biomarkers and down-regulation of endothelial cell biomarkers in in vivo and in vitro bleomycin-induced scleroderma models. Geniposide treatment suppressed these effects. Geniposide remedied bleomycin-induced dermal capillary loss and fibrosis in mice. The expression of key EndMT factors (Slug, Snail, and Twist) and the mTOR signaling pathway (mTOR and S6) were also attenuated by geniposide treatment. Conclusion: Geniposide had protective effects on endothelial cells in the bleomycin-induced scleroderma mouse model. These effects may occur via inhibition of the mTOR signaling pathway activation. The results suggested that geniposide could be a potential candidate drug for treatment of vascular damage in SSc patients. PMID:28386330

  10. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    SciTech Connect

    Lee, Hae-June; Yoon, Changhwan; Park, Do Joong; Kim, Yeo-Jung; Schmidt, Benjamin; Lee, Yoon-Jin; Tap, William D.; Eisinger-Mathason, T.S. Karin; Choy, Edwin; Kirsch, David G.; Simon, M. Celeste; and others

    2015-03-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm{sup 3} within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm{sup 3} for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

  11. Genistein inhibits TNF-α-induced endothelial inflammation through the protein kinase pathway A and improves vascular inflammation in C57BL/6 mice.

    PubMed

    Jia, Zhenquan; Babu, Pon Velayutham Anandh; Si, Hongwei; Nallasamy, Palanisamy; Zhu, Hong; Zhen, Wei; Misra, Hara P; Li, Yunbo; Liu, Dongmin

    2013-10-03

    Genistein, a soy isoflavone, has received wide attention for its potential to improve vascular function, but the mechanism of this effect is unclear. Here, we report that genistein at physiological concentrations (0.1 μM-5 μM) significantly inhibited TNF-α-induced adhesion of monocytes to human umbilical vein endothelial cells, a key event in the pathogenesis of atherosclerosis. Genistein also significantly suppressed TNF-α-induced production of adhesion molecules and chemokines such as sICAM-1, sVCAM-1, sE-Selectin, MCP-1 and IL-8, which play key role in the firm adhesion of monocytes to activated endothelial cells (ECs). Genistein at physiologically relevant concentrations didn't significantly induce antioxidant enzyme activities or scavenge free radicals. Further, blocking the estrogen receptors (ERs) in ECs didn't alter the preventive effect of genistein on endothelial inflammation. However, inhibition of protein kinase A (PKA) significantly attenuated the inhibitory effects of genistein on TNF-α-induced monocyte adhesion to ECs as well as the production of MCP-1 and IL-8. In animal study, dietary genistein significantly suppressed TNF-α-induced increase in circulating chemokines and adhesion molecules in C57BL/6 mice. Genistein treatment also reduced VCAM-1 and monocytes-derived F4/80-positive macrophages in the aorta of TNF-α-treated mice. In conclusion, genistein protects against TNF-α-induced vascular endothelial inflammation both in vitro and in vivo models. This anti-inflammatory effect of genistein is independent of the ER-mediated signaling machinery or antioxidant activity, but mediated via the PKA signaling pathway.

  12. EGCG protects endothelial cells against PCB 126-induced inflammation through inhibition of AhR and induction of Nrf2-regulated genes

    SciTech Connect

    Han, Sung Gu; Han, Seong-Su; Toborek, Michal; Hennig, Bernhard

    2012-06-01

    Tea flavonoids such as epigallocatechin gallate (EGCG) protect against vascular diseases such as atherosclerosis via their antioxidant and anti-inflammatory functions. Persistent and widespread environmental pollutants, including polychlorinated biphenyls (PCB), can induce oxidative stress and inflammation in vascular endothelial cells. Even though PCBs are no longer produced, they are still detected in human blood and tissues and thus considered a risk for vascular dysfunction. We hypothesized that EGCG can protect endothelial cells against PCB-induced cell damage via its antioxidant and anti-inflammatory properties. To test this hypothesis, primary vascular endothelial cells were pretreated with EGCG, followed by exposure to the coplanar PCB 126. Exposure to PCB 126 significantly increased cytochrome P450 1A1 (Cyp1A1) mRNA and protein expression and superoxide production, events which were significantly attenuated following pretreatment with EGCG. Similarly, EGCG also reduced DNA binding of NF-κB and downstream expression of inflammatory markers such as monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) after PCB exposure. Furthermore, EGCG decreased endogenous or base-line levels of Cyp1A1, MCP-1 and VCAM-1 in endothelial cells. Most of all, treatment of EGCG upregulated expression of NF-E2-related factor 2 (Nrf2)-controlled antioxidant genes, including glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in a dose-dependent manner. In contrast, silencing of Nrf2 increased Cyp1A1, MCP-1 and VCAM-1 and decreased GST and NQO1 expression, respectively. These data suggest that EGCG can inhibit AhR regulated genes and induce Nrf2-regulated antioxidant enzymes, thus providing protection against PCB-induced inflammatory responses in endothelial cells. -- Highlights: ► PCBs cause endothelial inflammation and subsequent atherosclerosis. ► Nutrition can modulate toxicity by environmental pollutants. ► We

  13. Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB1 receptors

    PubMed Central

    2011-01-01

    Background VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB) that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs) in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA) in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV) infection of brain endothelial cells using in vitro and in vivo approaches. Methods i) in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii) in vivo: CB1 receptor deficient mice (Cnr1-/-) infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. Results Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB1 receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB1 receptor deficient mice (Cnr1-/-), the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1-/- mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB1 receptor exacerbated

  14. Sesamin inhibits macrophage-induced vascular endothelial growth factor and matrix metalloproteinase-9 expression and proangiogenic activity in breast cancer cells.

    PubMed

    Lee, Chun-Chung; Liu, Ko-Jiunn; Wu, Yu-Chen; Lin, Sue-Jane; Chang, Ching-Chun; Huang, Tze-Sing

    2011-06-01

    Sesamin is a sesame component with antihypertensive and antioxidative activities and has recently aroused much interest in studying its potential anticancer application. Macrophage is one of the infiltrating inflammatory cells in solid tumor and may promote tumor progression via enhancement of tumor angiogenesis. In this study, we investigated whether sesamin inhibited macrophage-enhanced proangiogenic activity of breast cancer cell lines MCF-7 and MDA-MB-231. Using vascular endothelial cell capillary tube and network formation assays, both breast cancer cell lines exhibited elevated proangiogenic activities after coculture with macrophages or pretreatment with macrophage-conditioned medium. This elevation of proangiogenic activity was drastically suppressed by sesamin. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) induced by macrophages in both cell lines were also inhibited by sesamin. Nuclear levels of HIF-1α and NF-κB, important transcription factors for VEGF and MMP-9 expression, respectively, were obviously reduced by sesamin. VEGF induction by macrophage in MCF-7 cells was shown to be via ERK, JNK, phosphatidylinositol 3-kinase, and NF-κB-mediated pathways. These signaling molecules and additional p38(MAPK) were also involved in macrophage-induced MMP-9 expression. Despite such diverse pathways were induced by macrophage, only Akt and p38(MAPK) activities were potently inhibited by sesamin. Expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-α were substantially increased and involved in macrophage-induced VEGF and MMP-9 mRNA expression in MCF-7 cells. Sesamin effectively inhibited the expression of these cytokines to avoid the reinforced induction of VEGF and MMP-9. In conclusion, sesamin potently inhibited macrophage-enhanced proangiogenic activity of breast cancer cells via inhibition of VEGF and MMP-9 induction.

  15. Adoptive transfer of allogeneic liver sinusoidal endothelial cells specifically inhibits T-cell responses to cognate stimuli.

    PubMed

    Banshodani, Masataka; Onoe, Takashi; Shishida, Masayuki; Tahara, Hiroyuki; Hashimoto, Shinji; Igarashi, Yuka; Tanaka, Yuka; Ohdan, Hideki

    2013-01-01

    Although it is well known that liver allografts are often accepted by recipients, leading to donor-specific tolerance of further organ transplants, the underlying mechanisms remain unclear. We had previously used an in vitro model and showed that mouse liver sinusoidal endothelial cells (LSECs) selectively suppress allospecific T-cells across major histocompatibility complex (MHC) barriers. In the present study, we established an in vivo model for evaluating the immunomodulatory effects of allogeneic LSECs on corresponding T-cells. Allogeneic BALB/cA LSECs were injected intraportally into recombination activating gene 2 γ-chain double-knockout (RAG2/gc-KO, H-2(b)) mice lacking T, B, and natural killer (NK) cells. In order to facilitate LSEC engraftment, the RAG2/gc-KO mice were injected intraperitoneally with monocrotaline 2 days before the adoptive transfer of LSECs; this impaired the host LSECs, conferring a proliferative advantage to the transplanted LSECs. After orthotopic allogeneic LSEC engraftment, the RAG2/gc-KO mice were immune reconstituted intravenously with C57BL/6 splenocytes. After immune reconstitution, mixed lymphocyte reaction (MLR) assay using splenocytes from the recipients revealed that specific inhibition of host CD4(+) and CD8(+) T-cell proliferation was greater in response to allostimulation with irradiated BALB/cA splenocytes rather than to stimulation with irradiated third party SJL/jorllco splenocytes. This inhibitory effect was attenuated by administering anti-programmed death ligand 1 (PD-L1) monoclonal antibody during immune reconstitution in the above-mentioned mice, but not in RAG2/gc-KO mice engrafted with Fas ligand (FasL)-deficient BALB/cA LSECs. Furthermore, engraftment of allogeneic BALB/cA LSECs significantly prolonged the survival of subsequently grafted cognate allogeneic BALB/cA hearts in RAG2/gc-KO mice immune reconstituted with bone marrow transplantation from C57BL/6 mice. In conclusion, murine LSECs have been proven

  16. CCN1 Promotes VEGF Production in Osteoblasts and Induces Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-126 Expression in Rheumatoid Arthritis.

    PubMed

    Chen, Cheng-Yu; Su, Chen-Ming; Hsu, Chin-Jung; Huang, Chien-Chung; Wang, Shih-Wei; Liu, Shih-Chia; Chen, Wei-Cheng; Fuh, Lih-Jyh; Tang, Chih-Hsin

    2017-01-01

    Angiogenesis is the formation of new capillaries from preexisting vasculature. The perpetuation of angiogenesis plays a critical role in the pathogenesis of various disease states including rheumatoid arthritis (RA). Cysteine-rich 61 (Cyr61 or CCN1) is an important proinflammatory cytokine in RA. Here, we investigated the role of CCN1 in angiogenesis associated with vascular endothelial growth factor (VEGF) production and osteoblasts. We found higher expression of CCN1 and VEGF in synovial fluid from RA patients compared with healthy controls. CCN1 induced VEGF expression in osteoblasts and increased endothelial progenitor cells (EPCs) angiogenesis by inhibiting miR-126 via the protein kinase C-alpha (PKC-α) signaling pathway. CCN1 knockdown inhibited angiogenesis in both in vitro and in vivo models. Inhibition of CCN1 expression with lentiviral vectors expressing short hairpin RNA (shRNA) ameliorated articular swelling, cartilage erosion, and angiogenesis in the ankle joint of mice with collagen-induced arthritis (CIA). Our study is the first to describe how CCN1 promotes VEGF expression in osteoblasts and increased EPCs angiogenesis in RA disease. CCN1 may serve as a potential target for RA treatment. © 2016 American Society for Bone and Mineral Research.

  17. Chebulagic acid inhibits the LPS-induced expression of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation.

    PubMed

    Liu, Yueying; Bao, Luer; Xuan, Liying; Song, Baohua; Lin, Lin; Han, Hao

    2015-07-01

    Inflammatory response in the vasculature, including the overexpression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, has been demonstrated to increase the risk of thrombosis development. Chebulagic acid (CA) is a key chemical component in the traditional Mongolian anti-thrombotic drug Garidi-13, and has been suggested to exert anti-inflammatory and anti-infective effects. The present study aimed to evaluate the regulatory impact of CA on a number of biological processes, including lipopolysaccharide (LPS)-induced inflammation, LPS-promoted mitogen-activated protein kinase (MAPK) activation and the expression of toll-like receptor (TLR)4 in EA.hy926 human endothelial cells. The results indicated that CA significantly inhibited the LPS-induced upregulation of TNF-α and IL-1β in a dose- and time-dependent manner. Furthermore, LPS-activated MAPK signaling was inhibited by CA treatment in the EA.hy926 cells. However, TLR4, which serves a key function in LPS-induced inflammation as the receptor of LPS, was not regulated by the CA treatment. In summary, the results of the present study indicate that CA inhibits the LPS-induced promotion of TNF-α and IL-1β in endothelial cells by suppressing MAPK activation, which may contribute to the anti-thrombotic effect of Garidi-13.

  18. Gap junction-mediated transfer of miR-145-5p from microvascular endothelial cells to colon cancer cells inhibits angiogenesis.

    PubMed

    Thuringer, Dominique; Jego, Gaetan; Berthenet, Kevin; Hammann, Arlette; Solary, Eric; Garrido, Carmen

    2016-05-10

    Gap junctional communication between cancer cells and blood capillary cells is crucial to tumor growth and invasion. Gap junctions may transfer microRNAs (miRs) among cells. Here, we explore the impact of such a transfer in co-culture assays, using the antitumor miR-145 as an example. The SW480 colon carcinoma cells form functional gap junction composed of connexin-43 (Cx43) with human microvascular endothelial cells (HMEC). When HMEC are loaded with miR-145-5p mimics, the miR-145 level drastically increases in SW480. The functional inhibition of gap junctions, using either a gap channel blocker or siRNA targeting Cx43, prevents this increase. The transfer of miR-145 also occurs from SW480 to HMEC but not in non-contact co-cultures, excluding the involvement of soluble exosomes. The miR-145 transfer to SW480 up-regulates their Cx43 expression and inhibits their ability to promote angiogenesis. Our results indicate that the gap junctional communication can inhibit tumor growth by transferring miRs from one endothelial cell to neighboring tumor cells. This "bystander" effect could find application in cancer therapy.

  19. An anti-platelet-endothelial cell adhesion molecule-1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through the basement membrane

    PubMed Central

    1996-01-01

    Platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation of leukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-1 beta, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion of leukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-1 beta, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane. PMID:8691137

  20. Peptides based on alphaV-binding domains of erythrocyte ICAM-4 inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation.

    PubMed

    Kaul, Dhananjay K; Liu, Xiao-du; Zhang, Xiaoqin; Mankelow, Tosti; Parsons, Stephen; Spring, Frances; An, Xiuli; Mohandas, Narla; Anstee, David; Chasis, Joel Anne

    2006-11-01

    Growing evidence shows that adhesion molecules on sickle erythrocytes interact with vascular endothelium leading to vaso-occlusion. Erythrocyte intercellular adhesion molecule-4 (ICAM-4) binds alphaV-integrins, including alphaVbeta3 on endothelial cells. To explore the contribution of ICAM-4 to vascular pathology of sickle cell disease, we tested the effects of synthetic peptides, V(16)PFWVRMS (FWV) and T(91)RWATSRI (ATSR), based on alphaV-binding domains of ICAM-4 and capable of inhibiting ICAM-4 and alphaV-binding in vitro. For these studies, we utilized an established ex vivo microvascular model system that enables intravital microscopy and quantitation of adhesion under shear flow. In this model, the use of platelet-activating factor, which causes endothelial oxidant generation and endothelial activation, mimicked physiological states known to occur in sickle cell disease. Infusion of sickle erythrocytes into platelet-activating factor-treated ex vivo rat mesocecum vasculature produced pronounced adhesion of erythrocytes; small-diameter venules were sites of maximal adhesion and frequent blockage. Both FWV and ATSR peptides markedly decreased adhesion, and no vessel blockage was observed with either of the peptides, resulting in improved hemodynamics. ATSR also inhibited adhesion in unactivated microvasculature. Although infused fluoresceinated ATSR colocalized with vascular endothelium, pretreatment with function-blocking antibody to alphaVbeta3-integrin markedly inhibited this interaction. Our data strengthen the thesis that ICAM-4 on sickle erythrocytes binds endothelium via alphaVbeta3 and that this interaction contributes to vaso-occlusion. Thus peptides or small molecule mimetics of ICAM-4 may have therapeutic potential.

  1. Selective inhibition of endothelial and monocyte redox-sensitive genes by AGI-1067: a novel antioxidant and anti-inflammatory agent.

    PubMed

    Kunsch, Charles; Luchoomun, Jayraz; Grey, Janice Y; Olliff, Lynda K; Saint, Leigh B; Arrendale, Richard F; Wasserman, Martin A; Saxena, Uday; Medford, Russell M

    2004-03-01

    Atherosclerosis is a disease of oxidative stress and inflammation. AGI-1067 [butanedioic acid, mono[4-[[1-[[3,5-bis(1,1-dimethylethyl)-4-,hydroxyphenyl]thio]-1-methylethyl]thio]-2,6-bis (1,1-dimethylethyl)phenyl] ester] is a metabolically stable derivative of, yet pharmacologically distinct from, the antioxidant drug probucol. It is a member of a novel class of orally active, antioxidant, anti-inflammatory compounds termed vascular protectants and exhibits antiatherosclerotic properties in multiple animal models and in humans. To elucidate its antiatherosclerotic mechanisms, we have evaluated several cellular and molecular properties of AGI-1067 in vitro. AGI-1067 exhibited potent lipid peroxide antioxidant activity comparable with probucol yet demonstrated significantly enhanced cellular uptake over that observed with probucol. AGI-1067, but not probucol, inhibited basal levels of reactive oxygen species (ROS) in cultured primary human endothelial cells and both basal and hydrogen peroxide-induced levels of ROS in the promonocytic cell line, U937. Furthermore, AGI-1067 inhibited the inducible expression of the redox-sensitive genes, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1, in endothelial cells as well as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 production in peripheral blood mononuclear cells, whereas probucol had no effect. cDNA array hybridization experiments demonstrated that AGI-1067 selectively inhibited the expression of only a subset of TNF-alpha-responsive and nuclear factor-kappaB (NF-kappaB)-inducible genes in endothelial cells. The inhibitory effect of AGI-1067 on inducible VCAM-1 gene expression occurred at the transcriptional level, yet AGI-1067 had no effect on the activation of the redox-sensitive transcription factor NF-kappaB. These studies suggest that the anti-inflammatory and antiatherosclerotic properties of AGI-1067 may be due to selective inhibition of redox

  2. MiR-129-5p-mediated Beclin-1 suppression inhibits endothelial cell autophagy in atherosclerosis

    PubMed Central

    Geng, Zhaohua; Xu, Fei; Zhang, Yiguan

    2016-01-01

    Endothelial cell injury and subsequent death play an essential role in the pathogenesis of atherosclerosis. Autophagy of endothelial cells antagonizes the development of atherosclerosis, whereas the underlying molecular mechanisms are unclear. MicroRNA-129-5p (miR-129-5p) is a well-defined tumor suppressorin some types of cancer, while it is unknown whether miR-129-5p may also play a role in the development of atherosclerosis. Here, we addressed this question in the current study. We examined the levels of endothelial cell autophagy in ApoE (-/-) mice suppled with high-fat diet (HFD), a mouse model for atherosclerosis (simplified as HFD mice). We analyzed the levels of Beclin-1 and the levels of miR-129-5p in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-129-5p and 3’-UTR of Beclin-1 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-129-5p were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs). We found that HFD mice developed atherosclerosisin 12 weeks, while the control ApoE (-/-) mice that had received normal diet (simplified as CTL mice) did not. Compared to CTL mice, HFD mice had significantly lower levels of endothelial cell autophagy, resulting from decreases in Beclin-1 protein, but not mRNA. The decreases in Beclin-1 in endothelial cells were due to HFD-induced increases inmiR-129-5p, which suppressed the translation of Beclin-1 mRNA via 3’-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Together, these data suggest that upregulation of miR-129-5p by HFD may impair the protective effects of endothelial cell autophagy against development of atherosclerosis through suppressing protein translation of Beclin-1. PMID:27186312

  3. Pigment epithelium-derived factor attenuates myocardial fibrosis via inhibiting Endothelial-to-Mesenchymal Transition in rats with acute myocardial infarction

    PubMed Central

    Zhang, Hao; Hui, Hongliang; Li, Zhimin; Pan, Jiajun; Jiang, Xia; Wei, Tengteng; Cui, Huazhu; Li, Lei; Yuan, Xulong; Sun, Teng; Liu, Zhiwei; Zhang, Zhongming; Dong, Hongyan

    2017-01-01

    Endothelial mesenchymal transition (EndMT) plays a critical role in the pathogenesis and progression of interstitial and perivascular fibrosis after acute myocardial infarction (AMI). Pigment epithelium-derived factor (PEDF) is shown to be a new therapeutic target owing to its protective role in cardiovascular disease. In this study, we tested the hypothesis that PEDF is an endogenous inhibitor of EndMT and represented a novel mechanism for its protective effects against overactive cardiac fibrosis after AMI. Masson’s trichrome (MTC) staining and picrosirius red staining revealed decreased interstitial and perivascular fibrosis in rats overexpressing PEDF. The protective effect of PEDF against EndMT was confirmed by co-labeling of cells with the myofibroblast and endothelial cell markers. In the endothelial cells of microvessels in the ischemic myocardium, the inhibitory effect of PEDF against nuclear translocation of β-catenin was observed through confocal microscopic imaging. The correlation between antifibrotic effect of PEDF and inactivation of β-catenin was confirmed by co-transfecting cells with lentivirus carrying PEDF or PEDF RNAi and plasmids harboring β-catenin siRNA(r) or constitutive activation of mutant β-catenin. Taken together, these results establish a novel finding that PEDF could inhibit EndMT related cardiac fibrosis after AMI by a mechanism dependent on disruption of β-catenin activation and translocation. PMID:28167820

  4. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

    SciTech Connect

    Barnwell, J.W.; Ockenhouse, C.F.; Knowles, D.M. II

    1985-11-01

    Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. They show here the monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocyte,s and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an /sup 125/I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.

  5. Inhibition of B16 melanoma growth and metastasis in C57BL mice by vaccination with a syngeneic endothelial cell line

    PubMed Central

    Yoshiura, Kenta; Nishishita, Toshihide; Nakaoka, Takashi; Yamashita, Naohide; Yamashita, Naomi

    2009-01-01

    Background Key role of angiogenesis in tumor growth and metastasis based on accumulating evidence and recent progress of immunotherapy have led us to investigate vaccine therapy targeting tumor angiogenesis. Methods C57BL/6J mice were vaccinated with a syngeneic endothelial cell line Tpit/E by subcutaneous injection once a week. Prior to ninth vaccination, the mice were challenged with B16/F10 melanoma cells by subcutaneous inoculation on the back for the tumor growth model or by tail venous injection for the lung metastasis model. Development of subcutaneous tumor and lung metastasis was monitored by computed tomography scanning, which enabled accurate evaluation with the minimized sacrifice of mice. Results Vaccination with Tpit/E cells inhibited subcutaneous tumor growth and appearance of lung metastasis compared to control. Survival period was elongated in the Tpit/E vaccination in both of the two models. We also obtained hybridomas secreting specific antibodies to Tpit/E cells from a mouse vaccinated with the cells, indicating that specific immune response to the syngeneic endothelial cells was elicited. Conclusion These results suggest that vaccination with an autologous endothelial cell line may be effective against melanoma. PMID:19183492

  6. C1q/TNF-related protein-9 inhibits cytokine-induced vascular inflammation and leukocyte adhesiveness via AMP-activated protein kinase activation in endothelial cells.

