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  1. Allogeneic Mesenchymal Stem Cells Restore Endothelial Function in Heart Failure by Stimulating Endothelial Progenitor Cells

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    Courtney Premer

    2015-05-01

    Interpretation: These findings reveal a novel mechanism whereby allogeneic, but not autologous, MSC administration results in the proliferation of functional EPCs and improvement in vascular reactivity, which in turn restores endothelial function towards normal in patients with HF. These findings have significant clinical and biological implications for the use of MSCs in HF and other disorders associated with endothelial dysfunction.

  2. Equol-Stimulated Mitochondrial Reactive Oxygen Species Activate Endothelial Nitric Oxide Synthase and Redox Signaling in Endothelial Cells

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    Rowlands, David J.; Chapple, Sarah; Siow, Richard C.M.; Mann, Giovanni E.

    2011-01-01

    We reported previously that dietary isoflavones modulate arterial blood pressure in vivo and that the daidzein metabolite equol rapidly activates endothelial NO synthase (eNOS) via Akt and extracellular signal–regulated kinase 1/2– dependent signaling. In this study, we report the first evidence in human endothelial cells that acute stimulation of mitochondrial superoxide generation by equol (100 nmol/L) is required for eNOS activation. Scavengers of superoxide (superoxide dismutase and manganese [III] tetrakis[1-methyl-4-pyridyl]porphyrin) abrogated equol stimulated Akt and eNOS phosphorylation, and the mitochondrial complex I inhibitor rotenone inhibited Akt, extracellular signal–regulated kinase 1/2, and eNOS phosphorylation, as well as NO-mediated increases in intracellular cGMP. Equol also induced rapid alterations in F-actin fiber distribution, with depolymerization of F-actin with cytochalasin D abrogating equol-stimulated mitochondrial superoxide generation. Treatment of cells with pertussis toxin or inhibition of GPR30/epidermal growth factor receptor kinase transactivation prevented equol-induced activation of extracellular signal–regulated kinase 1/2 via c-Src, Akt, and eNOS. Moreover, inhibition of epidermal growth factor receptor kinase activation with AG-1478 abrogated equol-stimulated mitochondrial reactive oxygen species generation and subsequent kinase and eNOS activation. Our findings suggest that equol-stimulated mitochondrial reactive oxygen species modulate endothelial redox signaling and NO release involving transactivation of epidermal growth factor receptor kinase and reorganization of the F-actin cytoskeleton. Identification of these novel actions of equol may provide valuable insights for therapeutic strategies to restore endothelial function in cardiovascular disease. PMID:21300668

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

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    Suhaila Muid

    2016-07-01

    Full Text Available 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

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

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

    2006-10-24

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

  5. Effects of AMPK on high glucose stimulated apoptosis of endothelial cells via regulation of calcium influx

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    Ting LU

    2015-11-01

    Full Text Available Objective To investigate the inhibitory effect of adenosine monophosphate (AMP-dependent protein kinase (AMPK on high glucose-stimulated endothelial cell apoptosis and its mechanism. Methods MS-1 endothelial cells were cultured in vitro, and they were treated with AMPK agonist, AMPK inhibitor, 2-APB (a blocker of store operated Ca2+ channel (SOCC and (or high glucose, and a control group without any intervention were set up. TUNEL assay was performed to determine apoptotic cells. Laser scanning confocal microscopy was used to assess the Ca2+ influx into cells, and Western-blotting was performed to determine the expressions of Stim1 and Orai1 of the store operated Ca2+ channel (SOCC proteins. Results Apoptosis of endothelial cells was induced significantly, and the expressions of Stim1 and Orai1 were upregulated in high glucose group compared with that in control group (P<0.05. The rate of apoptosis of high glucose-induced endothelial cell was found to be increased in AMPK inhibitor group and decreased in AMPK agonist group, and the expressions of Stim1 and Orai1 were found to be down-regulated in AMPK agonist group as compared with that in high glucose group (P<0.05. Compared with the control group, high glucose stimulation significantly induced the Ca2+ influx to endothelial cells; compared with high glucose group, 2-APB significantly inhibited high glucose-induced Ca2+ influx to endothelial cells, and blocked the inducing effect of high-glucose on endothelial cell apoptosis. Compared with high glucose group, AMPK agonist significantly inhibited high glucose-induced cell Ca2+ influx. Conclusion By reducing the expressions of Stim1 and Orai1, AMPK may inhibit SOCC-mediated Ca2+ influx, and block the high glucose-stimulated endothelial cell apoptosis, thus play an important protective role in sustaining endothelial cell function. DOI: 10.11855/j.issn.0577-7402.2015.10.01