    PubMed

    Jung, Chang Hee; Lee, Min Jung; Kang, Yu Mi; Lee, Yoo La; Seol, So Mi; Yoon, Hae Kyeong; Kang, Sang-Wook; Lee, Woo Je; Park, Joong-Yeol

    2016-01-05

    Although recent studies have reported cardioprotective effects of C1q/TNF-related protein 9 (CTRP9), the closet adiponectin paralog, its role on cytokine-induced endothelial inflammation is unknown. We investigated whether CTRP9 prevented inflammatory cytokine-induced nuclear factor-kappa B (NF-κB) activation and inhibited the expression of adhesion molecules and a chemokine in the vascular endothelial cell. We used human aortic endothelial cells (HAECs) to examine the effects of CTRP9 on NF-κB activation and the expression of NF-κB-mediated genes, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1). Tumor necrosis factor alpha (TNFα) was used as a representative proinflammatory cytokine. In an adhesion assay using THP-1 cells, CTRP9 reduced TNFα-induced adhesion of monocytes to HAECs. Treatment with CTRP9 significantly decreased TNFα-induced activation of NF-κB, as well as the expression of ICAM-1, VCAM-1, and MCP-1. In addition, treatment with CTRP9 significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), the downstream target of AMPK. The inhibitory effect of CTRP9 on the expression of ICAM-1, VCAM-1, and MCP-1 and monocyte adhesion to HAECs was abolished after transfection with an AMPKα1-specific siRNA. Our study is the first to demonstrate that CTRP9 attenuates cytokine-induced vascular inflammation in endothelial cells mediated by AMPK activation.

  7. Cat's whiskers tea (Orthosiphon stamineus) extract inhibits growth of colon tumor in nude mice and angiogenesis in endothelial cells via suppressing VEGFR phosphorylation.

    PubMed

    Ahamed, Mohamed B Khadeer; Aisha, Abdalrahim F A; Nassar, Zeyad D; Siddiqui, Jamshed M; Ismail, Z; Omari, S M S; Parish, C R; Majid, A M S Abdul

    2012-01-01

    Cat's whiskers (Orthosiphon stamineus) is commonly used as Java tea to treat kidney stones including a variety of angiogenesis-dependent diseases such as tumorous edema, rheumatism, diabetic blindness, and obesity. In the present study, antitumor potential of standardized 50% ethanol extract of O. stamineus leaves (EOS) was evaluated against colorectal tumor in athymic mice and antiangiogenic efficacy of EOS was investigated in human umbilical vein endothelial cells (HUVEC). EOS at 100 mg/kg caused 47.62 ± 6.4% suppression in tumor growth, while at 200 mg/kg it caused 83.39 ± 4.1% tumor regression. Tumor histology revealed significant reduction in extent of vascularization. Enzyme-linked immunosorbent assay showed EOS (200 mg/kg) significantly reduced the vascular endothelial growth factor (VEGF) level in vitro (211 ± 0.26 pg/ml cell lysate) as well as in vivo (90.9 ± 2 pg/g tissue homogenate) when compared to the control (378 ± 5 and 135.5 ± 4 pg, respectively). However, EOS was found to be noncytotoxic to colon cancer and endothelial cells. In vitro, EOS significantly inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs). EOS suppressed VEGF-induced phosphorylation of VEGF receptor-2 in HUVECs. High performance liquid chromatography (HPLC) analysis of EOS showed high rosmarinic acid contents, whereas phytochemical analysis revealed high protein and phenolic contents. These results demonstrated that the antitumor activity of EOS may be due to its VEGF-targeted antiangiogenicity.

  8. Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    PubMed Central

    Passacquale, Gabriella; Phinikaridou, Alkystis; Warboys, Christina; Cooper, Margaret; Lavin, Begona; Alfieri, Alessio; Andia, Marcelo E; Botnar, Rene M; Ferro, Albert

    2015-01-01

    Background and Purpose There are conflicting data regarding whether netrin-1 retards or accelerates atherosclerosis progression, as it can lead either to monocyte repulsion from or retention within plaques depending on its cellular source. We investigated the effect of aspirin, which is widely used in cardiovascular prophylaxis, on the synthesis of different isoforms of netrin-1 by endothelial cells under pro-inflammatory conditions, and defined the net effect of aspirin-dependent systemic modulation of netrin-1 on atherosclerosis progression. Experimental Approach Netrin-1 synthesis was studied in vitro using human endothelial cells stimulated with TNF-α, with or without aspirin treatment. In vivo experiments were conducted in ApoE−/− mice fed with a high-fat diet (HFD), receiving either aspirin or clopidogrel. Key Results TNF-α-induced NF-κB activation up-regulated the nuclear isoform of netrin-1, while simultaneously reducing secreted netrin-1. Down-regulation of the secreted isoform compromised the chemorepellent action of the endothelium against monocyte chemotaxis. Aspirin counteracted TNF-α-mediated effects on netrin-1 synthesis by endothelial cells through COX-dependent inhibition of NF-κB and concomitant histone hyperacetylation. Administration of aspirin to ApoE−/− mice on HFD increased blood and arterial wall levels of netrin-1 independently of its effects on platelets, accompanied by reduced plaque size and content of monocytes/macrophages, compared with untreated or clopidogrel-treated mice. In vivo blockade of netrin-1 enhanced monocyte plaque infiltration in aspirin-treated ApoE−/− mice. Conclusions and Implications Aspirin counteracts down-regulation of secreted netrin-1 induced by pro-inflammatory stimuli in endothelial cells. The aspirin-dependent increase of netrin-1 in ApoE−/− mice exerts anti-atherogenic effects by preventing arterial accumulation of monocytes. PMID:25824964

  9. MiR-181c restrains nitration stress of endothelial cells in diabetic db/db mice through inhibiting the expression of FoxO1.

    PubMed

    Yang, Guangwei; Wu, Yuanbo; Ye, Shandong

    2017-04-22

    Endothelial dysfunction played an important role in the progression of diabetes mellitus (DM). miR-181c has been implicated in many diseases, including DM. However, the molecular mechanisms of miR-181c regulate this process remained poorly understood. Healthy ICR mice were divided into control group (n = 10) and db/db DM group (n = 10). The expression of miR-181c and FoxO1 were both investigated in diabetic db/db mice or high glucose-induced endothelial cells (MAECs and END-D). Here we found that down-regulation of miR-181c and the activation of FoxO1/iNOS were observed in mice and endothelial cells. Furthermore, we verified that miR-181c directly targeted and inhibited FoxO1 gene expression by targeting its 3'-UTR through luciferase reporter assay. Knockdown of FoxO1 reversed the up-regulation of iNOS, nitrotyrosine and the down-regulation of p-eNOS(Ser1177)/eNOS in high glucose (30 mM)-induced MAECs cells. In addition, over-expression of miR-181c could reverse the enhanced nitration stress induced by high glucose, while this effect could be attenuated by pcDNA-FoxO1 in MAECs. These results shown that miR-181c attenuated nitration stress through regulating FoxO1 expression and affecting endothelial cell function, which offering a new target for the development of preventive or therapeutic agents against DM.

  10. S-nitrosocaptopril interrupts adhesion of cancer cells to vascular endothelium by suppressing cell adhesion molecules via inhibition of the NF-кB and JAK/STAT signal pathways in endothelial cells.

    PubMed

    Lian, Shu; Lu, Yusheng; Cheng, Yunlong; Yu, Ting; Xie, Xiaodong; Liang, Haiyang; Ye, Yuying; Jia, Lee

    2016-11-15

    Inflammatory cytokines can induce the expression of cell adhesion molecules (CAMs) in endothelial cells. The induction may play an important role in attracting circulating tumor cells (CTCs) to endothelial cells. S-nitrosocaptopril (CapNO) is known to produce vasorelaxation and interfere the hetero-adhesion of CTCs to vascular endothelium via down-regulating the expression of CAMs. To elucidate the mechanisms underlying the inhibition of CapNO on CAMs, in this study, we examined the relationship between cytokines and CAMs expression and investigated the effects of CapNO on cytokine-induced NF-кB and JAK/STAT signal pathways. The activation of CAMs by cytokines was dependent on concentrations and reaction time of cytokines, and the combination of cytokines could produce a strong synergistic effect. IL-1β induced the expression of CAMs on endothelial cells by activating NF-кB and JAK/STAT pathways. CapNO inhibited IL-1β-stimulated NF-кB pathway by down-regulating IKK-α and inducing IкB-α directly. CapNO also inhibited JAK/STAT pathway by inhibiting JAK2 and STAT3 expressions. These effects bring about down-regulating CAMs expression on endothelial cells. These results suggest that CapNO may interrupt adhesion of cancer cells to endothelium by suppressing CAMs via inhibiting the NF-кB and JAK/STAT pathways in endothelial cells.

  11. Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability

    PubMed Central

    Wilkinson, Emma L.; Sidaway, James E.

    2016-01-01

    ABSTRACT Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes. PMID:27543060

  12. Up-regulation of CD81 inhibits cytotrophoblast invasion and mediates maternal endothelial cell dysfunction in preeclampsia

    PubMed Central

    Shen, Li; Diao, Zhenyu; Sun, Hai-Xiang; Yan, Gui-Jun; Wang, Zhiqun; Li, Ruo-Tian; Dai, Yimin; Wang, Jingmei; Li, Jie; Ding, Hailing; Zhao, Guangfeng; Zheng, Mingming; Xue, Pingping; Liu, Mo; Zhou, Yan; Hu, Yali

    2017-01-01

    Preeclampsia (PE) is initiated by abnormal placentation in the early stages of pregnancy, followed by systemic activation of endothelial cells of the maternal small arterioles in the late second or third trimester (TM) of pregnancy. During normal pregnancy, placental cytotrophoblasts (CTBs) invade the maternal uterine wall and spiral arteries, whereas this process is interrupted in PE. However, it is not known how the malformed placenta triggers maternal endothelial crisis and the associated manifestations. Here, we have focused on the association of CD81 with PE. CD81, a member of the tetraspanin superfamily, plays significant roles in cell growth, adhesion, and motility. The function of CD81 in human placentation and its association with pregnancy complications are currently unknown. In the present study, we have demonstrated that CD81 was preferentially expressed in normal first TM placentas and progressively down-regulated with gestation advance. In patients with early-onset severe PE (sPE), CD81 expression was significantly up-regulated in syncytiotrophoblasts (STBs), CTBs and the cells in the villous core. In addition, high levels of CD81 were observed in the maternal sera of patients with sPE. Overexpressing CD81 in CTBs significantly decreased CTB invasion, and culturing primary human umbilical vein endothelial cells (HUVECs) in the presence of a high dose of exogenous CD81 resulted in interrupted angiogenesis and endothelial cell activation in vitro. Importantly, the phenotype of human PE was mimicked in the CD81-induced rat model. PMID:28167787

  13. Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line.

    PubMed

    Konopacka, Agnieszka; Zielińska, Magdalena; Albrecht, Jan

    2008-05-01

    Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

  14. Notoginsenoside R1 inhibits oxidized low-density lipoprotein induced inflammatory cytokines production in human endothelial EA.hy926 cells.

    PubMed

    Su, Ping; Du, Shijing; Li, Hang; Li, Zhi; Xin, Wenfeng; Zhang, Wensheng

    2016-01-05

    Notoginsenoside R1 (NG-R1), a unique and main active ingredient of Panax notoginseng, has been described to exhibit anti-inflammatory activity. However, its protective effects against oxidized low-density lipoprotein (oxLDL)-induced inflammatory injury in vascular endothelial cells have not been clarified. In the present study, we have evaluated the anti-inflammatory effects of NG-R1 on oxLDL-induced endothelial cells and its possible molecular mechanism of action. Our results showed that NG-R1 treatment significantly attenuated oxLDL-induced expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. These effects were accompanied with suppression of oxLDL-induced activation of NF-κB and Mitogen-activated protein kinases (MAPK). Moreover, NG-R1 also increased in Peroxisome proliferator-activated receptor γ (PPARγ) protein expression and transcription levels, and attenuated oxLDL-induced suppression of PPARγ expression. The inhibition of NG-R1 on oxLDL-induced TNF-α and IL-1β productions can be reversed by PPARγ antagonist GW9662. In conclusion, these data suggested that NG-R1 could suppress oxLDL-induced inflammatory cytokines production via activating PPARγ, which subsequently inhibiting oxLDL-induced NF-κB and MAPK activation.

  15. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling

    PubMed Central

    Avanzato, D.; Genova, T.; Fiorio Pla, A.; Bernardini, M.; Bianco, S.; Bussolati, B.; Mancardi, D.; Giraudo, E.; Maione, F.; Cassoni, P.; Castellano, I.; Munaron, L.

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  16. Manassantin A and B isolated from Saururus chinensis inhibit TNF-alpha-induced cell adhesion molecule expression of human umbilical vein endothelial cells.

    PubMed

    Kwon, Oh Eok; Lee, Hyun Sun; Lee, Seung Woong; Chung, Mi Yeon; Bae, Ki Hwan; Rho, Mun-Chual; Kim, Young-Kook

    2005-01-01

    Leukocyte adhesion to the vascular endothelium is a critical initiating step in inflammation and atherosclerosis. We have herein studied the effect of manassantin A (1) and B (2), dineolignans, on interaction of THP-1 monocytic cells and human umbilical vein endothelial cells (HUVEC) and expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HUVEC. When HUVEC were pretreated with 1 and 2 followed by stimulation with TNF-alpha, adhesion of THP-1 cells to HUVEC decreased in dose-dependent manner with IC50 values of 5 ng/mL and 7 ng/mL, respectively, without cytotoxicity. Also, 1 and 2 inhibited TNF-alpha-induced up-regulation of ICAM-1, VCAM-1 and E-selectin. The present findings suggest that 1 and 2 prevent monocyte adhesion to HUVEC through the inhibition of ICAM-1, VCAM-1 and E-selectin expression stimulated by TNF-alpha, and may imply their usefulness for the prevention of atherosclerosis relevant to endothelial activation.

  17. Activation of the thromboxane A2 receptor by 8-isoprostane inhibits the pro-angiogenic effect of vascular endothelial growth factor in scleroderma

    PubMed Central

    Tsou, Pei-Suen; Amin, M. Asif; Campbell, Phil; Zakhem, George; Balogh, Beatrix; Edhayan, Gautam; Ohara, Ray A.; Schiopu, Elena; Khanna, Dinesh; Koch, Alisa E.; Fox, David A.

    2015-01-01

    The pathogenesis of scleroderma (SSc) includes components of autoimmunity, vascular dysfunction, and accumulation of extracellular matrix. 8-isoprostane, an oxidized lipid created by oxidative stress, activates the thromboxane A2 receptor (TXAR) and ROCK pathway. In this study we determined whether the TXAR was activated by 8-isoprostane in SSc endothelial cells (ECs), and whether this pathway inhibited VEGF-induced angiogenesis. Elevated 8-isoprostane was observed in plasma and conditioned media from SSc patients. SSc conditioned media inhibited EC tube formation, while addition of vitamin E, by reducing 8-isoprostane, increased tube formation. VEGF did not induce angiogenesis in SSc ECs, but vitamin E or TXAR inhibition restored its effect. The expression of TXAR, RhoA, and ROCK1/2 were elevated in SSc ECs. ROCK activity and 8-isoprostane-induced ROCK activation were significantly higher in SSc ECs while VEGF had no effect. The hyper-activation of the TXAR leads to inhibition of VEGF-induced angiogenesis, as inhibition of the TXAR pathway results in blockade of 8-isoprostane induced ROCK activation and restoration of VEGF activity. These results suggest that the TXAR pathway plays a crucial role in angiogenesis and that 8-isoprostane is not just a by-product of oxidative stress, but also plays a significant role in the impaired angiogenesis that characterizes SSc. PMID:26288351

  18. Kruppel-like factor 2 inhibit the angiogenesis of cultured human liver sinusoidal endothelial cells through the ERK1/2 signaling pathway

    SciTech Connect

    Zeng, Xiao-Qing; Li, Na; Pan, Du-Yi; Miao, Qing; Ma, Gui-Fen; Liu, Yi-Mei; Tseng, Yu-Jen; Li, Feng; Xu, Li-Li; Chen, Shi-Yao

    2015-09-04

    Kruppel-like factor 2 (KLF2) is a crucial anti-angiogenic factor. However, its precise role in hepatic angiogenesis induced by liver sinusoidal endothelial cells (LSECs) remain unclear. This study was aimed to evaluate the effect of KLF2 on angiogenesis of LSECs and to explore the corresponding mechanism. Cultured human LSECs were infected with different lentiviruses to overexpress or suppress KLF2 expression. The CCK-8 assay, transwell migration assay and tube formation test, were used to investigate the roles of KLF2 in the proliferation, migration and vessel tube formation of LSECs, respectively. The expression and phosphorylation of ERK1/2 were detected by western blot. We discovered that the up-regulation of KLF2 expression dramatically inhibited proliferation, migration and tube formation in treated LSECs. Correspondingly, down-regulation of KLF2 expression significantly promoted proliferation, migration and tube formation in treated LSECs. Additionally, KLF2 inhibited the phosphorylation of ERK1/2 pathway, followed by the function of KLF2 in the angiogenesis of LSECs disrupted. In conclusion, KLF2 suppressed the angiogenesis of LSECs through inhibition of cell proliferation, migration, and vessel tube formation. These functions of KLF2 may be mediated through the ERK1/2 signaling pathway. - Highlights: • Overexpression of KLF2 inhibits the proliferation and migration of LSECs. • Overexpression of KLF2 inhibits the angiogenesis of LSECs. • ERK1/2 signaling pathway involved in the anti-angiogenic process of KLF2 on LSECs.

  19. Soluble factors from the notochordal-rich intervertebral disc inhibit endothelial cell invasion and vessel formation in the presence and absence of pro-inflammatory cytokines

    PubMed Central

    Cornejo, M.C.; Cho, S.K.; Giannarelli, C.; Iatridis, J.C.; Purmessur, D.

    2015-01-01

    Background Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. Objective To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. Design NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNFα [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. Results NCCM (10 & 100%), CS (10 and 100 μg) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNFα. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNFα and CS 100 μg inhibited tubules in Basal conditions whereas CS 10 μg inhibited tubules with TNFα. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNFα. CS and Noggin had no effects on gene expression. Conclusions We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD. PMID:25534363

  20. Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

    PubMed Central

    Tang, Xu-dong; Zhou, Xin; Zhou, Ke-yuan

    2009-01-01

    Aim: To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7). Methods: Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed. Results: Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs. Conclusion: Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer. PMID:19349962

  1. OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

    SciTech Connect

    Omar, Hany A.; Arafa, El-Shaimaa A.; Salama, Samir A.; Arab, Hany H.; Wu, Chieh-Hsi; Weng, Jing-Ru

    2013-11-01

    Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to study the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy. - Highlights: • The antiangiogenic activity of OSU-A9 in HUVECs was explored. • OSU-A9 inhibited HUVECs proliferation, migration, invasion and tube formation. • OSU-A9

  2. Dicer Knockdown Inhibits Endothelial Cell Tumor Growth via MicroRNA 21a-3p Targeting of Nox-4*

    PubMed Central

    Gordillo, Gayle M.; Biswas, Ayan; Khanna, Savita; Pan, Xueliang; Sinha, Mithun; Roy, Sashwati; Sen, Chandan K.

    2014-01-01

    MicroRNAs (miR) are emerging as biomarkers and potential therapeutic targets in tumor management. Endothelial cell tumors are the most common soft tissue tumors in infants, yet little is known about the significance of miR in regulating their growth. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that post-transcriptional gene silencing of dicer, the enzyme that converts pre-miR to mature miR, can prevent tumor formation in vivo. Tumors were formed in eight of eight mice injected with EOMA cells transfected with control shRNA but formed in only four of ten mice injected with EOMA cells transfected with dicer shRNA. Tumors that formed in the dicer shRNA group were significantly smaller than tumors in the control group. This response to dicer knockdown was mediated by up-regulated miR 21a-3p activity targeting the nox-4 3′-UTR. EOMA cells were transfected with miR 21a-3p mimic and luciferase reporter plasmids containing either intact nox-4 3′-UTR or with mutation of the proposed 3′-UTR miR21a-3p binding sites. Mean luciferase activity was decreased by 85% in the intact compared with the site mutated vectors (p < 0.01). Attenuated Nox-4 activity resulted in decreased cellular hydrogen peroxide production and decreased production of oxidant-inducible monocyte chemoattractant protein-1, which we have previously shown to be critically required for endothelial cell tumor formation. These findings provide the first evidence establishing the significance of dicer and microRNA in promoting endothelial cell tumor growth in vivo. PMID:24497637

  3. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

    PubMed

    Lin, Jibin; He, Shaolin; Sun, Xinghui; Franck, Gregory; Deng, Yihuan; Yang, Dafeng; Haemmig, Stefan; Wara, A K M; Icli, Basak; Li, Dazhu; Feinberg, Mark W

    2016-09-01

    Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and

  4. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: L-glutamine inhibits the generation of L-arginine by cultured endothelial cells

    SciTech Connect

    Sessa, W.C.; Hecker, M.; Mitchell, J.A. Vane, J.R. )

    1990-11-01

    The mechanism by which L-glutamine (L-Gln) inhibits the release of endothelium-derived factor from bovine aortic cultured endothelial cells was investigated. The intracellular concentration of L-arginine (L-Arg) in Arg-depleted endothelial cells was inversely related to the level of L-Gln. Removal of L-Gln from the culture medium (usually containing L-Gln at 2 mM) abolished the inhibitory effect of the culture medium on L-Arg generation. L-Gln (0.2 and 2 mM) but not D-Gln inhibited the generation of L-Arg by both Arg-depleted and nondepleted endothelial cells. L-Gln did not interfere with the uptake of L-Arg or the metabolism of L-Arg-L-Phe to L-Arg but inhibited the formation of L-Arg from L-citrulline (L-Cit), L-Cit-L-Phe, and N{sup G}-monomethyl-L-arginine. L-Gln also inhibited the conversion of L-({sup 14}C)Cit to L-({sup 14}C)Arg by Arg-depleted endothelial cells. However, L-Gln did not inhibit the conversion of L-argininosuccinic acid to L-Arg by endothelial cell homogenates. Thus, L-Gln interferes with the conversion of L-Cit to L-Arg probably by acting on argininosuccinate synthetase rather than argininosuccinate lyase. L-Gln also inhibited the generation of L-Arg by the monocyte-macrophage cell line J774 but had no effect on the conversion of L-Cit to L-Arg by these cells. As the release of endothelium-derived relaxing factor from cultured and non-cultured endothelial cells is limited by the availability of L-Arg, endogenous L-Gln may play a regulatory role in the biosynthesis of endothelium-derived relaxing factor.

  5. Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

    SciTech Connect

    Maruyama, I.; Majerus, P.W.

    1987-05-01

    We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of /sup 125/I-thrombin-thrombomodulin complexes, but not /sup 125/I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of /sup 125/I-thrombin and diisopropylphosphoryl (DIP) /sup 125/I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C.

  6. Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration.

    PubMed

    Kim, Sung Hwan; Kim, Hyunjin; Ku, Hyeong Jun; Park, Jung Hyun; Cha, Hanvit; Lee, Seoyoon; Lee, Jin Hyup; Park, Jeen-Woo

    2016-12-01

    Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF), secreted by the retinal pigment epithelium (RPE), in pathological angiogenesis and the development of choroidal neovascularization (CNV) in age-related macular degeneration (AMD). RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA) is a competitive inhibitor of NADP(+)-dependent isocitrate dehydrogenase (IDH), which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS). Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

  7. Role of pigment epithelium-derived factor in the involution of hemangioma: Autocrine growth inhibition of hemangioma-derived endothelial cells

    SciTech Connect

    Kim, Kyung-Jin; Yun, Jang-Hyuk; Heo, Jong-Ik; Lee, Eun Hui; Min, Hye Sook; Choi, Tae Hyun; Cho, Chung-Hyun

    2014-11-14

    Highlights: • PEDF was expressed and induced during the involuting phase of IH. • PEDF inhibited the cell growth of the involuting HemECs in an autocrine manner. • PEDF suppression restored the impaired cell growth of the involuting HemECs. - Abstract: Hemangioma is a benign tumor derived from abnormal blood vessel growth. Unlike other vascular tumor counterparts, a hemangioma is known to proliferate during its early stage but it is followed by a stage of involution where regression of the tumor occurs. The critical onset leading to the involution of hemangioma is currently not well understood. This study focused on the molecular identities of the involution of hemangioma. We demonstrated that a soluble factor released from the involuting phase of hemangioma-derived endothelial cells (HemECs) and identified pigment epithelium-derived factor (PEDF) as an anti-angiogenic factor that was associated with the growth inhibition of the involuting HemECs. The growth inhibition of the involuting HemECs was reversed by suppression of PEDF in the involuting HemECs. Furthermore, we found that PEDF was more up-regulated in the involuting phase of hemangioma tissues than in the proliferating or the involuted. Taken together, we propose that PEDF accelerates the involution of hemangioma by growth inhibition of HemECs in an autocrine manner. The regulatory mechanism of PEDF expression could be a potential therapeutic target to treat hemangiomas.

  8. Cyclic phosphatidic acid inhibits alkyl-glycerophosphate-induced downregulation of histone deacetylase 2 expression and suppresses the inflammatory response in human coronary artery endothelial cells.

    PubMed

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    2014-01-01

    Activation of the endothelium by alkyl-glycerophosphate (AGP) has been implicated in the development of atherosclerosis. Our previous study suggested that cyclic phosphatidic acid (cPA) inhibits arterial wall remodeling in a rat model in vivo. However, the mechanisms through which specific target genes are regulated during this process remain unclear. Here, we examined whether cPA inhibited AGP-induced expression of class I histone deacetylases (HDACs, namely HDAC1, HDAC2, HDAC3, and HDAC8), which may affect subsequent transcriptional activity of target genes. Our experimental results showed that human coronary artery endothelial cells (HCAECs) expressed high levels of HDAC2 and low levels HDAC1, HDAC3, and HDAC8. Moreover, AGP treatment induced downregulation of HDAC2 expression in HCAECs. However, cotreatment with cPA inhibited this downregulation of HDAC2 expression. Interestingly, treatment with AGP increased the expression and secretion of endogenous interleukin (IL)-6 and IL-8; however, this effect was inhibited when HCAECs were cotreated with cPA or the synthetic peroxisome proliferator-activator receptor gamma (PPARγ) antagonist T0070907. Thus, our data suggested that cPA may have beneficial effects in inflammation-related cardiovascular disease by controlling HDAC2 regulation.

  9. Olive oil compounds inhibit the paracrine regulation of TNF-α-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression.

    PubMed

    Lamy, Sylvie; Ben Saad, Aroua; Zgheib, Alain; Annabi, Borhane

    2016-01-01

    The established causal relationship between the chronic inflammatory microenvironment, tumor development and cancer recurrence has provided leads for developing novel preventive strategies. Accumulating experimental, clinical and epidemiological data has provided support for the chemopreventive properties of olive oil compounds traditionally found within the Mediterranean diet. In this study, we investigated whether tyrosol (Tyr), hydroxytyrosol, oleuropein and oleic acid (OA), four compounds contained in extra virgin olive oil, can prevent tumor necrosis factor (TNF)-α-induced expression of cyclooxygenase (COX)-2 (an inflammation biomarker) in a human glioblastoma cell (U-87 MG) model. We found that Tyr and OA significantly inhibited TNF-α-induced COX-2 gene and protein expression, as well as PGE2 secretion. Both compounds also inhibited TNF-α-induced JNK and ERK phosphorylation, whereas only Tyr inhibited TNF-α-induced NF-κB phosphorylation. Paracrine-regulated migration of human brain microvascular endothelial cells (HBMECs) was assessed using growth factor-enriched conditioned media (CM) isolated from U-87 MG cells. We found that while PGE2 triggered HBMEC migration, the CM isolated from U-87 MG cells, where either COX-2 or NF-κB had been silenced or had been treated with Tyr or OA, exhibited decreased chemotactic properties. These observations demonstrate that olive oil compounds inhibit the effect of the chronic inflammatory microenvironment on glioblastoma progression through TNF-α actions and may be useful in cancer chemoprevention.

  10. Davallia bilabiata inhibits TNF-α-induced adhesion molecules and chemokines by suppressing IKK/NF-kappa B pathway in vascular endothelial cells.

    PubMed

    Yang, Rong-Chi; Chang, Cheng-Chieh; Sheen, Jer-Ming; Wu, Hsiao-Ting; Pang, Jong-Hwei S; Huang, Sheng-Teng

    2014-01-01

    Davallia bilabiata (D. bilabiata) is also called GuSuiBu in Taiwan and is used as a substitute for Drynaria fortunei J. Sm. It is often used for trauma and bone repair. The inhibitory effect of D. bilabiata on inflammatory activity has not been reported. In the present study, we aimed to study the mechanism of anti-inflammation of D. bilabiata on the adhesion of leukocytes to vascular endothelial cells. The results showed that D. bilabiata, at concentrations without cytotoxic effect, inhibited the adhesion of monocytes (THP-1) to the TNF-α-stimulated human umbilical vascular endothelial cells (HUVECs). D. bilabiata suppressed the expression of the adhesion molecules ICAM, VCAM, and E-selectin at both the mRNA and protein level. In addition, both of the TNF-α-induced mRNA and protein expression of chemokines including fractalkine/CX3CL1, MCP-1 and RANTES as well as the level of secreted soluble fractalkine were decreased by D. bilabiata. We also verified that D. bilabiata inhibited the TNF-α-induced nuclear translocation of NF-κB through the inhibitory process on the TNF-α-activated phosphorylation of IKKα, IKKβ, IκB and NF-κB. All together, we concluded that the D. bilabiata affected the canonical pathway of TNF-α-induced NF-κB activation and down-regulated cell adhesion molecules and chemokine expression through inhibition of the NF-κB/IκBα/IKK signaling pathway. These findings strongly indicated that D. bilabiata might be a promising alternative/adjunct treatment for inflammatory diseases, such as rheumatoid arthritis and osteoarthritis.

  11. Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II-mediated BMP4-oxidative stress cascade

    PubMed Central

    Lau, Yeh Siang; Tian, Xiao Yu; Mustafa, Mohd Rais; Murugan, Dharmani; Liu, Jian; Zhang, Yang; Lau, Chi Wai; Huang, Yu

    2013-01-01

    BACKGROUND AND PURPOSE Boldine is a potent natural antioxidant present in the leaves and bark of the Chilean boldo tree. Here we assessed the protective effects of boldine on endothelium in a range of models of diabetes, ex vivo and in vitro. EXPERIMENTAL APPROACH Vascular reactivity was studied in mouse aortas from db/db diabetic and normal mice. Reactive oxygen species (ROS) production, angiotensin AT1 receptor localization and protein expression of oxidative stress markers in the vascular wall were evaluated by dihydroethidium fluorescence, lucigenin enhanced-chemiluminescence, immunohistochemistry and Western blot respectively. Primary cultures of mouse aortic endothelial cells, exposed to high concentrations of glucose (30 mmol L−1) were also used. KEY RESULTS Oral treatment (20 mg kg−1day−1, 7 days) or incubation in vitro with boldine (1 μmol L−1, 12 h) enhanced endothelium-dependent aortic relaxations of db/db mice. Boldine reversed impaired relaxations induced by high glucose or angiotensin II (Ang II) in non-diabetic mouse aortas while it reduced the overproduction of ROS and increased phosphorylation of eNOS in db/db mouse aortas. Elevated expression of oxidative stress markers (bone morphogenic protein 4 (BMP4), nitrotyrosine and AT1 receptors) were reduced in boldine-treated db/db mouse aortas. Ang II-stimulated BMP4 expression was inhibited by boldine, tempol, noggin or losartan. Boldine inhibited high glucose-stimulated ROS production and restored the decreased phosphorylation of eNOS in mouse aortic endothelial cells in culture. CONCLUSIONS AND IMPLICATIONS Boldine reduced oxidative stress and improved endothelium-dependent relaxation in aortas of diabetic mice largely through inhibiting ROS overproduction associated with Ang II-mediated BMP4-dependent mechanisms. PMID:23992296

  12. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway.

    PubMed

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-07

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by N(ω)-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension.

  13. Functional inhibition of urea transporter UT-B enhances endothelial-dependent vasodilatation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway

    PubMed Central

    Sun, Yi; Lau, Chi-Wai; Jia, Yingli; Li, Yingjie; Wang, Weiling; Ran, Jianhua; Li, Fei; Huang, Yu; Zhou, Hong; Yang, Baoxue

    2016-01-01

    Mammalian urea transporters (UTs), UT-A and UT-B, are best known for their role in urine concentration. UT-B is especially distributed in multiple extrarenal tissues with abundant expression in vascular endothelium, but little is known about its role in vascular function. The present study investigated the physiological significance of UT-B in regulating vasorelaxations and blood pressure. UT-B deletion in mice or treatment with UT-B inhibitor PU-14 in Wistar-Kyoto rats (WKYs) and spontaneous hypertensive rats (SHRs) reduced blood pressure. Acetylcholine-induced vasorelaxation was significantly augmented in aortas from UT-B null mice. PU-14 concentration-dependently produced endothelium-dependent relaxations in thoracic aortas and mesenteric arteries from both mice and rats and the relaxations were abolished by Nω-nitro-L-arginine methyl ester. Both expression and phosphorylation of endothelial nitric oxide synthase (eNOS) were up-regulated and expression of arginase I was down-regulated when UT-B was inhibited both in vivo and in vitro. PU-14 induced endothelium-dependent relaxations to a similar degree in aortas from 12 weeks old SHRs or WKYs. In summary, here we report for the first time that inhibition of UT-B plays an important role in regulating vasorelaxations and blood pressure via up-regulation of L-arginine-eNOS-NO pathway, and it may become another potential therapeutic target for the treatment of hypertension. PMID:26739766

  14. Dlitiazem inhibits the oxidative stress induced by angiotensin II through growth hormone secretagogue receptor type 1a in human umbilicus vein endothelial cells.

    PubMed

    Zhou, Lingyun; Yang, Meng; Zuo, Shanru; Guan, Xiaofeng; Wang, Jianglin; Chen, Qingjie; Zuo, Xiaocong; Jia, Sujie; Guo, Ren

    2017-02-16

    Diltiazem has been used for post-transplant hypertension, but the mechanism underlying its protective effect of endothelial cells against angiotensin II (Ang II) - induced impairment remains unclear. Human umbilicus vein endothelial cells (HUVECs) were cultured and divided into seven groups: control, Ang II (10(-6)M), diltiazem (10(-6)M), [D-Lys3]-GHRP-6(25μM), diltiazem (10(-6)M)+Ang II (10(-6)M), losartan (10(-6)M)+Ang II (10(-6)M), [D-Lys3]-GHRP-6 (25μM) + Dil(10(-6)M)+Ang II (10(-6)M) groups. Nitric oxide (NO) production, intracellular reactive oxygen species (ROS) levels, protein and mRNA expressions of endothelial nitric oxide synthase (eNOS) and p47 phox subunit of NADPH were evaluated. Results indicated that pre- treatment with diltiazem significantly decreased the intracellular ROS levels and increased NO production. Treatment with 10(-6)M Ang II for 24h induced a significant decrease in the mRNA and protein levels of eNOS, which was significantly increased by the pre-incubated with diltiazem (10(-6)M). Treatment with 10(-6)M Ang II for 24h induced a significant increase in the mRNA and protein levels of p47 phox subunit of NADHP oxidase, which was significantly decreased by the pre-incubated with diltiazem. However, all of these protective roles of diltiazem were attenuated by pre-incubation of [D-Lys3]-GHRP-6. The results reveal that diltiazem inhibits the Ang II - induced oxidative stress in HUVECs, which may be partly mediated by GHSR1a.

  15. Inhibition of plaque neovascularization and intimal hyperplasia by specific targeting vascular endothelial growth factor with bevacizumab-eluting stent: an experimental study.

    PubMed

    Stefanadis, Christodoulos; Toutouzas, Konstantinos; Stefanadi, Elli; Lazaris, Andreas; Patsouris, Efstratios; Kipshidze, Nicholas

    2007-12-01

    Neovascularization is associated with destabilization of atheromatic plaques. Increased expression of vascular endothelial growth factor (VEGF) is important in the process of neovascularization. We assessed the effect of bevacizumab, a monoclonal antibody specific for VEGF, on neovascularization. We used 12 New Zealand rabbits under atherogenic diet for 3 weeks. We immersed a phosphorycholine coated stent into a solution of 4 ml bevacizumab according to previous studies. Twelve eluting stents and 12 non-eluting stents were implanted in the middle segment of the rabbit's iliac arteries. Follow-up angiography was performed at 4 weeks and tissues were obtained for histological analysis. The procedure of stent loading with bevacizumab and stent implantation was successful. There was no difference in angiographic measurements before, after implantation and at follow-up between the two groups. mean neointimal thickness (0.09+/-0.02 versus 0.12+/-0.02 mm, p<0.01), and mean neointimal area (1.08+/-0.09 versus 1.20+/-0.12 mm(2), p<0.01) were less in the bevacizumab treated segments. bevacizumab-treated arterial segments demonstrated significantly decreased microvessel density compared with the control group (1.69+/-0.06 CI: 1.65-1.73 versus 15.68+/-0.56 CI: 15.32-16.04 vessels per mm(2), p<0.001) and vegf expression was decreased in the media and adventitia of bevacizumab group. Endothelialization, inflammation and injury scores were similar between the two groups. These results suggest that bevacizumab-eluting stent implantation in rabbit iliac arteries is safe, and inhibits neovascularization without affecting the endothelialization.

  16. Confluence of Vascular Endothelial Cells Induces Cell Cycle Exit by Inhibiting p42/p44 Mitogen-Activated Protein Kinase Activity

    PubMed Central

    Viñals, Francesc; Pouysségur, Jacques

    1999-01-01

    Like other cellular models, endothelial cells in cultures stop growing when they reach confluence, even in the presence of growth factors. In this work, we have studied the effect of cellular contact on the activation of p42/p44 mitogen-activated protein kinase (MAPK) by growth factors in mouse vascular endothelial cells. p42/p44 MAPK activation by fetal calf serum or fibroblast growth factor was restrained in confluent cells in comparison with the activity found in sparse cells. Consequently, the induction of c-fos, MAPK phosphatases 1 and 2 (MKP1/2), and cyclin D1 was also restrained in confluent cells. In contrast, the activation of Ras and MEK-1, two upstream activators of the p42/p44 MAPK cascade, was not impaired when cells attained confluence. Sodium orthovanadate, but not okadaic acid, restored p42/p44 MAPK activity in confluent cells. Moreover, lysates from confluent 1G11 cells more effectively inactivated a dually phosphorylated active p42 MAPK than lysates from sparse cells. These results, together with the fact that vanadate-sensitive phosphatase activity was higher in confluent cells, suggest that phosphatases play a role in the down-regulation of p42/p44 MAPK activity. Enforced long-term activation of p42/p44 MAPK by expression of the chimera ΔRaf-1:ER, which activates the p42/p44 MAPK cascade at the level of Raf, enhanced the expression of MKP1/2 and cyclin D1 and, more importantly, restored the reentry of confluent cells into the cell cycle. Therefore, inhibition of p42/p44 MAPK activation by cell-cell contact is a critical step initiating cell cycle exit in vascular endothelial cells. PMID:10082542

  17. Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization

    PubMed Central

    Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

    2017-01-01

    Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV

  18. Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

    PubMed

    Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

    2017-01-01

    Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by (1)H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

  19. Alcohol consumption negates estrogen-mediated myocardial repair in ovariectomized mice by inhibiting endothelial progenitor cell mobilization and function.

    PubMed

    Mackie, Alexander R; Krishnamurthy, Prasanna; Verma, Suresh K; Thorne, Tina; Ramirez, Veronica; Qin, Gangjian; Abramova, Tatiana; Hamada, Hiromichi; Losordo, Douglas W; Kishore, Raj

    2013-06-21

    We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.

  20. Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis.

    PubMed

    Martin, Sophie; Giannone, Grégory; Andriantsitohaina, Ramaroson; Martinez, M Carmen

    2003-07-01

    1. Epidemiological studies have suggested that moderate consumption of natural dietary polyphenolic compounds might reduce the risk of cardiovascular disease and also protect against cancer. The present study investigates the effects of delphinidin, an anthocyanin present in red wine, on bovine aortic endothelial cells apoptosis. 2. Based on flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and detection of mitochondrial cytochrome c release, we show that delphinidin (10(-2) g l(-1)) alone had no effect either on necrosis or on apoptosis, but it significantly reduced apoptosis elicited by actinomycin D (1 micro g ml(-1), 24 h) and 7beta-hydroxycholesterol (10 micro g ml(-1), 18 h). 3. The protective effect of delphinidin was abolished by inhibitors of nitric oxide-synthase (NOS) (L-NA, 100 micro M and SMT, 100 micro M), guanylyl cyclase (ODQ, 100 micro M) and MAP kinase (PD98059, 30 micro M). 4. Western blot analysis and protein detection by confocal microscopy demonstrate that the antiapoptotic effect of delphinidin was associated with an increased endothelial NOS expression mediated by a MAP kinase pathway. 5. Finally, delphinidin alone had no effect on cytosolic-free calcium ([Ca(2+)](i)), but normalized the changes in [Ca(2+)](i) produced by actinomycin D towards the control values, suggesting that the antiapoptotic effect of delphinidin is associated with the maintenance of [Ca(2+)](i) in the physiological range. 6. All of the observed effects of delphinidin may preserve endothelium integrity, the alteration of which lead to pathologies including cardiovascular diseases, such as atherosclerosis, and is often associated with cancers. In conclusion, the protective effect of delphinidin against endothelial cell apoptosis contributes to understand the potential benefits of a consumption rich in polyphenols.

  1. Inhibitive effect of purple sweet potato leaf extract and its components on cell adhesion and inflammatory response in human aortic endothelial cells.

    PubMed

    Chao, Pi-Yu; Huang, Ya-Ping; Hsieh, Wen-Bin

    2013-01-01

    This study investigated the effects of purple sweet potato leaf extract (PSPLE) and its components, cyanidin and quercetin, on human aortic endothelial cells (HAECs) during the inflammatory process. HAECs were pretreated with 100 μg/mL PSPLE or 10 μM quercetin, cyanidin or aspirin for 18 h followed by TNF-α (2 ng/mL) for 6 h, and U937 cell adhesion was determined. Adhesion molecule expression and CD40 were evaluated; NFκB p65 protein localization and DNA binding were assessed. PSPLE, aspirin, cyanidin and quercetin significantly inhibited TNF-α-induced monocyte-endothelial cell adhesion (p < 0.05). Cyanidin, quercetin and PSPLE also significantly attenuated VCAM-1, IL-8 and CD40 expression, and quercetin significantly attenuated ICAM-1 and E-selectin expression (p < 0.05). Significant reductions in NFκB expression and DNA binding by aspirin, cyanidin and quercetin were also observed in addition to decreased expression of ERK1, ERK2 and p38 MAPK (p < 0.05). Thus, PSPLE and its components, cyanidin and quercetin, have anti-inflammatory effects through modulation of NFκB and MAPK signaling. Further in vivo studies are necessary to explore the possible therapeutic effects of PSPLE on atherosclerosis.

  2. Amla (Emblica officinalis Gaertn.) extract inhibits lipopolysaccharide-induced procoagulant and pro-inflammatory factors in cultured vascular endothelial cells.

    PubMed

    Rao, Theertham Pradyumna; Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Kato-Yasuda, Naomi; Suzuki, Koji

    2013-12-01

    Amla (Emblica officinalis Gaertn.) has been used for many centuries in traditional Indian Ayurvedic formulations for the prevention and treatment of many inflammatory diseases. The present study evaluated the anti-inflammatory and anticoagulant properties of amla fruit extract. The amla fruit extract potentially and significantly reduced lipopolysaccharide (LPS)-induced tissue factor expression and von Willebrand factor release in human umbilical vein endothelial cells (HUVEC) in vitro at clinically relevant concentrations (1-100 μg/ml). In a leucocyte adhesion model of inflammation, it also significantly decreased LPS-induced adhesion of human monocytic cells (THP-1) to the HUVEC, as well as reduced the expression of endothelial-leucocyte adhesion molecule-1 (E-selectin) in the target cells. In addition, the in vivo anti-inflammatory effects were evaluated in a LPS-induced endotoxaemia rat model. Oral administration of the amla fruit extract (50 mg/kg body weight) significantly decreased the concentrations of pro-inflammatory cytokines, TNF-α and IL-6 in serum. These results suggest that amla fruit extract may be an effective anticoagulant and anti-inflammatory agent.

  3. Sargaquinoic Acid Inhibits TNF-α-Induced NF-κB Signaling, Thereby Contributing to Decreased Monocyte Adhesion to Human Umbilical Vein Endothelial Cells (HUVECs).

    PubMed

    Gwon, Wi-Gyeong; Lee, Bonggi; Joung, Eun-Ji; Choi, Min-Woo; Yoon, Nayoung; Shin, Taisun; Oh, Chul-Woong; Kim, Hyeung-Rak

    2015-10-21

    Sargaquinoic acid (SQA) has been known for its antioxidant and anti-inflammatory properties. This study investigated the effects of SQA isolated from Sargassum serratifolium on the inhibition of tumor necrosis factor (TNF)-α-induced monocyte adhesion to human umbilical vein endothelial cells (HUVECs). SQA decreased the expression of cell adhesion molecules such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as chemotactic cytokines such as interleukin-8 and monocyte chemoattractant protein-1 in TNF-α-treated HUVECs. As a result, SQA prevented monocyte adhesion to TNF-α-induced adhesion. SQA also inhibited TNF-α-induced nuclear factor kappa B (NF-κB) translocation into the nucleus by preventing proteolytic degradation of inhibitor κB-α. Overall, SQA protects against TNF-α-induced vascular inflammation through inhibition of the NF-κB pathway in HUVECs. These data suggest that SQA may be used as a therapeutic agent for vascular inflammatory diseases such as atherosclerosis.

  4. 15-Deoxy-Δ12,14-Prostaglandin J2 Modifies Components of the Proteasome and Inhibits Inflammatory Responses in Human Endothelial Cells

    PubMed Central

    Marcone, Simone; Evans, Paul; Fitzgerald, Desmond J.

    2016-01-01

    15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an electrophilic lipid mediator derived from PGD2 with potent anti-inflammatory effects. These are likely to be due to the covalent modification of cellular proteins, via a reactive α,β-unsaturated carbonyl group in its cyclopentenone ring. This study was carried out to identify novel cellular target(s) for covalent modification by 15d-PGJ2 and to investigate the anti-inflammatory effects of the prostaglandin on endothelial cells (EC). The data presented here show that 15d-PGJ2 modifies and inhibits components of the proteasome and consequently inhibits the activation of the NF-κB pathway in response to TNF-α. This, in turn, inhibits the adhesion and migration of monocytes toward activated EC, by reducing the expression of adhesion molecules and chemokines in the EC. The effects are consistent with the covalent modification of 13 proteins in the 19S particle of the proteasome identified by mass spectrometry and the suppression of proteasome function, and were similar to the effects seen with a known proteasome inhibitor (MG132). The ubiquitin–proteasome system has been implicated in the regulation of several inflammatory processes and the observation that 15d-PGJ2 profoundly affects the proteasome functions in human EC suggests that 15d-PGJ2 may regulate the progression of inflammatory disorders such as atherosclerosis. PMID:27833612

  5. The dineolignan from Saururus chinensis, manassantin B, inhibits tumor-induced angiogenesis via downregulation of matrix metalloproteinases 9 in human endothelial cells.