  6. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

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    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  7. TNF-α stimulates endothelial palmitic acid transcytosis and promotes insulin resistance

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    Li, Wenjing; Yang, Xiaoyan; Zheng, Tao; Xing, Shasha; Wu, Yaogong; Bian, Fang; Wu, Guangjie; Li, Ye; Li, Juyi; Bai, Xiangli; Wu, Dan; Jia, Xiong; Wang, Ling; Zhu, Lin; Jin, Si

    2017-01-01

    Persistent elevation of plasma TNF-α is a marker of low grade systemic inflammation. Palmitic acid (PA) is the most abundant type of saturated fatty acid in human body. PA is bound with albumin in plasma and could not pass through endothelial barrier freely. Albumin-bound PA has to be transported across monolayer endothelial cells through intracellular transcytosis, but not intercellular diffusion. In the present study, we discovered that TNF-α might stimulate PA transcytosis across cardiac microvascular endothelial cells, which further impaired the insulin-stimulated glucose uptake by cardiomyocytes and promoted insulin resistance. In this process, TNF-α-stimulated endothelial autophagy and NF-κB signaling crosstalk with each other and orchestrate the whole event, ultimately result in increased expression of fatty acid transporter protein 4 (FATP4) in endothelial cells and mediate the increased PA transcytosis across microvascular endothelial cells. Hopefully the present study discovered a novel missing link between low grade systemic inflammation and insulin resistance. PMID:28304381

  8. Photodynamic therapy potentiates the paracrine endothelial stimulation by colorectal cancer

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    Lamberti, María Julia; Florencia Pansa, María; Emanuel Vera, Renzo; Belén Rumie Vittar, Natalia; Rivarola, Viviana Alicia

    2014-11-01

    Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death worldwide. Recurrence is a major problem and is often the ultimate cause of death. In this context, the tumor microenvironment influences tumor progression and is considered as a new essential feature that clearly impacts on treatment outcome, and must therefore be taken into consideration. Photodynamic therapy (PDT), oxygen, light and drug-dependent, is a novel treatment modality when CRC patients are inoperable. Tumor vasculature and parenchyma cells are both potential targets of PDT damage modulating tumor-stroma interactions. In biological activity assessment in photodynamic research, three-dimensional (3D) cultures are essential to integrate biomechanical, biochemical, and biophysical properties that better predict the outcome of oxygen- and drug-dependent medical therapies. Therefore, the objective of this study was to investigate the antitumor effect of methyl 5-aminolevulinic acid-PDT using a light emitting diode for the treatment of CRC cells in a scenario that mimics targeted tissue complexity, providing a potential bridge for the gap between 2D cultures and animal models. Since photodynamic intervention of the tumor microenvironment can effectively modulate the tumor-stroma interaction, it was proposed to characterize the endothelial response to CRC paracrine communication, if one of these two populations is photosensitized. In conclusion, we demonstrated that the dialogue between endothelial and tumor populations when subjected to lethal PDT conditions induces an increase in angiogenic phenotype, and we think that it should be carefully considered for the development of PDT therapeutic protocols.