    PubMed

    Liu, Zhaojie; Lu, Hong; Liu, Rong; Chen, Bin; Wang, Shan; Ma, Junchao; Fu, Jianjiang

    2014-08-01

    Manassantin B (MB) is a neolignan isolated from Saururus chinensis that exhibits a range of activities, including anti-inflammatory, antiseptic and antitumor activity. MB was recently found to affect cell adhesion and expression of several adhesion molecules. Based on the important roles of these adhesion molecules in angiogenesis, we evaluated a possible role for MB in tumor-induced angiogenesis in endothelial cells (ECs). In the present study, we found that MB blocked tumor-induced tube formation of ECs and significantly inhibited the invasion of ECs through the reconstituted basement membrane. MB suppressed the activity of matrix metalloproteinases (MMPs) and downregulated the expression of matrix metalloproteinases 9. Western blotting showed reduction of RUNX2 activation by MB. RUNX2 transcription factor assay and chromatin immunoprecipitation assay showed that the interaction between RUNX2 and target sequences in the matrix metalloproteinases 9 promoters was inhibited by MB. Our findings suggested that the inhibitory effects of MB on tumor-induced angiogenesis were caused by matrix metalloproteinases 9 inhibition, which was associated with the downregulation of RUNX2 transcriptional activity.

  6. Glossogyne tenuifolia Extract Inhibits TNF-α-Induced Expression of Adhesion Molecules in Human Umbilical Vein Endothelial Cells via Blocking the NF-kB Signaling Pathway.

    PubMed

    Hsuan, Chin-Feng; Hsu, Hsia-Fen; Tseng, Wei-Kung; Lee, Thung-Lip; Wei, Yu-Feng; Hsu, Kwan-Lih; Wu, Chau-Chung; Houng, Jer-Yiing

    2015-09-17

    Chronic inflammation plays a pivotal role in the development of atherosclerosis, where the pro-inflammatory cytokine-induced expression of endothelial adhesion molecules and the recruitment of monocytes are the crucial events leading to its pathogenesis. Glossogyne tenuifolia ethanol extract (GTE) is shown to have potent anti-inflammatory and antioxidant activities. We evaluated the effects of GTE and its major components, luteolin (lut), luteolin-7-glucoside (lut-7-g), and oleanolic acid (OA) on TNF-α-induced expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs). The results demonstrated that GTE, lut, and lut-7-g attenuated the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α-activated HUVECs, and inhibited the adhesion of monocytes to TNF-α-activated HUVECs. The TNF-α-induced mRNA expression of ICAM-1 and VCAM-1 was also suppressed, revealing their inhibitory effects at the transcriptional level. Furthermore, GTE, lut, and lut-7-g blocked the TNF-α-induced degradation of nuclear factor-kB inhibitor (IkB), an indicator of the activation of nuclear factor-kB (NF-kB). In summary, GTE and its bioactive components were effective in preventing the adhesion of monocytes to cytokine-activated endothelium by the inhibition of expression of adhesion molecules, which in turn is mediated through blocking the activation and nuclear translocation of NF-kB. The current results reveal the therapeutic potential of GTE in atherosclerosis.

  7. Activation of mTOR/p70S6 kinase by ANG II inhibits insulin-stimulated endothelial nitric oxide synthase and vasodilation

    PubMed Central

    Jang, Hyun-Ju; Martinez-Lemus, Luis A.; Sowers, James R.

    2012-01-01

    Elevated tissue levels of angiotensin II (ANG II) are associated with impairment of insulin actions in metabolic and cardiovascular tissues. ANG II-stimulated activation of mammalian target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) in cardiovascular tissues is implicated in cardiac hypertrophy and vascular remodeling. However, the role of ANG II-stimulated mTOR/p70S6K in vascular endothelium is poorly understood. In the present study, we observed that ANG II stimulated p70S6K in bovine aortic endothelial cells. ANG II increased phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser636/639 and inhibited the insulin-stimulated phosphorylation of endothelial nitric oxide synthase (eNOS). An inhibitor of mTOR, rapamycin, attenuated the ANG II-stimulated phosphorylation of p70S6K and phosphorylation of IRS-1 (Ser636/639) and blocked the ability of ANG II to impair insulin-stimulated phosphorylation of eNOS, nitric oxide production, and mesenteric-arteriole vasodilation. Moreover, point mutations of IRS-1 at Ser636/639 to Ala prevented the ANG II-mediated inhibition of insulin signaling. From these results, we conclude that activation of mTOR/p70S6K by ANG II in vascular endothelium may contribute to impairment of insulin-stimulated vasodilation through phosphorylation of IRS-1 at Ser636/639. This ANG II-mediated impairment of vascular actions of insulin may help explain the role of ANG II as a link between insulin resistance and hypertension. PMID:22028412

  8. Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2.

    PubMed Central

    Millanvoye-Van Brussel, Elisabeth; Topal, Gökce; Brunet, Annie; Do Pham, Thuc; Deckert, Valérie; Rendu, Francine; David-Dufilho, Monique

    2004-01-01

    The oxidation of plasma LDLs (low-density lipoproteins) is a key event in the pathogenesis of atherosclerosis. LPC (lysophosphatidylcholine) and oxysterols are major lipid constitutents of oxidized LDLs. In particular, 7-oxocholesterol has been found in plasma from cardiac patients and atherosclerotic plaque. In the present study, we investigated the ability of 7-oxocholesterol and LPC to regulate the activation of eNOS (endothelial nitric oxide synthase) and cPLA2 (cytosolic phospholipase A2) that synthesize two essential factors for vascular wall integrity, NO (nitric oxide) and arachidonic acid. In endothelial cells from human umbilical vein cords, both 7-oxocholesterol (150 microM) and LPC (20 microM) decreased histamine-induced NO release, but not the release activated by thapsigargin. The two lipids decreased NO release through a PI3K (phosphoinositide 3-kinase)-dependent pathway, and decreased eNOS phosphorylation. Their mechanisms of action were, however, different. The NO release reduction was dependent on superoxide anions in LPC-treated cells and not in 7-oxocholesterol-treated ones. The Ca2+ signals induced by histamine were abolished by LPC, but not by 7-oxocholesterol. The oxysterol also inhibited (i) the histamine- and thapsigargin-induced arachidonic acid release, and (ii) the phosphorylation of both cPLA2 and ERK1/2 (extracellular-signal-regulated kinases 1/2). The results show that 7-oxocholesterol inhibits eNOS and cPLA2 activation by altering a Ca2+-independent upstream step of PI3K and ERK1/2 cascades, whereas LPC desensitizes eNOS by interfering with receptor-activated signalling pathways. This suggests that 7-oxocholesterol and LPC generate signals which cross-talk with heterologous receptors, effects which could appear at early stage of atherosclerosis. PMID:14992685

  9. MicroRNA-200a is up-regulated in aged rats with erectile dysfunction and could attenuate endothelial function via SIRT1 inhibition

    PubMed Central

    Pan, Feng; Qiu, Xue-Feng; Yu, Wen; Zhang, Qi-Peng; Chen, Qun; Zhang, Chen-Yu; Chen, Yun; Pan, Lian-Jun; Zhang, Ai-Xia; Dai, Yu-Tian

    2016-01-01

    MiR-200a was shown to be upregulated in the corpus cavernosum (CC) of rats with aging-related erectile dysfunction (A-ED) in our previous study. Among its target genes, SIRT1 was also reported as a protective factor in erectile function by our groups previously. Thus, miR-200a might attenuate the erectile function in A-ED via SIRT1 inhibition. In the present study, three animal groups were included: aged rats with ED (group AE, n = 8), aged rats with normal erectile function (group AN, n = 8), and young rats as normal controls (group YN, n = 8). CCs from each group were collected for histological and molecular measurements to validate the dysregulation of miR-200a and SIRT1. After that, the cavernous endothelial cells (CECs) from CC of aged rats with normal erectile function were transfected with miR-200a in vitro. Then the expression of SIRT1 and molecules within the eNOS/NO/PKG pathway were measured to investigate whether the transfection could imitate the attenuated process of erectile function in the aged. As a result, miR-200a was upregulated while the SIRT1, the levels of eNOS and cGMP were all downregulated in the CCs from AE group. After transfection in vitro, the miR-200a was upregulated while the SIRT1 and levels of eNOS and cGMP were obviously downregulated. Finally, based on the results of our previous study, we further verify that up-regulation of miR-200a could participate in the mechanisms of A-ED via SIRT1 inhibition, and mainly attenuate endothelial function via influencing the eNOS/NO/PKGpathway. PMID:25966629

  10. The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis.

    PubMed

    Del Galdo, Sabrina; Vettel, Christiane; Heringdorf, Dagmar Meyer Zu; Wieland, Thomas

    2013-12-01

    Sphingosine-1-phosphate (S1P) is a multifunctional phospholipid inducing a variety of cellular responses in endothelial cells (EC). S1P responses are mediated by five G protein coupled receptors of which three types (S1P1R-S1P3R) have been described to be of importance in vascular endothelial cells (EC). Whereas the S1P1R regulates endothelial barrier function by coupling to Gαi and the monomeric GTPase Rac1, the signaling pathways involved in the S1P-induced regulation of angiogenesis are ill defined. We therefore studied the sprouting of human umbilical vein EC (HUVEC) in vitro and analyzed the activation of the RhoGTPases RhoA and RhoC. Physiological relevant concentrations of S1P (100-300nM) induce a moderate activation of RhoA and RhoC. Inhibition or siRNA-mediated depletion of the S1P2R preferentially decreased the activation of RhoC. Both manipulations caused an increase of sprouting in a spheroid based in vitro sprouting assay. Interestingly, a similar increase in sprouting was detected after effective siRNA-mediated knockdown of RhoC. In contrast, the depletion of RhoA had no influence on sprouting. Furthermore, suppression of the activity of G proteins of the Gα12/13 subfamily by adenoviral overexpression of the regulator of G protein signaling domain of LSC as well as siRNA-mediated knockdown of the Rho specific guanine nucleotide exchange factor leukemia associated RhoGEF (LARG) inhibited the S1P-induced activation of RhoC and concomitantly increased sprouting of HUVEC with similar efficacy. We conclude that the angiogenic sprouting of EC is suppressed via the S1P2R subtype. Thus, the increase in basal sprouting can be attributed to blocking of the inhibitory action of autocrine S1P stimulating the S1P2R. This inhibitory pathway involves the activation of RhoC via Gα12/13 and LARG, while the simultaneously occurring activation of RhoA is apparently dispensable here.

  11. Inhibition of tumour spheroid-induced prometastatic intravasation gates in the lymph endothelial cell barrier by carbamazepine: drug testing in a 3D model.

    PubMed

    Teichmann, Mathias; Kretschy, Nicole; Kopf, Sabine; Jarukamjorn, Kanokwan; Atanasov, Atanas G; Viola, Katharina; Giessrigl, Benedikt; Saiko, Philipp; Szekeres, Thomas; Mikulits, Wolfgang; Dirsch, Verena M; Huttary, Nicole; Krieger, Sigurd; Jäger, Walter; Grusch, Michael; Dolznig, Helmut; Krupitza, Georg

    2014-03-01

    Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment

  12. RhoA-mediated inhibition of vascular endothelial cell mobility: positive feedback through reduced cytosolic p21 and p27.

    PubMed

    Hsu, Yung-Ho; Chang, Chih-Cheng; Yang, Nian-Jie; Lee, Yi-Hsuan; Juan, Shu-Hui

    2014-10-01

    We previously identified that activation of the aryl hydrocarbon receptor (AhR) by 3-methylcholanthrene (3MC) exerts antiproliferative and antimigratory effects on human umbilical vein endothelial cells (HUVECs) through the upregulation of p21/p27 transcription and RhoA activation. In this study, we investigated the mechanisms of 3MC-mediated downregulation of cytosolic p21/ p27, and the effects of 3MC on RhoA activation and cell migration, in mouse cerebral vascular endothelial cells (MCVECs). Our results indicated that 3MC reduced the phosphorylation of p21/p27 through AhR/RhoA/PTEN-mediated PI3K/Akt inactivation, which reduced cytosolic p21/p27 retention, causing RhoA activation through positive feedback. Downregulation of p21/p27 by siRNA, and cytosolic p21/p27 by the nuclear export blocker leptomycin B, further reduced cell migration in the 3MC-treated cells. Reduced cytosolic p21/p27 expression led to reduced interaction between RhoA and the RhoA inhibitor p190RhoGAP, causing RhoA activation. Treatment with YS-49 activated PI3K/Akt, a downstream target of RhoA, to reduce RhoA/PTEN activation in the 3MC-treated cells, whereas treatment with wortmannin, a PI3K inhibitor, activated RhoA/PTEN. Gain- and loss-of-function analyses revealed that constitutively active (CA) Akt1, but not CA Akt2, inactivated RhoA and stimulated migratory activity. Considering the essential role of RhoA activation in cell migration, we evaluated the potential use of simvastatin, a RhoA inhibitor, as a therapeutic intervention in vivo using matrigel plug formation assays. Our results provide a molecular basis for the therapeutic application of simvastatin to reduce RhoA/PTEN activation, restore cytosolic levels of phosphorylated p21/p27, and induce angiogenic processes.

  13. Inhibition of the transcriptional repressor complex Bcl-6/BCoR induces endothelial sprouting but does not promote tumor growth

    PubMed Central

    Buchberger, Elisabeth; Payrhuber, Dietmar; Harchi, Miriam El; Zagrapan, Branislav; Scheuba, Katharina; Zommer, Anna; Bugyik, Edina; Dome, Balazs; Kral, Julia Barbara; Schrottmaier, Waltraud Cornelia; Schabbauer, Gernot; Petzelbauer, Peter; Gröger, Marion; Bilban, Martin; Brostjan, Christine

    2017-01-01

    The oncogenic potential of the transcriptional repressor Bcl-6 (B-cell lymphoma 6) was originally discovered in non-Hodgkin patients and the soluble Bcl-6 inhibitor 79-6 was developed to treat diffuse large B-cell lymphomas with aberrant Bcl-6 expression. Since we found Bcl-6 and its co-repressor BCoR (Bcl-6 interacting co-repressor) to be regulated in human microvascular endothelium by colorectal cancer cells, we investigated their function in sprouting angiogenesis which is central to tumor growth. Based on Bcl-6/BCoR gene silencing we found that the transcriptional repressor complex in fact constitutes an endogenous inhibitor of vascular sprouting by supporting the stalk cell phenotype: control of Notch target genes (HES1, HEY1, DLL4) and cell cycle regulators (cyclin A and B1). Thus, when endothelial cells were transiently transfected with Bcl-6 and/or BCoR siRNA, vascular sprouting was prominently induced. Comparably, when the soluble Bcl-6 inhibitor 79-6 was applied in the mouse retina model of physiological angiogenesis, endothelial sprouting and branching were significantly enhanced. To address the question whether clinical treatment with 79-6 might therefore have detrimental therapeutic effects by promoting tumor angiogenesis, mouse xenograft models of colorectal cancer and diffuse large B-cell lymphoma were tested. Despite a tendency to increased tumor vessel density, 79-6 therapy did not enhance tumor expansion. In contrast, growth of colorectal carcinomas was significantly reduced which is likely due to a combined 79-6 effect on cancer cells and tumor stroma. These findings may provide valuable information regarding the future clinical development of Bcl-6 inhibitors. PMID:27880939

  14. Inhibition of Hydrogen Peroxide-Induced Human Umbilical Vein Endothelial Cells Aging by Allicin Depends on Sirtuin1 Activation

    PubMed Central

    Lin, Xiao-Long; Liu, Yuanbo; Liu, Mihua; Hu, Huijun; Pan, Yongquan; Fan, Xiao-Juan; Hu, Xue-Mei; Zou, Wei-Wen

    2017-01-01

    Background The abnormal activity of Sirtuin 1 (Sirt1) is closely related to the aging of vascular endothelial cells. As a bioactive molecule, allicin has antioxidant, anti-inflammatory, and lipid-regulating mechanisms. However, few reports about the relationship of allicin and Sirt1 have been published. In this study, we aimed to elucidate the effect of allicin on Human Umbilical Vein Endothelial Cells (HUVECs) aging induced by hydrogen peroxide (H2O2) and the role of Sirt1 in this phenomenon. Material/Methods HUVEC were exposed to H2O2 to establish the aging model. The expression of protein and RNA were detected by Western blot and Reverse transcription-quantitative polymerase chain reaction. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability. Sirt1 enzyme activity assay was used to analyze enzymatic activity. Reactive oxygen species was detected by dichlorofluorescein diacetate (DCFH-DA). Cell aging was detected by Senescence β-Galactosidase (SA-β-gal) staining. Results Results of this study revealed that pretreating HUVECs with 5 ng/mL allicin before exposure to H2O2 resulted in increased cell viability and reduced reactive oxygen species generation. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that H2O2 attenuated the phosphorylation and activation of Sirt1 and increased the expression of plasminogen activator inhibitor-1(PAI-1) protein. Moreover, H2O2 also promoted HUVEC aging. These effects were significantly alleviated by 5 ng/mL allicin co-treatment. Furthermore, the anti-aging effects of allicin were abolished by the Sirt1 inhibitor nicotinamide (NAM). Conclusions Overall, the results demonstrated that allicin protects HUVECs from H2O2-induced oxidative stress and aging via the activation of Sirt1. PMID:28139552

  15. Aromatic hydrocarbon receptor inhibits lysophosphatidic acid-induced vascular endothelial growth factor-A expression in PC-3 prostate cancer cells

    SciTech Connect

    Wu, Pei-Yi; Lin, Yueh-Chien; Lan, Shun-Yan; Huang, Yuan-Li; Lee, Hsinyu

    2013-08-02

    Highlights: •LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT. •PI3K mediated LPA-induced VEGF-A expression. •AHR signaling inhibited LPA-induced VEGF-A expression in PC-3 cells. -- Abstract: Lysophosphatidic acid (LPA) is a lipid growth factor with multiple biological functions and has been shown to stimulate cancer cell secretion of vascular endothelial growth factor-A (VEGF-A) and trigger angiogenesis. Hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of HIF-1α and HIF-1β (also known as aromatic hydrocarbon receptor nuclear translocator (ARNT)) subunits, is an important regulator of angiogenesis in prostate cancer (PC) through the enhancement of VEGF-A expression. In this study, we first confirmed the ability of LPA to induce VEGF-A expression in PC-3 cells and then validated that LPA-induced VEGF-A expression was regulated by HIF-1α and ARNT through phosphatidylinositol 3-kinase activation. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with ARNT and was found to inhibit prostate carcinogenesis and vanadate-induced VEGF-A production. Since ARNT is a common dimerization partner of AHR and HIF-1α, we hypothesized that AHR might suppress LPA-induced VEGF-A expression in PC-3 cells by competing with HIF-1α for ARNT. Here we demonstrated that overexpression and ligand activation of AHR inhibited HIF-1-mediated VEGF-A induction by LPA treatment of PC-3 cells. In conclusion, our results suggested that AHR activation may inhibit LPA-induced VEGF-A expression in PC-3 cells by attenuating HIF-1α signaling, and subsequently, suppressing angiogenesis and metastasis of PC. These results suggested that AHR presents a potential therapeutic target for the prevention of PC metastasis.

  16. U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells

    SciTech Connect

    Shirotani, M.; Yui, Y.; Hattori, R.; Kawai, C. )

    1991-02-01

    The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.

  17. Autophagy Induction by Endothelial-Monocyte Activating Polypeptide II Contributes to the Inhibition of Malignant Biological Behaviors by the Combination of EMAP II with Rapamycin in Human Glioblastoma

    PubMed Central

    Ma, Jun; Meng, Fanjie; Li, Shuai; Liu, Libo; Zhao, Lini; Liu, Yunhui; Hu, Yi; Li, Zhen; Yao, Yilong; Xi, Zhuo; Teng, Hao; Xue, Yixue

    2015-01-01

    This study aims to investigate the effect of endothelial-monocyte activating polypeptide II (EMAP II) on human glioblastoma (GBM) cells and glioblastoma stem cells (GSCs) as well as its possible mechanisms. In this study, EMAP II inhibited the cell viability and decreased the mitochondrial membrane potential in human GBM cells and GSCs, and autophagy inhibitor 3-methyl adenine (3-MA) blocked these effects. Autophagic vacuoles were formed in these cells after EMAP II treatment and this phenomenon was blocked by 3-MA. In addition, the up-regulation of microtubule-associated protein-1 light chain-3 (LC3)-II and the down-regulation of autophagic degraded substrate p62/SQSTM1 caused by EMAP II were observed. Cells treated with EMAP-II inhibited the PI3K/Akt/mTOR signal pathway, and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) blocked the effect of EMAP II on the expression of LC3-II and p62/SQSTM1. Cells exposed to EMAP-II experienced mitophagy and ER stress. Furthermore, the inhibition of cell proliferation, migration and invasion of GBM cells and GSCs were more remarkable by the combination of EMAP II and rapamycin than either agent alone in vitro and in vivo. The current study demonstrated that the cytotoxicity of EMAP II in human GBM cells and GSCs was induced by autophagy, accompanied by the inhibition of PI3K/Akt/mTOR signal pathway, mitophagy and ER stress. The combination of EMAP II with rapamycin demonstrated the inhibitory effect on the malignant biological behaviors of human GBM cells and GSCs in vitro and in vivo. PMID:26648842

  18. Negative regulation of HLA-DR expression on endothelial cells by anti-blood group A/B antibody ligation and mTOR inhibition.

    PubMed

    Iwasaki, Kenta; Miwa, Yuko; Uchida, Kazuharu; Kodera, Yasuhiro; Kobayashi, Takaaki

    2017-02-01

    Donor-specific antibody (DSA), particularly against HLA class II, is a major cause of chronic antibody-mediated rejection (CAMR) after transplantation, although ABO-incompatible kidney transplantation has recently demonstrated favorable graft outcomes. The condition of no injury even in the presence of anti-donor antibody has been referred to as "accommodation", which would be one of the key factors for successful long-term graft survival. The purpose of this study was to analyze the beneficial effect of anti-blood group A/B antibody ligation on endothelial cells against HLA-DR antibody-mediated, complement-dependent cytotoxicity (CDC). Blood group A/B-expressing endothelial cells EA.hy926 or Human Umbilical Vein Endothelia Cells (HUVEC) were incubated with IFNγ in the presence or absence of anti-blood group A/B antibody or mTOR inhibitor (mTOR-i) for 48h. The effects on signaling pathway, HLA expression, complement regulatory factors, and CDC were investigated. Expression of HLA-DR on EA.hy926 or HUVEC were successfully elicited by IFNγ treatment, although little or no expression was observed in quiescent cells. Pre-incubation with anti-blood group A/B antibody had resistance to HLA-DR antibody-mediated CDC against IFNγ-treated cells in a concentration-dependent manner. This finding was ascribed to decreased expression of HLA-DR by post-translational regulation and increased expression of CD55/59, which was related to ERK and mTOR pathway inhibition. mTOR-i also inhibited HLA-DR expression by itself. Furthermore, the combination of mTOR-I and anti-blood group A/B ligation had an additive effect in preventing HLA-DR antibody-mediated CDC. Anti-blood group A/B antibody might play a preventive role in CAMR. Inhibition of the ERK and mTOR pathways may contribute to the development of a novel treatment in the maintenance period after transplantation.

  19. Apium graveolens Extract Inhibits Cell Proliferation and Expression of Vascular Endothelial Growth Factor and Induces Apoptosis in the Human Prostatic Carcinoma Cell Line LNCaP.

    PubMed

    Köken, Tülay; Koca, Buğra; Özkurt, Mete; Erkasap, Nilüfer; Kuş, Gökhan; Karalar, Mustafa

    2016-12-01

    Apium graveolens has been shown to inhibit the growth of a variety of cancer tissues. In this study, we investigated the anticancer effect of A. graveolens on the human prostatic carcinoma cell line LNCaP. LNCaP cells were treated with increasing concentrations of an ethanolic extract of A. graveolens ranging from 1000 to 3000 μg/mL, and viability was determined after 24 and 48 h using the XTT cell proliferation assay. The levels of cleaved poly (ADP-ribose) polymerase (PARP), one of the best biomarkers of apoptosis, were analyzed. Finally, quantitative gene expression analysis of vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis, was performed using real-time reverse transcription-polymerase chain reaction. A. graveolens extract inhibited cell viability in both a time- and dose-dependent manner. Data from cleaved PARP assays suggested that A. graveolens caused induction of apoptosis in these cells. Treatment of cells with A. graveolens also resulted in downregulation of VEGF expression. This study showed that the antiproliferative effect exerted by an ethanolic extract of A. graveolens is triggered by induction of apoptosis. We also demonstrated that VEGF expression was downregulated by treatment with A. graveolens extract.

  20. Prophylactic Administration of Fucoidan Represses Cancer Metastasis by Inhibiting Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloproteinases (MMPs) in Lewis Tumor-Bearing Mice

    PubMed Central

    Huang, Tse-Hung; Chiu, Yi-Han; Chan, Yi-Lin; Chiu, Ya-Huang; Wang, Hang; Huang, Kuo-Chin; Li, Tsung-Lin; Hsu, Kuang-Hung; Wu, Chang-Jer

    2015-01-01

    Fucoidan, a heparin-like sulfated polysaccharide, is rich in brown algae. It has a wide assortment of protective activities against cancer, for example, induction of hepatocellular carcinoma senescence, induction of human breast and colon carcinoma apoptosis, and impediment of lung cancer cells migration and invasion. However, the anti-metastatic mechanism that fucoidan exploits remains elusive. In this report, we explored the effects of fucoidan on cachectic symptoms, tumor development, lung carcinoma cell spreading and proliferation, as well as expression of metastasis-associated proteins in the Lewis lung carcinoma (LLC) cells-inoculated mice model. We discovered that administration of fucoidan has prophylactic effects on mitigation of cachectic body weight loss and improvement of lung masses in tumor-inoculated mice. These desired effects are attributed to inhibition of LLC spreading and proliferation in lung tissues. Fucoidan also down-regulates expression of matrix metalloproteinases (MMPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and vascular endothelial growth factor (VEGF). Moreover, the tumor-bearing mice supplemented with fucoidan indeed benefit from an ensemble of the chemo-phylacticity. The fact is that fucoidan significantly decreases viability, migration, invasion, and MMPs activities of LLC cells. In summary, fucoidan is suitable to act as a chemo-preventative agent for minimizing cachectic symptoms as well as inhibiting lung carcinoma metastasis through down-regulating metastatic factors VEGF and MMPs. PMID:25854641

  1. Growth-inhibiting effect of tumor necrosis factor on human umbilical vein endothelial cells is enhanced with advancing age in vitro

    SciTech Connect

    Shimada, Y.; Kaji, K.; Ito, H.; Noda, K.; Matsuo, M. )

    1990-01-01

    We have examined the effects of in vitro aging on the growth capacity of human umbilical vein endothelial cells (HUVECs) under the influence of tumor necrosis factor (TNF) with or without interferon-gamma (IFN-gamma). The growth and colony-forming abilities of control cells were impaired with advancing age in vitro, especially at later stages (more than 70-80% of life span completed). It was found that treatment with TNF inhibited growth and colony-forming efficiency at any in vitro age. The effects of TNF were shown to increase with increasing in vitro age, as reflected by a more pronounced increase in doubling times, a decrease in saturation density, and a reduction in colony-forming efficiency. However, the characteristics of TNF receptors, including the dissociation constant, and the number of TNF-binding sites per cell-surface area remained rather constant. The effect of TNF was augmented by IFN-gamma at a dose that alone affected growth and colony formation only slightly. The augmentation by IFN-gamma was also found to depend on in vitro age; the synergy with TNF in the deterioration of colony-forming ability was observed only in aged cells. These results suggest that the intrinsic responsiveness of HUVECs to growth-inhibiting factors, as well as to growth-stimulating factors, changes during aging in vitro.

  2. Prophylactic administration of fucoidan represses cancer metastasis by inhibiting vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in Lewis tumor-bearing mice.

    PubMed

    Huang, Tse-Hung; Chiu, Yi-Han; Chan, Yi-Lin; Chiu, Ya-Huang; Wang, Hang; Huang, Kuo-Chin; Li, Tsung-Lin; Hsu, Kuang-Hung; Wu, Chang-Jer

    2015-04-03

    Fucoidan, a heparin-like sulfated polysaccharide, is rich in brown algae. It has a wide assortment of protective activities against cancer, for example, induction of hepatocellular carcinoma senescence, induction of human breast and colon carcinoma apoptosis, and impediment of lung cancer cells migration and invasion. However, the anti-metastatic mechanism that fucoidan exploits remains elusive. In this report, we explored the effects of fucoidan on cachectic symptoms, tumor development, lung carcinoma cell spreading and proliferation, as well as expression of metastasis-associated proteins in the Lewis lung carcinoma (LLC) cells-inoculated mice model. We discovered that administration of fucoidan has prophylactic effects on mitigation of cachectic body weight loss and improvement of lung masses in tumor-inoculated mice. These desired effects are attributed to inhibition of LLC spreading and proliferation in lung tissues. Fucoidan also down-regulates expression of matrix metalloproteinases (MMPs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and vascular endothelial growth factor (VEGF). Moreover, the tumor-bearing mice supplemented with fucoidan indeed benefit from an ensemble of the chemo-phylacticity. The fact is that fucoidan significantly decreases viability, migration, invasion, and MMPs activities of LLC cells. In summary, fucoidan is suitable to act as a chemo-preventative agent for minimizing cachectic symptoms as well as inhibiting lung carcinoma metastasis through down-regulating metastatic factors VEGF and MMPs.

  3. Fluvoxamine inhibits some inflammatory genes expression in LPS/stimulated human endothelial cells, U937 macrophages, and carrageenan-induced paw edema in rat

    PubMed Central

    Rafiee, Laleh; Hajhashemi, Valiollah; Javanmard, Shaghayegh Haghjooy

    2016-01-01

    Objective(s): Fluvoxamine is a well-known selective serotonin reuptake inhibitor (SSRI); Despite its anti-inflammatory effect, little is known about the precise mechanisms involved. In our previous work, we found that IP administration of fluvoxamine produced a noticeable anti-inflammatory effect in carrageenan-induced paw edema in rats. In this study, we aimed to evaluate the effect of fluvoxamine on the expression of some inflammatory genes like intercellular adhesion molecule (ICAM1), vascular cell adhesion molecule (VCAM1), cyclooxygenases2 (COX2), and inducible nitric oxide synthase (iNOS). Materials and Methods: An in vitro model of LPS stimulated human endothelial cells and U937 macrophages were used. Cells were pretreated with various concentrations of fluvoxamine, from 10-8 M to 10-6 M. For in vivo model, fluvoxamine was administered IP at doses of 25 and 50 mg/kg-1, before injection of carrageenan. At the end of experiment, the expression of mentioned genes were measured by quantitative real time (RT)-PCR in cells and in paw edema in rat. Results: The expression of ICAM1, VCAM1, COX2, and iNOS was significantly decreased by fluvoxamine in endothelial cells, macrophages, and in rat carrageenan-induced paw edema. Our finding also confirmed that IP injection of fluvoxamine inhibits carrageenan-induced inflammation in rat paw edema. Conclusion: The results of present study provide further evidence for the anti-inflammatory effect of fluvoxamine. This effect appears to be mediated by down regulation of inflammatory genes. Further studies are needed to evaluate the complex cellular and molecular mechanisms of immunomodulatory effect of fluvoxamine. PMID:27803785

  4. Mesenchymal stem cell-conditioned medium prevents radiation-induced liver injury by inhibiting inflammation and protecting sinusoidal endothelial cells.

    PubMed

    Chen, Yi-Xing; Zeng, Zhao-Chong; Sun, Jing; Zeng, Hai-Ying; Huang, Yan-; Zhang, Zhen-Yu

    2015-07-01

    Current management of radiation-induced liver injury is limited. Sinusoidal endothelial cell (SEC) apoptosis and inflammation are considered to be initiating events in hepatic damage. We hypothesized that mesenchymal stem cells (MSCs) possess anti-apoptotic and anti-inflammatory actions during hepatic irradiation, acting via paracrine mechanisms. This study aims to examine whether MSC-derived bioactive components are protective against radiation-induced liver injury in rats. MSC-conditioned medium (MSC-CM) was generated from rat bone marrow-derived MSCs. The effect of MSC-CM on the viability of irradiated SECs was examined by flow cytometric analysis. Activation of the Akt and ERK pathways was analyzed by western blot. MSC-CM was also delivered to Sprague-Dawley rats immediately before receiving liver irradiation, followed by testing for pathological features, changes in serum hyaluronic acid, ALT, and inflammatory cytokine levels, and liver cell apoptosis. MSC-CM enhanced the viability of irradiated SECs in vitro and induced Akt and ERK phosphorylation in these cells. Infusion of MSC-CM immediately before liver irradiation provided a significant anti-apoptotic effect on SECs and improved the histopathological features of injury in the irradiated liver. MSC-CM also reduced the secretion and expression of inflammatory cytokines and increased the expression of anti-inflammatory cytokines. MSC-derived bioactive components could be a novel therapeutic approach for treating radiation-induced liver injury.

  5. Vascular endothelial-targeted therapy combined with cytotoxic chemotherapy induces inflammatory intratumoral infiltrates and inhibits tumor relapses after surgery.

    PubMed

    Judy, Brendan F; Aliperti, Louis A; Predina, Jarrod D; Levine, Daniel; Kapoor, Veena; Thorpe, Philip E; Albelda, Steven M; Singhal, Sunil

    2012-04-01

    Surgery is the most effective therapy for cancer in the United States, but disease still recurs in more than 40% of patients within 5 years after resection. Chemotherapy is given postoperatively to prevent relapses; however, this approach has had marginal success. After surgery, recurrent tumors depend on rapid neovascular proliferation to deliver nutrients and oxygen. Phosphatidylserine (PS) is exposed on the vascular endothelial cells in the tumor microenvironment but is notably absent on blood vessels in normal tissues. Thus, PS is an attractive target for cancer therapy after surgery. Syngeneic mice bearing TC1 lung cancer tumors were treated with mch1N11 (a novel mouse chimeric monoclonal antibody that targets PS), cisplatin (cis), or combination after surgery. Tumor relapses and disease progression were decreased 90% by combination therapy compared with a 50% response rate for cis alone (P = .02). Mice receiving postoperative mch1N11 had no wound-related complications or added systemic toxicity in comparison to control animals. Mechanistic studies demonstrated that the effects of mch1N11 were associated with a dense infiltration of inflammatory cells, particularly granulocytes. This strategy was independent of the adaptive immune system. Together, these data suggest that vascular-targeted strategies directed against exposed PS may be a powerful adjunct to postoperative chemotherapy in preventing relapses after cancer surgery.

  6. Olive oil phenolic compounds inhibit homocysteine-induced endothelial cell adhesion regardless of their different antioxidant activity.

    PubMed

    Manna, Caterina; Napoli, Daniela; Cacciapuoti, Giovanna; Porcelli, Marina; Zappia, Vincenzo

    2009-05-13

    In this study, we examine the effect of extra virgin olive oil phenolic compounds on homocysteine-induced endothelial dysfunction and whether the protective effects are related to their different scavenging activities. Structurally related compounds have been assayed for their ability to reduce homocysteine-induced monocyte adhesion as well as the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in EA.hy.926 cells. As well-known, among the selected phenolic compounds, hydroxytyrosol, homovanillyl alcohol, and the hydroxycinnamic acid derivatives caffeic and ferulic acid display high scavenging activities, while tyrosol and p-coumaric acid are poorly active. All of the tested compounds, approaching potential in vivo concentrations, significantly reduce homocysteine-induced cell adhesion and ICAM-1 expression. Interestingly, we report the first evidence that monophenols tyrosol and p-coumaric acid are selectively protective only in homocysteine-activated cells, while they are ineffective in reducing ICAM-1 expression induced by TNFalpha. Finally, we report the synergistic effect of o-diphenolic and monophenolic compounds.

  7. Physiological electric fields control the G1/S phase cell cycle checkpoint to inhibit endothelial cell proliferation.

    PubMed

    Wang, Entong; Yin, Yili; Zhao, Min; Forrester, John V; McCaig, Colin D

    2003-03-01

    Vascular endothelial cell (VEC) proliferation is a key event in angiogenesis and is tightly regulated. Electric potential differences exist around the vascular endothelium and give rise to endogenous electric fields (EFs), whether these EFs influence VEC proliferation is unclear. We exposed cultured VECs to applied EFs of physiological strengths for up to 72 h. EF at 50 or 100 mV/mm did not influence cell proliferation, but at 200 mV/mm, cell density, cell growth rate, and mitosis index decreased significantly. EF-induced reduction in VEC proliferation was not due to increased apoptosis, because caspase apoptosis inhibitor Z-VAD-FMK (20 microM), had no effect on this response. Rather, EF responses were mediated via decreased entry of cells into S phase from G1 phase, as shown by flow cytometry. Western blot showed that EFs decreased G1-specific cyclin E expression and increased cyclin/cyclin-dependent kinase complex inhibitor p27kipl expression. Thus EFs controlled VEC proliferation through induction of cell cycle arrest at G1 by down-regulation of cyclin E expression and up-regulation of p27kipl expression, rather than by promoting apoptosis. If control of the cell cycle by endogenous EFs extends beyond VECs, this would be of widespread biological significance in vivo.

  8. Ghrelin inhibits AngII -induced expression of TNF-α, IL-8, MCP-1 in human umbilical vein endothelial cells

    PubMed Central

    Deng, Bin; Fang, Fang; Yang, Tianlu; Yu, Zaixin; Zhang, Bin; Xie, Xiumei

    2015-01-01

    Aim: Ghrelin, a gastric peptide, is involved in several metabolic and cardiovascular processes. Emerging evidence indicates the potential involvement of ghrelin in low-grade inflammatory diseases such as atherosclerosis and hypertension. Cytokine-induced inflammation is critical in these pathological states. The growth hormone secretagogue receptor (GHSR) has been identified in blood vessels, so we predict that ghrelin might inhibit proinflammatory responses in human umbilical vein endothelial cells (HUVECs). The aim of this study is to examine the effect of ghrelin on angiotension II (AngII)-induced expression of TNF-α, MCP-1, IL-8 in HUVECs. Method: HUVECs were pretreated with ghrelin, with or without the specific antagonist of GHSR [D-Lys3]-GHRP-6, the selective inhibitor of nuclear factor-kappaB (NF-κB) PDTC, and the selective inhibitor of extracellular signal-regulated kinase (ERK1/2) PD98059. The cells were finally treated with AngII. The expression of TNF-α, MCP-1, IL-8 was examined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The activity of ERK1/2 and NF-κB was analyzed by Western blot. Result: our study showed that ghrelin inhibited AngII -induced expression of IL-8, TNF-α and MCP-1 in the HUVECs via GHSR pathway in concentration- and time-dependent manners. We also found that ghrelin inhibited AngII -induced activation of ERK1/2 and NF-κB. Conclusions: these results suggest that Ghrelin may play novel antiinflammatory and immunoregulatory roles in HUVECs. PMID:25785032

  9. Salvianolic Acid B Inhibits Hydrogen Peroxide-Induced Endothelial Cell Apoptosis through Regulating PI3K/Akt Signaling

    PubMed Central

    Liu, Chen-Li; Xie, Li-Xia; Li, Min; Durairajan, Siva Sundara Kumar; Goto, Shinya; Huang, Jian-Dong

    2007-01-01

    Background Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H2O2) is implicated in the pathogenesis of cerebrovascular disorders. Methodology and Principal Findings By examining the effect of Sal B on H2O2-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H2O2-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H2O2 induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H2O2 and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H2O2-induced apoptosis, suggesting that Sal B prevents H2O2-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway. Significance Our findings provide the first evidence that H2O2 induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H2O2-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway. PMID:18091994

  10. Cytotoxic T lymphocyte-dependent tumor growth inhibition by a vascular endothelial growth factor-superantigen conjugate

    SciTech Connect

    Sun, Qingwen; Jiang, Songmin; Han, Baohui; Sun, Tongwen; Li, Zhengnan; Zhao, Lina; Gao, Qiang; Sun, Jialin

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer We construct and purify a fusion protein VEGF-SEA. Black-Right-Pointing-Pointer VEGF-SEA strongly repressed the growth of murine solid sarcoma 180 (S180) tumors. Black-Right-Pointing-Pointer T cells driven by VEGF-SEA were accumulated around tumor cells bearing VEGFR by mice image model. Black-Right-Pointing-Pointer VEGF-SEA can serve as a tumor targeting agent and sequester CTLs into the tumor site. Black-Right-Pointing-Pointer The induced CTLs could release the cytokines, perforins and granzyme B to kill the tumor cells. -- Abstract: T cells are major lymphocytes in the blood and passengers across the tumor vasculature. If these T cells are retained in the tumor site, a therapeutic potential will be gained by turning them into tumor-reactive cytotoxic T lymphocytes (CTLs). A fusion protein composed of human vascular endothelial growth factor (VEGF) and staphylococcal enterotoxin A (SEA) with a D227A mutation strongly repressed the growth of murine solid sarcoma 180 (S180) tumors (control versus VEGF-SEA treated with 15 {mu}g, mean tumor weight: 1.128 g versus 0.252 g, difference = 0.876 g). CD4{sup +} and CD8{sup +} T cells driven by VEGF-SEA were accumulated around VEGFR expressing tumor cells and the induced CTLs could release the tumoricidal cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Meanwhile, intratumoral CTLs secreted cytolytic pore-forming perforin and granzyme B proteins around tumor cells, leading to the death of tumor cells. The labeled fusion proteins were gradually targeted to the tumor site in an imaging mice model. These results show that VEGF-SEA can serve as a tumor targeting agent and sequester active infiltrating CTLs into the tumor site to kill tumor cells, and could therefore be a potential therapeutical drug for a variety of cancers.

  11. Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts

    PubMed Central

    Sziksz, Erna; Pap, Domonkos; Balicza-Himer, Leonóra; Boros, Szilvia; Magda, Balázs; Vannay, Ádám; Kis-Petik, Katalin; Fekete, Andrea; Peti-Peterdi, János; Szabó, Attila J.

    2016-01-01

    Background Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD). Methods Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining. Results The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands. Conclusions Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity. PMID:26502369

  12. Suppression of Retinal Neovascularization in vivo by Inhibition of Vascular Endothelial Growth Factor (VEGF) Using Soluble VEGF-Receptor Chimeric Proteins

    NASA Astrophysics Data System (ADS)

    Aiello, Lloyd Paul; Pierce, Eric A.; Foley, Eliot D.; Takagi, Hitoshi; Chen, Helen; Riddle, Lavon; Ferrara, Napoleone; King, George L.; Smith, Lois E. H.

    1995-11-01

    The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-um section averaged 47% ± 4% (P < 0.001) and 37% ± 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66

  13. Ethanol extract of propolis protects endothelial cells from oxidized low density lipoprotein-induced injury by inhibiting lectin-like oxidized low density lipoprotein receptor-1-mediated oxidative stress.

    PubMed

    Fang, Yongqi; Li, Jinguo; Ding, Mingde; Xu, Xiaoyan; Zhang, Jiajun; Jiao, Peng; Han, Ping; Wang, Jiafu; Yao, Shutong

    2014-12-01

    Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1), as the primary oxidized low-density lipoprotein (ox-LDL) receptor on endothelial cells, plays a crucial role in endothelial injury, which is a driving force in the initiation and development of atherosclerosis. Our previous studies have shown that ethanol extract of propolis (EEP) promotes reverse cholesterol transport and inhibits atherosclerotic lesion development. However, the protective effects of EEP against ox-LDL-induced injury in endothelial cells and the underlying mechanisms are still unknown. This study was designed to test the hypothesis that EEP attenuates ox-LDL-induced endothelial oxidative injury via modulation of LOX-1-mediated oxidative stress. Our results showed that exposure of human umbilical vein endothelial cells (HUVECs) to ox-LDL (100 mg/L) led to the decrease in cell viability and increase in lactate dehydrogenase (LDH) release, caspase-3 activation, and apoptosis, whereas pretreatment with EEP (7.5, 15 and 30 mg/L) protected against such damages in a dose-dependent manner. In addition, EEP mitigated ox-LDL uptake by HUVECs and attenuated ox-LDL-upregulated LOX-1 expression both at the mRNA and protein levels. Moreover, EEP suppressed the ox-LDL-induced oxidative stress as assessed by decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, reactive oxygen species (ROS), and malondialdehyde (MDA) generation as well as increased antioxidant enzyme activities. Similar results were observed in the anti-LOX-1 antibody or diphenyleneiodonium (DPI)-pretreated HUVECs. These data indicate that EEP may protect HUVECs from ox-LDL-induced injury and that the mechanism at least partially involves its ability to inhibit endothelial LOX-1 upregulation and subsequent oxidative stress.

  14. Indomethacin and cyclosporin a inhibit in vitro ischemia-induced expression of ICAM-1 and chemokines in human brain endothelial cells.

    PubMed

    Zhang, W; Smith, C; Monette, R; Hutchison, J; Stanimirovic, D B

    2000-01-01

    Brain inflammation has been implicated in the development of brain edema and secondary brain damage in ischemia and trauma. Mechanisms involved in leukocyte infiltration across the blood-brain barrier are still unknown. In this study, we show that human cere-bromicrovascular endothelial cells (HCEC) subjected to a 4 h in vitro ischemia (hypoxia + glucose deprivation) followed by a 4-24 h recovery express elevated levels of ICAM-1, IL-8, and MCP-1 mRNAs (semi-quantitative RT-PCR) and secrete increased amounts of the immunoreactive chemokines IL-8 and MCP-1 (ELISA). The ischemia-induced expression of ICAM-1 in HCEC, and the expression/release of IL-8 and MCP-1 in HCEC were abolished by the non-steroid anti-inflammatory drug, indomethacin (100-300 microM). The immunosuppressant cyclosporin A (50 microM) partially reduced the ischemia-stimulated IL-8 and MCP-1 secretion by HCEC. Both indomethacin and cyclosporin A also inhibited the ischemia-induced neutrophil chemotaxis elicited by HCEC media. The study indicates that in vitro ischemia augments the expression of adhesion molecules and leukocyte chemoattractants at the site of the BBB. This ischemic pro-inflammatory activation of HCEC may constitute a key event in initiating post-ischemic inflammation, and it can be suppressed by the anti-inflammatory drugs, indomethacin and cyclosporin A.

  15. Erythropoietin inhibits osmotic swelling of retinal glial cells by Janus kinase- and extracellular signal-regulated kinases1/2-mediated release of vascular endothelial growth factor.

    PubMed

    Krügel, K; Wurm, A; Linnertz, R; Pannicke, T; Wiedemann, P; Reichenbach, A; Bringmann, A

    2010-02-17

    The volume homeostasis of retinal glial cells is mediated by an autocrine purinergic mechanism of ion channel opening which is activated in response to a decrease in the extracellular osmolarity. Here, we show that erythropoietin (EPO) prevents the osmotic swelling of glial somata in retinal slices and of isolated glial cells from control and diabetic rats, with a half-maximal effect at approximately 0.01 nM. The downstream signaling evoked by EPO includes a release of vascular endothelial growth factor from the cells which was blocked by Janus kinase and extracellular signal-regulated kinases (ERK)1/2 inhibitors. Transactivation of kinase insert domain-containing receptor/fms-like tyrosine kinase 1 (KDR/flk-1) evokes a calcium-dependent, exocytotic release of glutamate, followed by activation of group I/II metabotropic glutamate receptors which results in calcium-independent release of ATP and adenosine from the cells. The final step in this cascade is the activation of adenosine A(1) receptors which results in protein kinase A- and phosphoinositide 3-kinase-mediated opening of potassium and chloride channels. EPO receptor protein was immunohistochemically localized to the inner retina and photoreceptor inner segments. In isolated glial cells, EPO receptor protein is selectively localized to fibers which traverse the inner nuclear layer in situ. Inhibition of glial swelling might contribute to the neuroprotective action of EPO in the retina under pathological conditions.

  16. Cyclosporin-A inhibits constitutive nitric oxide synthase activity and neuronal and endothelial nitric oxide synthase expressions after spinal cord injury in rats.

    PubMed

    Diaz-Ruiz, Araceli; Vergara, Paula; Perez-Severiano, Francisca; Segovia, Jose; Guizar-Sahagún, Gabriel; Ibarra, Antonio; Ríos, Camilo

    2005-02-01

    Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca2+/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.