  9. The response of VEGF-stimulated endothelial cells to angiostatic molecules is substrate-dependent

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    Polverini Peter J

    2005-10-01

    Full Text Available Abstract Background The microenvironment surrounding cells can exert multiple effects on their biological responses. In particular the extracellular matrix surrounding cells can profoundly influence their behavior. It has been shown that the extracellular matrix composition in tumors is vastly different than that found in normal tissue with increased amounts of certain matrices such as collagen I. It has been previously demonstrated that VEGF stimulation of endothelial cells growing on type I collagen results in the induction of bcl-2 expression and enhanced endothelial cell survival. We sought to investigate whether this increased endothelial cell survival resulted in the failure of angiostatic molecules to inhibit angiogenesis. Results We now demonstrate that VEGF-induced survival on collagen I impairs the ability of three known angiostatic molecules, TSP-1, IP-10 and endostatin to inhibit endothelial cell proliferation. Apoptosis of endothelial cells, growing on collagen I, induced by TSP-1 and IP-10 was also inhibited following VEGF stimulation. In contrast, endostatin induced apoptosis in these same cells. Further analysis determined that endostatin did not decrease the expression of bcl-2 nor did it increase activation of caspase-3 in the presence of VEGF. Alternatively, it appeared that in the presence of VEGF, endostatin induced the activation of caspase-8 in endothelial cells grown on collagen I. Furthermore, only endostatin had the ability to inhibit VEGF-induced sprout formation in collagen I gels. Conclusion These data suggest that TSP-1, IP-10 and endostatin inhibit endothelial cells via different mechanisms and that only endostatin is effective in inhibiting angiogenic activities in the presence of collagen I. Our results suggest that the efficacy of angiostatic treatments may be impaired depending on the context of the extracellular matrix within the tumor environment and thus could impede the efficacy of angiostatic therapies.

  10. Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.

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    Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

    2014-03-01

    Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO

  11. Tumour necrosis factor-alpha stimulates expression of TNF-alpha converting enzyme in endothelial cells.

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    Bzowska, Monika; Jura, Natalia; Lassak, Adam; Black, Roy A; Bereta, Joanna

    2004-07-01

    Tumor necrosis factor-alpha converting enzyme (ADAM17) is a major metalloproteinase involved in the shedding of several membrane-bound cytokines and cytokine receptors. Interplay of cytokines and their soluble receptors might be an important regulatory element in the network of interactions responsible for maintaining homeostasis in the immune system. ADAM17 thus has the potential to participate in a broad range of immune reactions. We studied the mechanisms of ADAM17 activation in endothelial cells and found that pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma) and growth factors (epidermal growth factor, vascular endothelial growth factor) are able to upregulate transcription of ADAM17 and expression of ADAM17 protein. This process might constitute an important mechanism of regulation of ADAM17 activity. Stimulation of transcription, rather than increased ADAM17 mRNA stability, was responsible for increased levels of ADAM17 mRNA. Importantly, the increase in ADAM17 was accompanied by increased shedding of TNF-Receptor I (p55) in tumor necrosis factor-alpha-stimulated endothelial cells. Therefore, ADAM17-dependent depletion of membrane-bound tumor necrosis factor receptors from endothelial cells might constitute a mechanism of self-protection in states of prolonged immunostimulation.

  12. Increased Vitreous Shedding of Microparticles in Proliferative Diabetic Retinopathy Stimulates Endothelial Proliferation

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    Chahed, Sadri; Leroyer, Aurélie S.; Benzerroug, Mounir; Gaucher, David; Georgescu, Adriana; Picaud, Serge; Silvestre, Jean-Sébastien; Gaudric, Alain; Tedgui, Alain; Massin, Pascale; Boulanger, Chantal M.