  17. Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

    PubMed

    Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

    2016-01-01

    Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.

  18. Sargachromenol protects against vascular inflammation by preventing TNF-α-induced monocyte adhesion to primary endothelial cells via inhibition of NF-κB activation.

    PubMed

    Gwon, Wi-Gyeong; Joung, Eun-Ji; Kwon, Mi-Sung; Lim, Su-Jin; Utsuki, Tadanobu; Kim, Hyeung-Rak

    2017-01-01

    Vascular inflammation is a key factor in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the protective effects of sargachromenol (SCM) against tumor necrosis factor (TNF)-α-induced vascular inflammation. SCM decreased the expression of cell adhesion molecules, including intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs), resulted in reduced adhesion of monocytes to HUVECs. SCM also decreased the production of monocyte chemoattractant protein-1 and matrix metalloproteinase-9 in TNF-α-induced HUVECs. Additionally, SCM inhibited activation of nuclear factor kappa B (NF-κB) induced by TNF-α through preventing the degradation of inhibitor kappa B. Moreover, SCM reduced the production of reactive oxygen species in TNF-α-treated HUVECs. Overall, SCM alleviated vascular inflammation through the regulation of NF-κB activation and through its intrinsic antioxidant activity in TNF-α-induced HUVECs. These results indicate that SCM may have potential application as a therapeutic agent against vascular inflammation.

  19. Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts.

    PubMed

    Fu, Yunhe; Hu, Xiaoyu; Cao, Yongguo; Zhang, Zecai; Zhang, Naisheng

    2015-12-01

    Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses.

  20. MiR-590-5p inhibits colorectal cancer angiogenesis and metastasis by regulating nuclear factor 90/vascular endothelial growth factor A axis

    PubMed Central

    Zhou, Qingxin; Zhu, Yuekun; Wei, Xiaoli; Zhou, Jianhua; Chang, Liang; Sui, Hong; Han, Yu; Piao, Daxun; Sha, Ruihua; Bai, Yuxian

    2016-01-01

    Altered expression of microRNA-590-5p (miR-590-5p) is involved in tumorigenesis, however, its role in colorectal cancer (CRC) remains to be determined. In this study, we focused on examining the effects of different expression levels of miR-590-5p in cancer cells and normal cells. Results showed that there are lower expression levels of miR-590-5p in human CRC cells and tissues than in normal control cells and tissues. Similarly, in our xenograft mouse model, knockdown of miR-590-5p promoted the progression of CRC. However, an overexpression of miR-590-5p in the mice inhibited angiogenesis, tumor growth, and lung metastasis. Nuclear factor 90 (NF90), a positive regulator of vascular endothelial growth factor (VEGF) mRNA stability and protein synthesis, was shown to be a direct target of miR-590-5p. The overexpression of NF90 restored VEGFA expression and rescued the loss of tumor angiogenesis caused by miR-590-5p. Conversely, the NF90-shRNA attenuated the increased tumor progression caused by the miR-590-5p inhibitor. Clinically, the levels of miR-590-5p were inversely correlated with those of NF90 and VEGFA in CRC tissues. Furthermore, knockdown of NF90 lead to a reduction of pri-miR-590 and an increase of mature miR-590-5p, suggesting a negative feedback loop between miR-590-5p and NF90. Collectively, these data establish miR-590-5p as an anti-onco-miR that inhibits CRC angiogenesis and metastasis through a new mechanism involving NF90/VEGFA signaling axis, highlighting the potential of miR-590-5p as a target for human CRC therapy. PMID:27735951

  1. Soluble epoxide hydrolase inhibition improves coronary endothelial function and prevents the development of cardiac alterations in obese insulin-resistant mice

    PubMed Central

    Roche, Clothilde; Besnier, Marie; Cassel, Roméo; Harouki, Najah; Coquerel, David; Guerrot, Dominique; Nicol, Lionel; Loizon, Emmanuelle; Remy-Jouet, Isabelle; Morisseau, Christophe; Mulder, Paul; Ouvrard-Pascaud, Antoine; Madec, Anne-Marie; Richard, Vincent

    2015-01-01

    This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in insulin resistance, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 10 mg/l in drinking water) were compared with those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 wk in FVB mice subjected to a high-fat diet (HFD; 60% fat) for 16 wk. Mice on control chow diet (10% fat) and nontreated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice, but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids, and LDL cholesterol and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the nitric oxide synthase and cytochrome P-450 epoxygenase inhibitors l-NA and MSPPOH on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis, and inflammation and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the end-diastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that sEH inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese insulin-resistant mice. PMID:25724490

  2. Enhanced peripheral dopamine impairs post-ischemic healing by suppressing angiotensin receptor type 1 expression in endothelial cells and inhibiting angiogenesis.

    PubMed

    Sarkar, Chandrani; Ganju, Ramesh K; Pompili, Vincent J; Chakroborty, Debanjan

    2017-02-01

    Increased circulating catecholamines have been linked with cardiovascular anomalies as well as with peripheral vascular diseases. Although the roles of epinephrine and norepinephrine have received considerable attention, the role of the other catecholamine, dopamine, has been less studied. Since dopamine is a potent endogenous inhibitor of angiogenesis and as angiogenesis is essential for ischemic healing, we therefore studied the role played by dopamine during ischemic healing using dopamine D2 receptor knockout (KOD2) mice. Although concentration of dopamine and its rate-limiting enzyme, tyrosine hydroxylase, was considerably high in the muscle tissues of wild-type and KOD2 mice with unilateral hind limb ischemia (HLI), recovery was significantly faster in the KOD2 mice compared to the wild-type controls, thereby indicating that peripheral dopamine might have a role in this healing process. In addition, we observed significant differences in post-ischemic angiogenesis between these two groups. Our study further revealed that elevated dopamine independently suppressed activation of local tissue-based renin-angiotensin system (RAS), a critical growth factor system stimulating angiogenesis in ischemia. Angiotensin II (ATII) and its receptor, angiotensin receptor type 1 (AT1R), are the key players in RAS-mediated angiogenesis. Dopamine acting through its D2 receptors in endothelial cells inhibited ATII-mediated angiogenesis by suppressing the expression of AT1R in these cells. This study thus for the first time demonstrates the role played by dopamine in prolonging post-ischemic recovery. Therefore, pharmacological intervention inhibiting the action of dopamine holds promise as future therapeutic strategy for the treatment of HLI and other peripheral arterial diseases.

  3. Equol Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice by Inhibiting Endoplasmic Reticulum Stress via Activation of Nrf2 in Endothelial Cells

    PubMed Central

    Shi, Linying; Qin, Li; Zhang, Qianyong; Mi, Mantian

    2016-01-01

    The development of atherosclerosis is closely related to excessive endoplasmic reticulum stress (ERs). Equol reportedly protects against cardiovascular disease; however, the underlying mechanism for this protection remains unknown. Herein, the mechanisms contributing to the atheroprotective effect of equol were addressed using apolipoprotein E knockout (apoE-/-) mice fed a high-fat diet (HFD) with or without equol. Equol intervention reduced atherosclerotic lesions in the aorta in HFD-fed apoE-/- mice. Plasma lipid analysis showed that equol intervention reduced triglycerides, total cholesterol and LDL-cholesterol and increased HDL-cholesterol. Additionally, equol administration decreased lipid accumulation in the liver. Simultaneously, equol treatment inhibited cell apoptosis induced by t-BHP and thapsigargin in human umbilical vein endothelial cells (HUVECs). Furthermore, equol treatment attenuated palmitate, t-BHP or thapsigargin-induced upregulation of ER stress markers, including p-PERK, p-eIF2α, GRP78, ATF6 and CHOP proteins expression. The same tendency was also observed in aortic lysates in apoE-/- mice fed with equol plus HFD compared with HFD alone. Moreover, equol treatment dose dependently activated the Nrf2 signaling pathway under oxidative stress. Additionally, elevation of Nrf2 induction was found in aortic lysates in apoE-/- mice fed with a HFD diet containing equol compared with a HFD diet without equol. Importantly, Nrf2 siRNA interference induced CHOP and attenuated the effect of equol to inhibit t-BHP mediated CHOP induction, furthermore, abrogated cell apoptosis induced by t-BHP, suggesting a role for Nrf2 in the protective effect of equol in HUVECs. Collectively, these findings implicate that the improvement of atherosclerosis by equol through attenuation of ER stress is mediated, at least in part, by activating the Nrf2 signaling pathway. PMID:27907038

  4. Hydroxycarbamide decreases sickle reticulocyte adhesion to resting endothelium by inhibiting endothelial lutheran/basal cell adhesion molecule (Lu/BCAM) through phosphodiesterase 4A activation.

    PubMed

    Chaar, Vicky; Laurance, Sandrine; Lapoumeroulie, Claudine; Cochet, Sylvie; De Grandis, Maria; Colin, Yves; Elion, Jacques; Le Van Kim, Caroline; El Nemer, Wassim

    2014-04-18

    Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

  5. TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells.

    PubMed

    Sasi, Sharath P; Song, Jin; Park, Daniel; Enderling, Heiko; McDonald, J Tyson; Gee, Hannah; Garrity, Brittany; Shtifman, Alexander; Yan, Xinhua; Walsh, Kenneth; Natarajan, Mohan; Kishore, Raj; Goukassian, David A

    2014-05-16

    TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1-5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3-5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general.

  6. TNF-TNFR2/p75 Signaling Inhibits Early and Increases Delayed Nontargeted Effects in Bone Marrow-derived Endothelial Progenitor Cells*

    PubMed Central

    Sasi, Sharath P.; Song, Jin; Park, Daniel; Enderling, Heiko; McDonald, J. Tyson; Gee, Hannah; Garrity, Brittany; Shtifman, Alexander; Yan, Xinhua; Walsh, Kenneth; Natarajan, Mohan; Kishore, Raj; Goukassian, David A.

    2014-01-01

    TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1–5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3–5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general. PMID:24711449

  7. Inhibition of nitric oxide and prostaglandins, but not endothelial-derived hyperpolarizing factors, reduces blood flow and aerobic energy turnover in the exercising human leg.

    PubMed

    Mortensen, Stefan P; González-Alonso, José; Damsgaard, Rasmus; Saltin, Bengt; Hellsten, Ylva

    2007-06-01

    Prostaglandins, nitric oxide (NO) and endothelial-derived hyperpolarizing factors (EDHFs) are substances that have been proposed to be involved in the regulation of skeletal muscle blood flow during physical activity. We measured haemodynamics, plasma ATP at rest and during one-legged knee-extensor exercise (19 +/- 1 W) in nine healthy subjects with and without intra-arterial infusion of indomethacin (Indo; 621 +/- 17 microg min(-1)), Indo + N(G)-monomethyl-L-arginine (L-NMMA; 12.4 +/- 0.3 mg min(-1)) (double blockade) and Indo + L-NMMA + tetraethylammonium chloride (TEA; 12.4 +/- 0.3 mg min(-1)) (triple blockade). Double and triple blockade lowered leg blood flow (LBF) at rest (P<0.05), while it remained unchanged with Indo. During exercise, LBF and vascular conductance were 2.54 +/- 0.10 l min(-1) and 25 +/- 1 mmHg, respectively, in control and they were lower with double (33 +/- 3 and 36 +/- 4%, respectively) and triple (26 +/- 4 and 28 +/- 3%, respectively) blockade (P<0.05), while there was no difference with Indo. The lower LBF and vascular conductance with double and triple blockade occurred in parallel with a lower O(2) delivery, cardiac output, heart rate and plasma [noradrenaline] (P<0.05), while blood pressure remained unchanged and O(2) extraction and femoral venous plasma [ATP] increased. Despite the increased O(2) extraction, leg was 13 and 17% (triple and double blockade, respectively) lower than control in parallel to a lower femoral venous temperature and lactate release (P<0.05). These results suggest that NO and prostaglandins play important roles in skeletal muscle blood flow regulation during moderate intensity exercise and that EDHFs do not compensate for the impaired formation of NO and prostaglandins. Moreover, inhibition of NO and prostaglandin formation is associated with a lower aerobic energy turnover and increased concentration of vasoactive ATP in plasma.

  8. Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells.

    PubMed

    Matsui, Takanori; Higashimoto, Yuichiro; Taira, Junichi; Yamagishi, Sho-ichi

    2013-11-15

    Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10(-7) M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.

  9. N-3 Polyunsaturated Fatty Acids Prevent Diabetic Retinopathy by Inhibition of Retinal Vascular Damage and Enhanced Endothelial Progenitor Cell Reparative Function

    PubMed Central

    Tikhonenko, Maria; Lydic, Todd A.; Opreanu, Madalina; Li Calzi, Sergio; Bozack, Svetlana; McSorley, Kelly M.; Sochacki, Andrew L.; Faber, Matthew S.; Hazra, Sugata; Duclos, Shane; Guberski, Dennis; Reid, Gavin E.; Grant, Maria B.; Busik, Julia V.

    2013-01-01

    Objective The vasodegenerative phase of diabetic retinopathy is characterized by not only retinal vascular degeneration but also inadequate vascular repair due to compromised bone marrow derived endothelial progenitor cells (EPCs). We propose that n-3 polyunsaturated fatty acid (PUFA) deficiency in diabetes results in activation of the central enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM) and that ASM represents a molecular metabolic link connecting the initial damage in the retina and the dysfunction of EPCs. Research Design and Methods Type 2 diabetic rats on control or docosahexaenoic acid (DHA)-rich diet were studied. The number of acellular capillaries in the retinas was assessed by trypsin digest. mRNA levels of interleukin (IL)-1β, IL-6, intracellular adhesion molecule (ICAM)-1 in the retinas from diabetic animals were compared to controls and ASM protein was assessed by western analysis. EPCs were isolated from blood and bone marrow and their numbers and ability to form colonies in vitro, ASM activity and lipid profiles were determined. Results DHA-rich diet prevented diabetes-induced increase in the number of retinal acellular capillaries and significantly enhanced the life span of type 2 diabetic animals. DHA-rich diet blocked upregulation of ASM and other inflammatory markers in diabetic retina and prevented the increase in ASM activity in EPCs, normalized the numbers of circulating EPCs and improved EPC colony formation. Conclusions In a type 2 diabetes animal model, DHA-rich diet fully prevented retinal vascular pathology through inhibition of ASM in both retina and EPCs, leading to a concomitant suppression of retinal inflammation and correction of EPC number and function. PMID:23383097

  10. Salvianolic acid A inhibits angiotensin II-induced proliferation of human umbilical vein endothelial cells by attenuating the production of ROS

    PubMed Central

    Yang, Luan-luan; Li, Dong-ye; Zhang, Yan-bin; Zhu, Man-yi; Chen, Dan; Xu, Tong-da

    2012-01-01

    Aim: To investigate the action of salvianolic acid A (SalA) on angiotensin II (Ang II)-induced proliferation of human umbilical vein endothelial cells (HUVECs) and the possible signaling pathways mediating this action. Methods: Cell proliferation was examined with MTT assay. The expression levels of Src phosphorylation (phospho-Src), Akt phosphorylation (phospho-Akt), and NADPH oxidase 4 (Nox4) in HUVECs were determined by Western blot. The production of reactive oxygen species (ROS) was estimated using fluorescence-activated cell sorting (FACS). Results: SalA (6.25–50 μmol/L) did not affect the viability of HUVECs. Treatment of HUVECs with Ang II (1 μmol/L) markedly increased the cell viability; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) prevented Ang II-induced increase of the cell viability in a concentration-dependent manner. Treatment of HUVECs with Ang II (1 μmol/L) markedly up-regulated the protein expression levels of phospho-Src, phospho-Akt (473) and Nox4; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) blocked all the effects in a concentration-dependent manner. Treatment of HUVECs with Ang II (1 μmol/L) dramatically increased ROS production in HUVECs; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) blocked the ROS production in a concentration-dependent manner. Conclusion: SalA inhibits Ang II-induced proliferation of HUVECs via reducing the expression levels of phospho-Src and phospho-Akt (473), thereby attenuating the production of ROS. PMID:22101169

  11. The ω-3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down-regulation

    PubMed Central

    Cui, Pei H; Petrovic, Nenad; Murray, Michael

    2011-01-01

    BACKGROUND AND PURPOSE Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation. EXPERIMENTAL APPROACH Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids. KEY RESULTS The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive. CONCLUSIONS AND IMPLICATIONS The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation. PMID:21077851

  12. Protocatechuic aldehyde inhibits TNF-α-induced fibronectin expression in human umbilical vein endothelial cells via a c-Jun N-terminal kinase dependent pathway.

    PubMed

    Tong, Yue-Feng; Liu, Yong; Hu, Zhi-Xing; Li, Zhe-Cheng; A, Agula

    2016-01-01

    Fibronectin (FN) is one of the most important extracellular matrix proteins and plays an important role in the pathogenesis of atherosclerosis (AS). The aim of the present study was to evaluate the effect of a potent, water-soluble antioxidant, protocatechuic aldehyde (PA), which is derived from the Chinese herb Salvia miltiorrhiza, on the expression of FN in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-α (TNF-α). The pharmacological effects of PA on the production of FN were investigated using ELISA and western blot analysis. In addition, ELISA and western blot analysis were used to examine the activation and suppression of the mitogen-activated protein kinase (MAPK) pathways and nuclear factor (NF)-κB in TNF-α-stimulated HUVECs, in order to explore the underlying pharmacological mechanism of PA. The inhibitory effect of PA on the total generation of reactive oxygen species (ROS) in TNF-α-stimulated HUVECs was assessed using 2',7'-dichlorofluorescein diacetate. Pretreatment of HUVECs with PA (0.15, 0.45 and 1.35 mM) for 18 h markedly attenuated the TNF-α-stimulated FN surface expression and secretion in a dose-dependent manner. Intracellular ROS generation and the expression of extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) were significantly induced by TNF-α (2 ng/ml) in HUVECs. TNF-α-induced ROS generation and JNK activation were inhibited by PA in a concentration-dependent manner. By contrast, ERK1/2 and p38 activation was not significantly affected by PA. Pretreatment of HUVECs with PA for 18 h markedly attenuated TNF-α-stimulated NF-κB activation. In conclusion, the present findings suggest that PA inhibits TNF-α-induced FN expression in HUVECs through a mechanism that involves ROS/JNK and NF-κB.

  13. Down-regulation of dihydrofolate reductase inhibits the growth of endothelial EA.hy926 cell through induction of G1 cell cycle arrest via up-regulating p53 and p21(waf1/cip1) expression.

    PubMed

    Fei, Zhewei; Gao, Yong; Qiu, Mingke; Qi, Xianqin; Dai, Yuxin; Wang, Shuqing; Quan, Zhiwei; Liu, Yingbin; Ou, Jingmin

    2016-03-01

    Folic acid supplementation may meliorate cardiovascular disease risk by improving vascular endothelial structure and function. However, the underlying mechanisms are still lack of a global understanding. To be used, folic acid must be converted to 7,8-dihydrofolate by dihydrofolate reductase to generate one-carbon derivatives serving as important cellular cofactors in the synthesis of nucleotides and amino acids required for cell growth. Therefore, this study explored the effect of dihydrofolate reductase knockdown on endothelial EA.hy926 cell growth and the mechanism involved. We found that down-regulation of dihydrofolate reductase inhibited EA.hy926 cell proliferation, and induced G1 phase arrest. Meanwhile, the expression of regulators necessary for G1/S phase transition, such as cyclin-dependent kinases CDK2, CDK4 and CDK6, were remarkably down-regulated; by contrast, the cell cycle inhibitors p21(waf/cip1), p27(Kip1) and p53 were significantly up-regulated after dihydrofolate reductase knockdown. Furthermore, supplementation of 5-methyltetrahydrofolate to the dihydrofolate reductase knockdown cells could weaken the inhibitory effect of dihydrofolate reductase knockdown on cell proliferation, simultaneously, inducing the expression of p53 and p21(waf/cip1) falling back moderately. Our findings suggest that attenuating dihydrofolate reductase may cause imbalanced expression of cell cycle regulators, especially up-regulation of p53-p21(waf/cip1) pathway, leading to G1 cell cycle arrest, thereby inhibiting the growth of endothelial EA.hy926 cells.

  14. Lycopene inhibits cyclic strain-induced endothelin-1 expression through the suppression of reactive oxygen species generation and induction of heme oxygenase-1 in human umbilical vein endothelial cells.

    PubMed

    Sung, Li-Chin; Chao, Hung-Hsing; Chen, Cheng-Hsien; Tsai, Jen-Chen; Liu, Ju-Chi; Hong, Hong-Jye; Cheng, Tzu-Hurng; Chen, Jin-Jer

    2015-06-01

    Lycopene is the most potent active antioxidant among the major carotenoids, and its use has been associated with a reduced risk for cardiovascular disease (CVD). Endothelin-1 (ET-1) is a powerful vasopressor synthesized by endothelial cells and plays a crucial role in the pathophysiology of CVD. However, the direct effects of lycopene on vascular endothelial cells have not been fully described. This study investigated the effects of lycopene on cyclic strain-induced ET-1 gene expression in human umbilical vein endothelial cells (HUVECs) and identified the signal transduction pathways that are involved in this process. Cultured HUVECs were exposed to cyclic strain (20% in length, 1 Hz) in the presence or absence of lycopene. Lycopene inhibited strain-induced ET-1 expression through the suppression of reactive oxygen species (ROS) generation through attenuation of p22(phox) mRNA expression and NAD(P)H oxidase activity. Furthermore, lycopene inhibited strain-induced ET-1 secretion by reducing ROS-mediated extrace-llular signal-regulated kinase (ERK) phosphorylation. Conversely, lycopene treatment enhanced heme oxygenase-1 (HO-1) gene expression through the activation of phosphoinositide 3-kinase (PI3K)/Akt pathway, followed by induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation; in addition, HO-1 silencing partially inhibited the repressive effects of lycopene on strain-induced ET-1 expression. In summary, our study showed, for the first time, that lycopene inhibits cyclic strain-induced ET-1 gene expression through the suppression of ROS generation and induction of HO-1 in HUVECs. Therefore, this study provides new valuable insight into the molecular pathways that may contribute to the proposed beneficial effects of lycopene on the cardiovascular system.

  15. Paraoxonase-1 inhibits oxidized low-density lipoprotein-induced metabolic alterations and apoptosis in endothelial cells: a nondirected metabolomic study.

    PubMed

    García-Heredia, Anabel; Marsillach, Judit; Rull, Anna; Triguero, Iris; Fort, Isabel; Mackness, Bharti; Mackness, Michael; Shih, Diana M; Joven, Jorge; Camps, Jordi

    2013-01-01

    We studied the influence of PON1 on metabolic alterations induced by oxidized LDL when incubated with endothelial cells. HUVEC cells were incubated with native LDL, oxidized LDL, oxidized LDL plus HDL from wild type mice, and oxidized LDL plus HDL from PON1-deficient mice. Results showed alterations in carbohydrate and phospholipid metabolism and increased apoptosis in cells incubated with oxidized LDL. These changes were partially prevented by wild type mouse HDL, but the effects were less effective with HDL from PON1-deficient mice. Our results suggest that PON1 may play a significant role in endothelial cell survival by protecting cells from alterations in the respiratory chain induced by oxidized LDL. These results extend current knowledge on the protective role of HDL and PON1 against oxidation and apoptosis in endothelial cells.

  16. Chronic vagus nerve stimulation attenuates vascular endothelial impairments and reduces the inflammatory profile via inhibition of the NF-κB signaling pathway in ovariectomized rats.