    2010-01-01

    OBJECTIVE Diabetic retinopathy is associated with progressive retinal capillary activation and proliferation, leading to vision impairment and blindness. Microparticles are submicron membrane vesicles with biological activities, released following cell activation or apoptosis. We tested the hypothesis that proangiogenic microparticles accumulate in vitreous fluid in diabetic retinopathy. RESEARCH DESIGN AND METHODS Levels and cellular origin of vitreous and plasma microparticles from control (n = 26) and diabetic (n = 104) patients were analyzed by flow cytometry, and their proangiogenic activity was assessed by in vitro thymidine incorporation and neovessel formation in subcutaneous Matrigel plugs in mice. RESULTS Microparticles of endothelial, platelet, photoreceptor, and microglial origin were identified in vitreous samples. Levels of photoreceptor and microglial microparticles were undetectable in plasmas but were comparable in diabetic and control vitreous samples. Vitreous platelet and endothelial microparticles levels were increased in diabetic patients and decreased following panretinal laser photocoagulation or intravitreal antivascular endothelial growth factor injection in proliferative diabetic retinopathy (PDR). The ratio of vitreous to plasma microparticle levels was calculated to estimate local formation versus potential plasma leakage. In PDR, the endothelial microparticles ratio—but not that for platelet—was greater than 1.0, indicating local formation of endothelial microparticles from retinal vessels and permeation of platelet microparticles from plasma. Isolated vitreous microparticles stimulated by 1.6-fold endothelial proliferation and increased new vessel formation in mice. CONCLUSIONS The present study demonstrates that vitreous fluid contains shed membrane microparticles of endothelial, platelet, and retinal origin. Vitreous microparticles levels are increased in patients with diabetic retinopathy, where they could contribute to disease

  13. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

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    Bai, Huai; Forrester, John V; Zhao, Min

    2011-07-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells

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    Kaori Yama

    2015-04-01

    Full Text Available Epalrestat (EPS is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs, an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

  15. Quinapril treatment increases insulin-stimulated endothelial function and adiponectin gene expression in patients with type 2 diabetes

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    Hermann, Thomas S; Li, Weijie; Dominguez, Helena

    2005-01-01

    OBJECTIVE: Angiotensin-converting enzyme inhibitors reduce cardiovascular mortality and improve endothelial function in type 2 diabetic patients. We hypothesized that 2 months of quinapril treatment would improve insulin-stimulated endothelial function and glucose uptake in type 2 diabetic subjects...... and simultaneously increase the expression of genes that are pertinent for endothelial function and metabolism. METHODS: Twenty-four type 2 diabetic subjects were randomized to receive 2 months of quinapril 20 mg daily or no treatment in an open parallel study. Endothelium-dependent and -independent vasodilation...... occlusion plethysmography. Gene expression was measured by real-time PCR. RESULTS: Quinapril treatment increased insulin-stimulated endothelial function in the type 2 diabetic subjects (P = 0.005), whereas forearm glucose uptake was unchanged. Endothelial function was also increased by quinapril (P = 0...

  16. Palmitate-stimulated monocytes induce adhesion molecule expression in endothelial cells via IL-1 signaling pathway.

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    Shikama, Yosuke; Aki, Nanako; Hata, Akiko; Nishimura, Miho; Oyadomari, Seiichi; Funaki, Makoto