    PubMed

    Li, Ping; Liu, Huaipu; Sun, Peng; Wang, Xiaoyu; Wang, Chen; Wang, Ling; Wang, Tinghuai

    2016-02-01

    Vagus nerve stimulation (VNS), a method for activating cholinergic anti-inflammatory pathways, could suppress endothelial activation and minimize tissue injury during inflammation. The aim of this study was to investigate the effects of chronic VNS on endothelial impairments and the inflammatory profile in ovariectomized (OVX) rats. Sprague-Dawley rats (7-8 months old) were randomly assigned to the following four groups: sham-OVX, OVX, OVX+sham-VNS, and OVX+VNS. Throughout the experimental period, the OVX+VNS group received VNS for 3h (20.0 Hz, 1.0 mA, and 10.00 ms pulse width) at the same time every other day. After 12 weeks of VNS, blood samples and thoracic aortas were collected for further analyses. Light microscopy and electron microscopy analyses showed that chronic VNS prevented endothelial swelling, desquamation and even necrosis in the OVX rats. In addition, it obviously improved endothelial function in the OVX rats by restoring the endothelial nitric oxide synthase (e-NOS) and serum endothelin-1 level. Increased expression of cell adhesion molecules (VCAM-1, ICAM-1 and E-selectin) in the thoracic aortas and increases in the levels of circulating cytokines (TNF-α, IL-6, MCP-1, and CINC/KC) were also observed in the OVX rats. Chronic VNS significantly restored these detrimental changes partly by increasing the ACh concentrations in vascular walls and blocking NF-κB pathway activity. The results of this in vivo study have shown that the administration of chronic VNS during, in the early stage of estrogen deficiency, protects OVX rats from endothelial impairments and the inflammatory profile. These findings indicate that activation of the vagus nerve could be a promising supplemental therapy for reducing the risks of suffering from further CVDs in postmenopausal women.

  17. Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1.

    PubMed

    Petry, Andreas; BelAiba, Rachida S; Weitnauer, Michae; Görlach, Agnes

    2012-11-01

    Disruption of endothelial homeostasis results in endothelial dysfunction, characterised by a dysbalance between nitric oxide (NO) and reactive oxygen species (ROS) levels often accompanied by a prothrombotic and proproliferative state. The serine protease thrombin not only is instrumental in formation of the fibrin clot, but also exerts direct effects on the vessel wall by activating proliferative and angiogenic responses. In endothelial cells, thrombin can induce NO as well as ROS levels. However, the relative contribution of these reactive species to the angiogenic response towards thrombin is not completely clear. Since plasminogen activator inhibitor-1 (PAI-1), a direct target of the proangiogenic transcription factors hypoxia-inducible factors (HIFs), exerts prothrombotic and proangiogenic activities we investigated the role of ROS and NO in the regulation of HIF-1α, PAI-1 and capillary formation in response to thrombin. Thrombin enhanced the formation of NO as well as ROS generation involving the GTPase Rac1 in endothelial cells. Rac1-dependent ROS formation promoted induction of HIF-1α, PAI-1 and capillary formation by thrombin, while NO reduced ROS bioavailability and subsequently limited induction of HIF-1α, PAI-1 and the angiogenic response. Importantly, thrombin activation of Rac1 was diminished by NO, but enhanced by ROS. Thus, our findings show that capillary formation induced by thrombin via Rac1-dependent activation of HIF-1 and PAI-1 is limited by the concomitant release of NO which reduced ROS bioavailability. Rac1 activity is sensitive to ROS and NO, thereby playing an essential role in fine tuning the endothelial response to thrombin.

  18. Folic Acid Attenuates Vascular Endothelial Cell Injury Caused by Hypoxia via the Inhibition of ERK1/2/NOX4/ROS Pathway.

    PubMed

    Cheng, Fei; Lan, Jun; Xia, Wenhao; Tu, Chang; Chen, Benfa; Li, Shicheng; Pan, Weibiao

    2016-06-01

    Coronary artery disease is a disease with high morbidity and mortality, in which vascular endothelial dysfunction plays an important role. Hypoxia leads to the inflammation and oxidative stress in endothelial cells, which results in the endothelial injury. The present study was designed to investigate the protective effect and mechanism of folic acid on hypoxia-induced injury in human umbilical vein endothelial cells (HUVEC). Cell counting Kit was used to detect cell survival rate, and apoptotic cells were detected by Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured using dichloro-dihydro-fluorescein diacetate staining. Western blot was used to determine the protein expressions of extracellular signal protein kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2), NOX4 subunit of NAPDH and endothelial nitric oxide synthase (eNOS). Folic acid significantly increased the cell survival rate and decreased the apoptosis of HUVECs treated with folic acid compared with hypoxia-treated HUVEC. Folic acid also decreased ROS level, while it increased the nitrite content in HUVECs. In addition, folic acid decreased protein expressions of NOX4 and p-ERK1/2, while it increased the protein expression of eNOS in HUVECs. Furthermore, N-acetyl cysteine (NAC), the antioxidant, had similar effect on the cell survival rate and the apoptosis. In addition, DPI (NOX4 inhibitor) and U0126 (ERK1/2 inhibitor) rather than NAC decreased the protein expression of NOX4. NAC, DPI, and U0126 increased the protein expression of eNOS. Furthermore, U0126 rather than DPI and NAC decreased the protein expression of p-ERK1/2. Taken together, the results suggested that hypoxia decreased the cell survival rate and induced apoptosis via ERK1/2/NOX4/ROS pathway, which could be the target of folic acid in protecting the HUVECs from injury caused by hypoxia.

  19. Clematichinenoside inhibits VCAM-1 and ICAM-1 expression in TNF-α-treated endothelial cells via NADPH oxidase-dependent IκB kinase/NF-κB pathway.

    PubMed

    Yan, Simin; Zhang, Xu; Zheng, Haili; Hu, Danhong; Zhang, Yongtian; Guan, Qinghua; Liu, Lifang; Ding, Qilong; Li, Yunman

    2015-01-01

    Proinflammatory cytokine TNF-α-induced adhesion of leukocytes to endothelial cells plays a critical role in the early stage of atherosclerosis. Oxidative stress and redox-sensitive transcription factors are implicated in the process. Thus, compounds that mediate intracellular redox status and regulate transcription factors are of great therapeutic interest. Clematichinenoside (AR), a triterpene saponin isolated from the root of Clematis chinensis Osbeck, was previously demonstrated to have anti-inflammatory and antioxidative properties. However, little is known about the exact mechanism underlying these actions. Thus we performed a detailed study on its effect on leukocytes-endothelial cells adhesion with TNF-α-stimulated human umbilical vein endothelial cells (HUVECs) and cell-free systems. First, we found that AR reduced TNF-α-induced VCAM-1 and ICAM-1 expression and their promoter activity, inhibited translocation of p65 and phosphorylation of IκBα, suppressed IκB kinase-β (IKK-β) activity, lowered O2(∙-) and H2O2 levels, tackled p47(phox) translocation, and decreased NOX4 NADPH oxidase expression. Second, we showed that AR exhibited no direct free radical scavenging ability in cell-free systems at concentrations that were used in intact cells. Besides, AR had no direct effect on the activity of IKK-β that was extracted from TNF-α-stimulated HUVECs. We also found that p47 translocation, NOX4 expression, and reactive oxygen species (ROS) levels were up-regulated before IκB phosphorylation in TNF-α-induced HUVECs. Moreover, TNF-α-enhanced IKK-β activity was also inhibited by (polyethylene glycol) PEG-catalase, N-acetylcysteine (NAC), and vitamin E. In conclusion, these results suggest that AR reduces VCAM-1 and ICAM-1 expression through NADPH oxidase-dependent IKK/NF-κB pathways in TNF-α-induced HUVECs, which finally suppress monocyte-HUVECs adhesion. This compound is potentially beneficial for early-stage atherosclerosis.

  20. Cinnamaldehyde inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion molecules in endothelial cells by suppressing NF-kappaB activation: effects upon IkappaB and Nrf2.

    PubMed

    Liao, Being-Chyuan; Hsieh, Chia-Wen; Liu, Yen-Chin; Tzeng, Tsai-Teng; Sun, Yung-Wei; Wung, Being-Sun

    2008-06-01

    The production of adhesion molecules and subsequent attachment of leukocytes to endothelial cells (ECs) are critical early events in atherogenesis. These adhesion molecules thus play an important role in the development of this disease. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of cinnamaldehyde, a Cinnamomum cassia Presl-specific diterpene. In our current study, we have examined the effects of both cinnamaldehyde and extracts of C. cassia on cytokine-induced monocyte/human endothelial cell interactions. We find that these compounds inhibit the adhesion of TNFalpha-induced monocytes to endothelial cells and suppress the expression of the cell adhesion molecules, VCAM-1 and ICAM-1, at the transcriptional level. Moreover, in TNFalpha-treated ECs, the principal downstream signal of VCAM-1 and ICAM-1, NF-kappaB, was also found to be abolished in a time-dependent manner. Interestingly, cinnamaldehyde exerts its anti-inflammatory effects by blocking the degradation of the inhibitory protein IkappaB-alpha, but only in short term pretreatments, whereas it does so via the induction of Nrf2-related genes, including heme-oxygenase-1 (HO-1), over long term pretreatments. Treating ECs with zinc protoporphyrin, a HO-1 inhibitor, partially blocks the anti-inflammatory effects of cinnamaldehyde. Elevated HO-1 protein levels were associated with the inhibition of TNFalpha-induced ICAM-1 expression. In addition to HO-1, we also found that cinnamaldehyde can upregulate Nrf2 in nuclear extracts, and can increase ARE-luciferase activity and upregulate thioredoxin reductase-1, another Nrf2-related gene. Moreover, cinnamaldehyde exposure rapidly reduces the cellular GSH levels in ECs over short term treatments but increases these levels after 9 h exposure. Hence, our present findings indicate that cinnamaldehyde suppresses TNF-induced singling pathways via two distinct mechanisms that are activated by different pretreatment periods.

  1. SIRT1 inhibits releases of HMGB1 and HSP70 from human umbilical vein endothelial cells caused by IL-6 and the serum from a preeclampsia patient and protects the cells from death.

    PubMed

    Yin, Yongxiang; Feng, Yaling; Zhao, Hua; Zhao, Ziyu; Yua, Hua; Xu, Jianjuan; Che, Haisha

    2017-04-01

    Preeclampsia (PE), a pregnancy-specific disorder, is associated with inappropriate maternal inflammatory response, oxidative stress, and vascular endothelial cell dysfunction and damage. Releases of high mobility group box-1 (HMGB1) and heat-shock protein 70 (HSP70) into serum are considered to participate in the pathogenesis of PE. The deacetylase, sirtuin 1 (SIRT1), has protective effects against inflammation, apoptosis, and oxidative stress in various pathological conditions. We established a PE mouse model by injection of phosphatidylserine/dioleoyl-phosphatidycholine compounds, followed by measurement of the SIRT1 protein level in the placenta via Western blotting and the serum HMGB1 and HSP70 concentrations via enzyme-linked immunosorbent assay (ELISA). SIRT1 was down-regulated in the placenta of PE mice, in accompany with increased serum HMGB1 and HSP70 concentrations. We incubated human umbilical vein endothelial cells (HUVECs) with IL-6 and the serum from a PE patient individually to mimic status of vein endothelial cells in PE. Western blot and Immunofluorescent assays showed that HMGB1 and HSP70 protein levels were decreased in the cells, but they were increased in the cell medium based on ELISA. These suggested that HMGB1 and HSP70 were forced to be released from the cells. SIRT1 knockdown promoted the releases of HMGB1 and HSP70, whereas its over-expression inhibited the releases. Moreover, SIRT1 protected the cells from death. Collectively, SIRT1 inhibits the releases of HMGB1 and HSP70 from HUVECs caused by IL-6 and the serum from PE patient and protects the cells from death, thus SIRT1 is probably a potentially protective factor in placenta against PE.

  2. Metformin and Resveratrol Inhibited High Glucose-Induced Metabolic Memory of Endothelial Senescence through SIRT1/p300/p53/p21 Pathway

    PubMed Central

    Gao, Haiyang; Xu, Ruixia; Teng, Siyong; Wu, Yongjian

    2015-01-01

    Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as “metabolic memory.” Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how “metabolic memory” would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of “metabolic memory” of cellular senescence (senescent “memory”). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent “memory.” In contrast, senescent “memory” was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of “metabolic memory.” Furthermore, we found that RSV or MET treatment prevented senescent “memory” by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent “memory.” In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT

  3. Xiang-Qi-Tang and its active components exhibit anti-inflammatory and anticoagulant properties by inhibiting MAPK and NF-κB signaling pathways in LPS-treated rat cardiac microvascular endothelial cells.

    PubMed

    He, Chang-Liang; Yi, Peng-Fei; Fan, Qiao-Jia; Shen, Hai-Qing; Jiang, Xiao-Lin; Qin, Qian-Qian; Song, Zhou; Zhang, Cui; Wu, Shuai-Cheng; Wei, Xu-Bin; Li, Ying-Lun; Fu, Ben-Dong

    2013-04-01

    Xiang-Qi-Tang (XQT) is a Chinese herbal formula containing Cyperus rotundus, Astragalus membranaceus and Andrographis paniculata. Alpha-Cyperone (CYP), astragaloside IV (AS-IV) and andrographolide (AND) are the three major active components in this formula. XQT may modulate the inflammatory or coagulant responses. We therefore assessed the effects of XQT on lipopolysaccharide (LPS)-induced inflammatory model of rat cardiac microvascular endothelial cells (RCMECs). XQT, CYP, AS-IV and AND inhibited the production of tumor necrosis factor alpha (TNF-α), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1), and up-regulated the mRNA expression of Kruppel-like factor 2 (KLF2). XQT and CYP inhibited the secretion of tissue factor (TF). To further explore the mechanism, we found that XQT, or its active components CYP, AS-IV and AND significantly inhibited extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 phosphorylation protein expression as well as decreased the phosphorylation levels of nuclear factor κB (NF-κB) p65 proteins in LPS-stimulated RCMECs. These results suggested that XQT and its active components inhibited the expression of inflammatory and coagulant mediators via mitogen-activated protein kinase (MAPKs) and NF-κB signaling pathways. These findings may contribute to future research on the action mechanisms of this formula, as well as therapy for inflammation- or coagulation-related diseases.

  4. Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells.

    PubMed

    Chang, Hebron C; Yang, Hsin-Ling; Pan, Jih-Hao; Korivi, Mallikarjuna; Pan, Jian-You; Hsieh, Meng-Chang; Chao, Pei-Min; Huang, Pei-Jane; Tsai, Ching-Tsan; Hseu, You-Cheng

    2016-01-01

    Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50-200 μg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways.

  5. Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells

    PubMed Central

    Chang, Hebron C.; Yang, Hsin-Ling; Pan, Jih-Hao; Korivi, Mallikarjuna; Pan, Jian-You; Hsieh, Meng-Chang; Chao, Pei-Min; Huang, Pei-Jane; Tsai, Ching-Tsan; Hseu, You-Cheng

    2016-01-01

    Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50–200 μg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways. PMID:26823953

  6. Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signaling

    PubMed Central

    Das, Amlan; Gopalakrishnan, Bhavani; Voss, Oliver H.; Doseff, Andrea I.; Villamena, Frederick A.

    2012-01-01

    Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanisms by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone alpha-phenyl N-tert-butyl nitrone (PBN) against H2O2-induced cytoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H2O2–mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H2O2. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H2O2–treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade. PMID:22580046

  7. Human aortic smooth muscle cell-derived exosomal miR-221/222 inhibits autophagy via a PTEN/Akt signaling pathway in human umbilical vein endothelial cells.

    PubMed

    Li, Luocheng; Wang, Zhiwei; Hu, Xiaoping; Wan, Ting; Wu, Hongbing; Jiang, Wanli; Hu, Rui

    2016-10-14

    Dysregulation of autophagy in endothelial cells plays a vital role in cardiovascular dysfunction and atherosclerosis. Accumulating evidence shows that miRNAs regulate autophagy in various cell types by targeting autophagy-related genes. In the present study, we found that a co-culture of human umbilical vein endothelial cells (HUVECs) with human aortic smooth muscle cells (HAoSMCs) inhibited autophagy activity in HUVECs. Furthermore, we isolated exosomes secreted by HAoSMCs, and confirmed that the exosomes contain miR-221/222. We investigated the role of miR-221/222 transferred by HAoSMC-derived exosomes in HUVECs. These exosomes induced an increase of miR-221/222 expression and a down-regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in HUVECs. Dual luciferase reporter assays revealed that miR-221/222 could bind to the 3'UTR of PTEN, which implied that PTEN was a direct target of miR-221/222. The expression of PTEN could be down-regulated by miR-221/222 over-expression. Then, we detected the expression of PTEN, LC3, ATG5, SQSTM1/p62, Beclin-1, Akt, and phospho-Akt in HUVECs transfected with miR-221/222 mimics and inhibitors. Our results demonstrated that miR-221/222 overexpression inhibited the expression of PTEN and subsequently activated Akt signaling, and eventually down-regulated the expression of LC3II, ATG5 and Beclin-1, and elevated the expression of SQSTM1/p62. This phenomenon can be reversed by the transfection of miR-221/222 inhibitors. These data suggested that miR-221/222 from HAoSMC-derived exosomes inhibited autophagy in HUVECs by modulating PTEN/Akt signaling pathway.

  8. HIV-1 Tat protein increases the permeability of brain endothelial cells by both inhibiting occludin expression and cleaving occludin via matrix metalloproteinase-9.

    PubMed

    Xu, Ruifen; Feng, Xuyang; Xie, Xin; Zhang, Jin; Wu, Daocheng; Xu, Lixian

    2012-02-03

    Brain homeostasis is maintained by the blood-brain barrier (BBB), which prevents the entrance of circulating molecules and immune cells into the central nervous system. The BBB is formed by specialized brain endothelial cells that are connected by tight junctions (TJ). Previous studies have proven that the Tat protein of human immunodeficiency virus type 1 (HIV-1) alters TJ protein expression. However, the mechanisms by which the alterations occur have not been characterized in detail. In this study, primary human brain microvascular endothelial cells (HBMEC) were exposed to recombinant HIV-1 Tat protein, and the effects on occludin were observed. Tat treatment decreased occludin mRNA and protein levels. This effect was partially abrogated by addition of the RhoA inhibitor C3 exoenzyme and the p160-Rho-associated coiled kinase (ROCK) inhibitor Y-27632. Meanwhile, Tat also induced MMP-9 expression. RNA interference targeting MMP-9 reduced both the paracellular permeability of Tat-treated HBMEC and the concentration of soluble occludin in supernatants from the cells. Taken together, these results show that the HIV-1 Tat protein disrupts BBB integrity, at least in part by decreasing the production of occludin.

  9. Berberine inhibits tumor necrosis factor-α-induced expression of inflammatory molecules and activation of nuclear factor-κB via the activation of AMPK in vascular endothelial cells.

    PubMed

    Liu, Su-Jian; Yin, Cai-Xia; Ding, Ming-Chao; Wang, Yi-Zhong; Wang, Hong

    2015-10-01

    Berberine, which is a well‑known drug used in traditional medicine, has been demonstrated to exert diverse pharmacological effects, including anti‑inflammatory effects. However, whether berberine can affect the production of inflammatory molecules in vascular endothelial cells remains to be elucidated. Therefore, the present study aimed to determine the effects of berberine, and the underlying molecular mechanisms of these effects. The effect of berberine on tumor necrosis factor (TNF)‑α‑induced inflammatory molecule expression was examined in cultured human aortic endothelial cells (HAECs). The HAECs were stimulated with TNF‑α and incubated with or without berberine. The activation of nuclear factor (NF)‑κB and adenosine monophosphate‑activated protein kinase (AMPK) were analyzed using western blotting, and the protein secretion of intercellular adhesion molecule (ICAM)‑1 and monocyte chemoattractant protein (MCP)‑1 was measured using ELISA kits. The mRNA expression levels of ICAM‑1 and MCP‑1 were analyzed using reverse transcription‑quantitative polymerase chain reaction. The results of the present study demonstrated that berberine significantly inhibited the TNF‑α‑induced expression of ICAM‑1 and MCP‑1, as well as the activation of NF‑κB in the HAECs. These effects were attenuated following co‑treatment with AMPK inhibitor compound C, or specific small interfering RNAs. In conclusion, the results of the present study indicated that berberine inhibits the TNF‑α‑induced expression of ICAM‑1 and MCP‑1, and the activation of NF‑κB in HAECs in vitro, possibly through the AMPK‑dependent pathway.

  10. Inhibiting Extracellular Vesicle Release from Human Cardiosphere Derived Cells with Lentiviral Knockdown of nSMase2 Differentially Effects Proliferation and Apoptosis in Cardiomyocytes, Fibroblasts and Endothelial Cells In Vitro

    PubMed Central

    Young, Rebeccah F.; Ashraf, Hashmat; Canty, John M.

    2016-01-01

    Numerous studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes may contribute to improved cardiac function. However, it has not yet been demonstrated by a genetic approach that exosome release contributes to the therapeutic effect of transplanted CDCs. By employing a lentiviral knockdown (KD) strategy against neutral spingomyelinase 2 (nSMase2), a crucial gene in exosome secretion, we have defined the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblast, endothelial cell and primary cardiomyocyte proliferation, cell death, migration and angiogenesis using a series of in vitro coculture assays. We found that secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 lentiviral KD and shRNAi expression was stable and constitutive. hCDC exosome release contributed to the angiogenic and pro-migratory effects of hCDCs on HUVECs, decreased proliferation of fibroblasts, and decreased apoptosis of cardiomyocytes. These in vitro reactions support a role for exosome secretion as a paracrine mechanism of stem cell-mediated cardiac repair in vivo. Importantly, we have established a novel tool to test constitutive inhibition of exosome secretion in stem cell populations in animal models of cardiac disease. PMID:27806113

  11. Fcγ Receptor-induced Soluble Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Production Inhibits Angiogenesis and Enhances Efficacy of Anti-tumor Antibodies*

    PubMed Central

    Justiniano, Steven E.; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M.; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D.; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P.; Byrd, John C.; Tridandapani, Susheela

    2013-01-01

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy. PMID:23902770

  12. Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

    PubMed

    Justiniano, Steven E; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P; Byrd, John C; Tridandapani, Susheela

    2013-09-13

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

  13. Soluble mediators produced by the crosstalk between microvascular endothelial cells and dengue-infected primary dermal fibroblasts inhibit dengue virus replication and increase leukocyte transmigration.

    PubMed

    Bustos-Arriaga, José; Mita-Mendoza, Neida K; Lopez-Gonzalez, Moises; García-Cordero, Julio; Juárez-Delgado, Francisco J; Gromowski, Gregory D; Méndez-Cruz, René A; Fairhurst, Rick M; Whitehead, Stephen S; Cedillo-Barrón, Leticia

    2016-04-01

    When dengue virus (DENV)-infected mosquitoes use their proboscis to probe into human skin during blood feeding, both saliva and virus are released. During this process, cells from the epidermis and dermis layers of the skin, along with small blood vessels, may get exposed to or infected with DENV. In these microenvironments of the skin, the presence of DENV initiates a complex interplay among the DENV-infected and non-infected neighboring cells at the initial bite site. Previous studies suggested that DENV-infected human dermal fibroblasts (HDFs) participate in the immune response against DENV by secreting soluble mediators of innate immunity. In the present study, we investigated whether DENV-infected HDFs activate human dermal microvascular endothelial cells (HDMECs) in co-cultures. Our results suggest that co-cultures of DENV-infected HDFs and HDMECs elicit soluble mediators that are sufficient to reduce viral replication, activate HDMECs, and induce leukocyte migration through HDMEC monolayers. These effects were partly dependent on HDF donor and DENV serotype, which may provide novel insights into the natural variation in host susceptibility to DENV disease.

  14. α-Solanine inhibits vascular endothelial growth factor expression by down-regulating the ERK1/2-HIF-1α and STAT3 signaling pathways.

    PubMed

    Wen, Zhengde; Huang, Chaohao; Xu, Yaya; Xiao, Yuwu; Tang, Lili; Dai, Juji; Sun, Hongwei; Chen, Bicheng; Zhou, Mengtao

    2016-01-15

    In tumors, vascular endothelial growth factor (VEGF) contributes to angiogenesis, vascular permeability, and tumorigenesis. In our previous study, we found that α-solanine, which is widespread in solanaceae, has a strong anti-cancer effect under normoxia. However, it is unknown whether α-solanine has a similar effect under hypoxia. We used cobalt chloride (CoCl2) to mimic hypoxia in vitro. HIF-1α, which is almost undetectable under normoxia, was significantly increased. Simultaneously, another regulator of VEGF, STAT3, was also significantly activated by CoCl2. We utilized α-solanine in co-culture with CoCl2. α-solanine decreased the expression of VEGF and loss of E-cadherin. α-solanine also suppressed the activation of phospho-ERK1/2 (p-ERK1/2), HIF-1α, and STAT3 signaling. The results provide new evidence that α-solanine has a strong anti-cancer effect via the ERK1/2-HIF-1α and STAT3 signaling pathways and suggest that it may be a potential new drug.

  15. Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

    PubMed

    Beierle, Elizabeth A; Ma, Xiaojie; Stewart, Jerry E; Megison, Michael; Cance, William G; Kurenova, Elena V

    2014-03-01

    Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma.

  16. Anticancer effects of novel thalidomide analogs in A549 cells through inhibition of vascular endothelial growth factor and matrix metalloproteinase-2.

    PubMed

    El-Aarag, Bishoy; Kasai, Tomonari; Masuda, Junko; Agwa, Hussein; Zahran, Magdy; Seno, Masaharu

    2017-01-01

    Lung cancer is one of the major causes of cancer-related mortality worldwide, and non-small-cell lung cancer is the most common form of lung cancer. Several studies had shown that thalidomide has potential for prevention and therapy of cancer. Therefore, the current study aimed to investigate the antitumor effects of two novel thalidomide analogs in human lung cancer A549 cells. The antiproliferative, antimigratory, and apoptotic effects in A549 cells induced by thalidomide analogs were examined. In addition, their effects on the expression of mRNAs encoding vascular endothelial growth factor165 (VEGF165) and matrix metalloproteinase-2 (MMP-2) were evaluated. Their influence on the tumor volume in nude mice was also determined. Results revealed that thalidomide analogs exhibited antiproliferative, antimigratory, and apoptotic activities with more pronounced effect than thalidomide drug. Furthermore, analogs 1 and 2 suppressed the expression levels of VEGF165 by 42% and 53.2% and those of MMP-2 by 45% and 52%, respectively. Thalidomide analogs 1 and 2 also reduced the tumor volume by 30.11% and 53.52%, respectively. Therefore, this study provides evidence that thalidomide analogs may serve as a new therapeutic option for treating lung cancer.

  17. Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C6 glioma cells and RAW 264.7 macrophages.

    PubMed Central

    Chen, B. C.; Lee, C. M.; Lin, W. W.

    1996-01-01

    1. Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P2Y and P2U purinoceptors, rat C6 glioma cells have P2U purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide-specific phospholipase C (PI-PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto-ATPase inhibitors were studied in these three cell types. 2. In CPAE, suramin, at 3-100 microM, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA2 values of 5.5 +/- 0.3 and 4.4 +/- 0.4, respectively. Reactive blue, at 1-3 microM, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 microM. PPADS, at 10 microM, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 microM. In contrast, a dose-dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. 3. In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA2 values of 4.8 +/- 0.5 and 5.8 +/- 0.7, respectively. 4. In C6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. 5. The ecto-ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto-ATPase present in CPAE, C6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. 6. This study indicates that PPADS, suramin and reactive blue ar ecto-ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist-mediated PI responses. Reactive blue is a more potent antagonist than suramin on P2Y, P2U and pyrimidinoceptors, and PPADS is a weak antagonist for P2Y

  18. A novel LMP1 antibody synergizes with mitomycin C to inhibit nasopharyngeal carcinoma growth in vivo through inducing apoptosis and downregulating vascular endothelial growth factor.

    PubMed

    Mao, Yuan; Zhang, Da-Wei; Wen, Juan; Cao, Qing; Chen, Ren-Jie; Zhu, Jin; Feng, Zhen-Qing

    2012-01-01

    Combined therapy emerges as an attractive strategy for cancer treatment. The aim of this study was to investigate the inhibitory effects of mitomycin C (MMC) combined with a novel antibody fragment (Fab) targeting latent membrane protein 1 (LMP1) on nasopharyngeal carcinoma (NPC) xenograft nude mice. The inhibitory rates of MMC (2 mg/kg), Fab (4 mg/kg), MMC (2 mg/kg) + Fab (4 mg/kg), and MMC (1 mg/kg) + Fab (4 mg/kg) were 20.1%, 7.3%, 42.5% and 40.5%, respectively. Flow cytometry analysis showed that the apoptotic rate of xenograft tumor cells in the MMC and Fab combination group was 28 ± 4.12%, significantly higher than the MMC (2 mg/kg) group (P < 0.01). Immunohistochemical staining showed that VEGF expression in NPC xenografts was significantly inhibited in the combination group compared to the Fab (4 mg/kg) group (P < 0.05). In conclusion, both MMC and Fab could inhibit NPC xenograft tumor growth in vivo and combination therapy showed apparent synergistic anti-tumor effects, which may be due to the induction of tumor cell apoptosis and the downregulation of VEGF expression. These results suggest that the novel combined therapy utilizing traditional chemotherapeutics and antibody-targeted therapy could be a promising strategy for the treatment of NPC.

  19. 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

  20. The anti-diabetic drug metformin inhibits vascular endothelial growth factor expression via the mammalian target of rapamycin complex 1/hypoxia-inducible factor-1α signaling pathway in ELT-3 cells.

    PubMed

    Tadakawa, Mari; Takeda, Takashi; Li, Bin; Tsuiji, Kenji; Yaegashi, Nobuo

    2015-01-05

    The aim of this study was to elucidate whether metformin can regulate the expression of vascular endothelial growth factor (VEGF) in rat-derived uterine leiomyoma cells (ELT-3 cells). In vitro studies were conducted using ELT-3 cells. Under normoxic conditions, metformin suppressed VEGF protein levels in the supernatant and cells in a dose-dependent manner. In hypoxia-mimicking conditions, VEGF and hypoxia-inducible factor-1α (HIF-1α) proteins were both highly expressed and were suppressed by the metformin treatment. Metformin did not affect HIF-1α mRNA levels, which indicated that its effects occurred at the post-translational level. Metformin inhibited mammalian target of rapamycin complex 1 (mTORC1) activity by phosphorylating the mTORC1 component raptor. This study revealed the anti-angiogenic activity of metformin in ELT-3 cells by suppressing the expression of VEGF via the mTORC1/HIF-1α pathway. These results indicate that metformin may represent an effective alternative in the future treatment of uterine leiomyomas.

  1. Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model.

    PubMed

    Liu, Chung-Pin; Dai, Zen-Kong; Huang, Chein-Heng; Yeh, Jwu-Lai; Wu, Bin-Nan; Wu, Jiunn-Ren; Chen, Ing-Jun

    2014-06-01

    This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 μM), KMUP-1 (0.1-100 μM) inhibited ET-1 (0.01 μM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1-10 μM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH

  2. Endothelial glucocorticoid receptor suppresses atherogenesis- Brief Report

    PubMed Central

    Zhang, Xinbo; Rotllan, Noemi; Feng, Yan; Zhou, Han; Fernández-Hernando, Carlos; Yu, Jun; Sessa, William C.

    2015-01-01

    Objective The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. Approach and Results Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the two groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery and aortic sinus as well as a heightened inflammatory milieu as evidence by increased macrophage recruitment in the lesions. Conclusions These data suggest the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model. PMID:25810297

  3. 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.

  4. [Vascular endothelial Barrier Function].

    PubMed

    Ivanov, A N; Puchinyan, D M; Norkin, I A

    2015-01-01

    Endothelium is an important regulator of selective permeability of the vascular wall for different molecules and cells. This review summarizes current data on endothelial barrier function. Endothelial glycocalyx structure, its function and role in the molecular transport and leukocytes migration across the endothelial barrier are discussed. The mechanisms of transcellular transport of macromolecules and cell migration through endothelial cells are reviewed. Special section of this article addresses the structure and function of tight and adherens endothelial junction, as well as their importance for the regulation of paracellular transport across the endothelial barrier. Particular attention is paid to the signaling mechanism of endothelial barrier function regulation and the factors that influence on the vascular permeability.

  5. Eldecalcitol prevents endothelial dysfunction in postmenopausal osteoporosis model rats.

    PubMed

    Serizawa, Kenichi; Yogo, Kenji; Tashiro, Yoshihito; Takeda, Satoshi; Kawasaki, Ryohei; Aizawa, Ken; Endo, Koichi

    2016-02-01

    Postmenopausal women have high incidence of cardiovascular events as estrogen deficiency can cause endothelial dysfunction. Vitamin D is reported to be beneficial on endothelial function, but it remains controversial whether vitamin D is effective for endothelial dysfunction under the treatment for osteoporosis in postmenopausal women. The aim of this study was to evaluate the endothelial protective effect of eldecalcitol (ELD) in ovariectomized (OVX) rats. ELD (20  ng/kg) was orally administrated five times a week for 4 weeks from 1 day after surgery. After that, flow-mediated dilation (FMD) as an indicator of endothelial function was measured by high-resolution ultrasound in the femoral artery of living rats. ELD ameliorated the reduction of FMD in OVX rats. ELD inhibited the increase in NOX4, nitrotyrosine, and p65 and the decrease in dimer/monomer ratio of nitric oxide synthase in OVX rat femoral arteries. ELD also prevented the decrease in peroxisome proliferator-activated receptor gamma (PPARγ) in femoral arteries and cultured endothelial cells. Although PPARγ is known to inhibit osteoblastogenesis, ELD understandably increased bone mineral density of OVX rats without increase in PPARγ in bone marrow. These results suggest that ELD prevented the deterioration of endothelial function under condition of preventing bone loss in OVX rats. This endothelial protective effect of ELD might be exerted through improvement of endothelial nitric oxide synthase uncoupling, which is mediated by an antioxidative effect through normalization of vascular PPARγ/NF-κB signaling.

  6. The function of vascular endothelial growth factor.

    PubMed

    Nieves, Bonnie J; D'Amore, Patricia A; Bryan, Brad A

    2009-01-01

    Vascular endothelial growth factor (VEGF) is considered the master regulator of angiogenesis during growth and development, as well as in disease states such as cancer, diabetes, and macular degeneration. This review details our current understanding of VEGF signaling and discusses the benefits and unexpected side effects of promising anti-angiogenic therapeutics that are currently being used to inhibit neovacularization in tumors.

  7. Inhibition of the NF-κB pathway by R65 ribozyme gene via adeno-associatedvirus serotype 9 ameliorated oxidized LDL induced human umbilical vein endothelial cell injury

    PubMed Central

    Zhai, Hui; Chen, Qing-Jie; Gao, Xiao-Ming; Ma, Yi-Tong; Chen, Bang-Dang; Yu, Zi-Xiang; Li, Xiao-Mei; Liu, Fen; Xiang, Yang; Xie, Jia; Yang, Yi-Ning

    2015-01-01

    Objective: NF-κB signaling plays a central role in the regulation of inflammatory responses in atherosclerosis. R65 ribozyme gene suppresses activation of NF-κB pathway, therefore we studied whether R65 gene therapy can ameliorate oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) injury. Methods and results: Recombinant adeno-associated virus serotype 9 (rAVV9) vector was used to transfect the R65 ribozyme gene (rAVV9-R65) into HUVECs then following ox-LDL stimulation, expression of NF-κB p65 and p50 subunits, inflammatory mediators and cell apoptosis were examined. First, rAVV9-enhanced green fluorescent protein (eGFP)-R65 at 1×107 v.g./cell multiplicity of infection reached a long-lasting and significant increase in R65 gene expression. Second, ox-LDL treatment led to time- and dose-dependent activation of NF-κB pathway, and enhanced inflammatory response and cell death evidenced by increased expression of nuclear NF-κB p65 and p50 subunits, greater production of tumor necrosis factor α, interleukin-6 and von willebrand factor and 20.57% increasedapoptotic HUVECs. Third, over-expression ofR65 gene was 2-fold increased in HUVECs attenuated ox-LDL induced unclear accumulation and expression of p65 subunit and ameliorated inflammation and cell death (all P < 0.05). Conclusion: rAAV9-mediated R65 ribozyme gene transfection in cultured HUVECs effectively inhibits ox-LDL induced activation of NF-κB and production of inflammatory cytokines and prevents cell apoptosis. PMID:26617700

  8. MicroRNA-34a regulation of endothelial senescence

    SciTech Connect

    Ito, Takashi; Yagi, Shusuke; Yamakuchi, Munekazu

    2010-08-06

    Research highlights: {yields} MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. {yields} MiR-34a expression increases during endothelial cell senescence and in older mice. {yields} SIRT1 is a miR-34a target gene in endothelial cells. {yields} SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells. -- Abstract: Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelial cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.

  9. Polyphenols in preventing endothelial dysfunction.

    PubMed

    Biegańska-Hensoldt, Sylwia; Rosołowska-Huszcz, Danuta

    2017-03-27

    One of the main causes of mortality in developed countries is atherosclerosis. The pathogenesis of atherosclerosis is associated with endothelial dysfunction. Consumption of food rich in natural antioxidants including polyphenols significantly improves endothelial cells functions. Polyphenols have a beneficial effect on the human body and play an important part in protecting the cardiovascular system. Polyphenols present in food have antioxidant, anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. Catechins cause an increase in the activity of endothelial nitric oxide synthase (eNOS) and increased production of nitric oxide (NO) and decrease in blood pressure. Catechins also reduce platelet adhesion, lower the concentration of C-reactive protein and tumor necrosis factor alpha and interleukin-6. Resveratrol inhibits NADPH oxidase expression, increases the expression of eNOS and NO production as well as decreases the expression of proinflammatory cytokines, and also lowers the concentration of the soluble forms of adhesion molecules - sICAM-1 and sVCAM-1 in blood. Quercetin reduces the blood level of low density lipoprotein cholesterol, lowers blood pressure, reduces the concentration of C-reactive protein and F2-isoprostane level. Curcumin has antagonistic activity to homocysteine. Curcumin increases the expression of eNOS and reduces oxidative DNA damage in rat cardiomyocytes. Numerous attempts are taken for improving the bioavailability of polyphenols in order to increase their use in the body.

  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. TNF-induced endothelial barrier disruption: beyond actin and Rho.

    PubMed

    Marcos-Ramiro, B; García-Weber, D; Millán, J

    2014-12-01

    The decrease of endothelial barrier function is central to the long-term inflammatory response. A pathological alteration of the ability of endothelial cells to modulate the passage of cells and solutes across the vessel underlies the development of inflammatory diseases such as atherosclerosis and multiple sclerosis. The inflammatory cytokine tumour necrosis factor (TNF) mediates changes in the barrier properties of the endothelium. TNF activates different Rho GTPases, increases filamentous actin and remodels endothelial cell morphology. However, inhibition of actin-mediated remodelling is insufficient to prevent endothelial barrier disruption in response to TNF, suggesting that additional molecular mechanisms are involved. Here we discuss, first, the pivotal role of Rac-mediated generation of reactive oxygen species (ROS) to regulate the integrity of endothelial cell-cell junctions and, second, the ability of endothelial adhesion receptors such as ICAM-1, VCAM-1 and PECAM-1, involved in leukocyte transendothelial migration, to control endothelial permeability to small molecules, often through ROS generation. These adhesion receptors regulate endothelial barrier function in ways both dependent on and independent of their engagement by immune cells, and orchestrate the crosstalk between leukocyte transendothelial migration and endothelial permeability during inflammation.

  12. p38 mitogen-activated protein kinase mediates sidestream cigarette smoke-induced endothelial permeability.

    PubMed

    Low, Brad; Liang, Mei; Fu, Jian

    2007-07-01

    Second-hand smoke is associated with increased risk of cardiovascular diseases. So far, little is known about the signaling mechanisms of second-hand smoke-induced vascular dysfunction. Endothelial junctions are fundamental structures important for maintaining endothelial barrier function. Our study showed that sidestream cigarette smoke (SCS), a major component of second-hand smoke, was able to disrupt endothelial junctions and increase endothelial permeability. Sidestream cigarette smoke stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and myosin light chain (MLC). A selective inhibitor of p38 MAPK (SB203580) prevented SCS-induced loss of endothelial barrier integrity as evidenced by transendothelial resistance measurements. Resveratrol, an antioxidant that was able to inhibit SCS-induced p38 MAPK and MLC phosphorylation, also protected endothelial cells from the damage. Thus, p38 MAPK mediates SCS-induced endothelial permeability. Inhibition of p38 MAPK may have therapeutic potential for second-hand smoke-induced vascular injury.

  13. 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

  14. Dietary phosphorus acutely impairs endothelial function.

    PubMed

    Shuto, Emi; Taketani, Yutaka; Tanaka, Rieko; Harada, Nagakatsu; Isshiki, Masashi; Sato, Minako; Nashiki, Kunitaka; Amo, Kikuko; Yamamoto, Hironori; Higashi, Yukihito; Nakaya, Yutaka; Takeda, Eiji

    2009-07-01

    Excessive dietary phosphorus may increase cardiovascular risk in healthy individuals as well as in patients with chronic kidney disease, but the mechanisms underlying this risk are not completely understood. To determine whether postprandial hyperphosphatemia may promote endothelial dysfunction, we investigated the acute effect of phosphorus loading on endothelial function in vitro and in vivo. Exposing bovine aortic endothelial cells to a phosphorus load increased production of reactive oxygen species, which depended on phosphorus influx via sodium-dependent phosphate transporters, and decreased nitric oxide production via inhibitory phosphorylation of endothelial nitric oxide synthase. Phosphorus loading inhibited endothelium-dependent vasodilation of rat aortic rings. In 11 healthy men, we alternately served meals containing 400 mg or 1200 mg of phosphorus in a double-blind crossover study and measured flow-mediated dilation of the brachial artery before and 2 h after the meals. The high dietary phosphorus load increased serum phosphorus at 2 h and significantly decreased flow-mediated dilation. Flow-mediated dilation correlated inversely with serum phosphorus. Taken together, these findings suggest that endothelial dysfunction mediated by acute postprandial hyperphosphatemia may contribute to the relationship between serum phosphorus level and the risk for cardiovascular morbidity and mortality.

  15. 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

  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. 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.

  18. Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

    SciTech Connect

    Lin, Ming-Chung; Chen, Chia-Ling; Yang, Tsan-Tzu; Choi, Pui-Ching; Hsing, Chung-Hsi; Lin, Chiou-Feng

    2012-12-01

    An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

  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. HDL and endothelial protection

    PubMed Central

    Tran-Dinh, A; Diallo, D; Delbosc, S; Varela-Perez, L Maria; Dang, QB; Lapergue, B; Burillo, E; Michel, JB; Levoye, A; Martin-Ventura, JL; Meilhac, O

    2013-01-01

    High-density lipoproteins (HDLs) represent a family of particles characterized by the presence of apolipoprotein A-I (apoA-I) and by their ability to transport cholesterol from peripheral tissues back to the liver. In addition to this function, HDLs display pleiotropic effects including antioxidant, anti-apoptotic, anti-inflammatory, anti-thrombotic or anti-proteolytic properties that account for their protective action on endothelial cells. Vasodilatation via production of nitric oxide is also a hallmark of HDL action on endothelial cells. Endothelial cells express receptors for apoA-I and HDLs that mediate intracellular signalling and potentially participate in the internalization of these particles. In this review, we will detail the different effects of HDLs on the endothelium in normal and pathological conditions with a particular focus on the potential use of HDL therapy to restore endothelial function and integrity. PMID:23488589

  1. 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

  2. Magic roundabout, a tumor endothelial marker: expression and signaling.

    PubMed

    Seth, Pankaj; Lin, Yanfeng; Hanai, Jun-ichi; Shivalingappa, Venkatesha; Duyao, Mabel P; Sukhatme, Vikas P

    2005-07-01

    Molecular signals that guide blood vessels to specific paths are not fully deciphered, but are thought to be similar to signals that mediate neuronal guidance. These cues are not only critical for normal blood vessel development, but may also play a major role in tumor angiogenesis. In this study, we have demonstrated the tumor endothelial specific expression of a Robo family member, magic roundabout (MRB), functionally characterized its role in endothelial cell migration and defined a signaling pathway that might mediate this function. We show that MRB is differentially over-expressed in tumor endothelial cells versus normal adult endothelial cells in numerous solid tumors. Moreover, over-expression of MRB in endothelial cells activates MRB in a ligand-independent fashion, and activation of MRB via Slit2, a putative ligand, results in inhibition of VEGF and FGF induced migration. We also demonstrate that MRB induced inhibition of endothelial migration is partially mediated by the Ras-Raf-Mek-Erk signaling pathway. We therefore hypothesize that expression of MRB is involved in regulating the migration of endothelial cells during tumor angiogenesis.

  3. 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

  4. Hydrogen-Rich Medium Attenuated Lipopolysaccharide-Induced Monocyte-Endothelial Cell Adhesion and Vascular Endothelial Permeability via Rho-Associated Coiled-Coil Protein Kinase.

    PubMed

    Xie, Keliang; Wang, Weina; Chen, Hongguang; Han, Huanzhi; Liu, Daquan; Wang, Guolin; Yu, Yonghao

    2015-07-01

    Sepsis is the leading cause of death in critically ill patients. In recent years, molecular hydrogen, as an effective free radical scavenger, has been shown a selective antioxidant and anti-inflammatory effect, and it is beneficial in the treatment of sepsis. Rho-associated coiled-coil protein kinase (ROCK) participates in junction between normal cells, and regulates vascular endothelial permeability. In this study, we used lipopolysaccharide to stimulate vascular endothelial cells and explored the effects of hydrogen-rich medium on the regulation of adhesion of monocytes to endothelial cells and vascular endothelial permeability. We found that hydrogen-rich medium could inhibit adhesion of monocytes to endothelial cells and decrease levels of adhesion molecules, whereas the levels of transepithelial/endothelial electrical resistance values and the expression of vascular endothelial cadherin were increased after hydrogen-rich medium treatment. Moreover, hydrogen-rich medium could lessen the expression of ROCK, as a similar effect of its inhibitor Y-27632. In addition, hydrogen-rich medium could also inhibit adhesion of polymorphonuclear neutrophils to endothelial cells. In conclusion, hydrogen-rich medium could regulate adhesion of monocytes/polymorphonuclear neutrophils to endothelial cells and vascular endothelial permeability, and this effect might be related to the decreased expression of ROCK protein.

  5. 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

  6. You're Only as Old as Your Arteries: Translational Strategies for Preserving Vascular Endothelial Function with Aging

    PubMed Central

    Kaplon, Rachelle E.; Gioscia-Ryan, Rachel A.; LaRocca, Thomas J.

    2014-01-01

    Endothelial dysfunction develops with age and increases the risk of age-associated vascular disorders. Nitric oxide insufficiency, oxidative stress, and chronic low-grade inflammation, induced by upregulation of adverse cellular signaling processes and imbalances in stress resistance pathways, mediate endothelial dysfunction with aging. Healthy lifestyle behaviors preserve endothelial function with aging by inhibiting these mechanisms, and novel nutraceutical compounds that favorably modulate these pathways hold promise as a complementary approach for preserving endothelial health. PMID:24985329

  7. Vascular incompetence in dialysis patients--protein-bound uremic toxins and endothelial dysfunction.

    PubMed

    Jourde-Chiche, Noémie; Dou, Laetitia; Cerini, Claire; Dignat-George, Françoise; Brunet, Philippe

    2011-01-01

    Patients with chronic kidney disease (CKD) have a much higher risk of cardiovascular diseases than the general population. Endothelial dysfunction, which participates in accelerated atherosclerosis, is a hallmark of CKD. Patients with CKD display impaired endothelium-dependent vasodilatation, elevated soluble biomarkers of endothelial dysfunction, and increased oxidative stress. They also present an imbalance between circulating endothelial populations reflecting endothelial injury (endothelial microparticles and circulating