    2015-03-01

    Increased intake of saturated fatty acids (SFAs), such as palmitate (Pal), is linked to a higher risk of type 2 diabetes and cardiovascular disease. Although recent studies have investigated the direct effects of SFAs on inflammatory responses in vascular endothelial cells, it remains unknown whether SFAs also induce these responses mediated by circulating cells. In this study, especially focused on adhesion molecules and monocytes, we investigated the indirect effects of Pal on expression and release of ICAM-1 and E-selectin in vascular endothelial cells. Phorbol 12-myristate 13-acetate (PMA)-treated THP-1 (pTHP-1) cells and human monocytes were stimulated with various free fatty acids (FFAs). SFAs, but not unsaturated fatty acids (UFAs), increased interleukin (IL)-1β secretion and decreased IL-1 receptor antagonist (IL-1Ra) secretion, resulting in an increase in the IL-1β/IL-1Ra secretion ratio. UFAs dose-dependently inhibited the increase in IL-1β secretion and decrease in IL-1Ra secretion induced by Pal. Moreover, in human aortic and vein endothelial cells, expression and release of ICAM-1 and E-selectin were induced by treatment with conditioned medium collected from Pal-stimulated pTHP-1 cells and human monocytes, but not by Pal itself. The up-regulated expression and release of adhesion molecules by the conditioned medium were mostly abolished by recombinant human IL-1Ra supplementation. These results suggest that the Pal-induced increase in the ratio of IL-1β/IL-1Ra secretion in monocytes up-regulates endothelial adhesion molecules, which could enhance leukocyte adhesion to endothelium. This study provides further evidence that IL-1β neutralization through receptor antagonism may be useful for preventing the onset and development of cardiovascular disease. © 2014 Wiley Periodicals, Inc., A Wiley Company.

  17. Analysis of biological effects in human endothelial cells after stimulated microgravity

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    Min, Zhang; Sun, Yeqing; Xu, Dan

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

  18. hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells.

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    Schanz, Andrea; Lukosz, Margarete; Hess, Alexandra P; Baston-Büst, Dunja M; Krüssel, Jan S; Heiss, Christian

    2015-08-01

    Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation. Copyright © 2015. Published by Elsevier Ireland Ltd.

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

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    Xi Ling Liu

    2016-01-01

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

  20. Citrus Polyphenol Hesperidin Stimulates Production of Nitric Oxide in Endothelial Cells while Improving Endothelial Function and Reducing Inflammatory Markers in Patients with Metabolic Syndrome

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    Rizza, Stefano; Muniyappa, Ranganath; Iantorno, Micaela; Kim, Jeong-a; Chen, Hui; Pullikotil, Philomena; Senese, Nicoletta; Tesauro, Manfredi; Lauro, Davide; Cardillo, Carmine

    2011-01-01

    Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. Design, Setting, and Interventions: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). Main Outcome Measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H2O2. Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption. PMID:21346065

  1. Κ-opioid receptor stimulation improves endothelial function in hypoxic pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Qi Wu

    Full Text Available The present study was designed to investigate the effect of κ-opioid receptor stimulation with U50,488H on endothelial function and underlying mechanism in rats with hypoxic pulmonary hypertension (HPH. Chronic hypoxia-induced HPH was simulated by exposing the rats to 10% oxygen for 2 wk. After hypoxia, mean pulmonary arterial pressure (mPAP, right ventricular pressure (RVP and right ventricular hypertrophy index (RVHI were measured. Relaxation of pulmonary artery in response to acetylcholine (ACh was determined. Expression and activity of endothelial nitric oxide (NO synthase (eNOS and inducible NO synthase (iNOS with NO production, total antioxidant capacity (T-AOC, gp91(phox expression and nitrotyrosine content were measured. The effect of U50,488H administration during chronic hypoxia was investigated. Administration of U50,488H significantly decreased mPAP and right ventricular hypertrophy as evidenced by reduction in RVP and RVHI. These effects were mediated by κ-opioid receptor. In the meantime, treatment with U50,488H significantly improved endothelial function as evidenced by enhanced relaxation in response to ACh. Moreover, U50,488H resulted in a significant increase in eNOS phosphorylation, NO content in serum, and T-AOC in pulmonary artery of HPH rats. In addition, the activity of eNOS was enhanced, but the activity of iNOS was attenuated in the pulmonary artery of chronic hypoxic rats treated with U50,488H. On the other hand, U50,488H markedly blunted HPH-induced elevation of gp91(phox expression and nitrotyrosine content in pulmonary artery, and these effects were blocked by nor-BNI, a selective κ-opioid receptor antagonist. These data suggest that κ-opioid receptor stimulation with U50,488H improves endothelial function in rats with HPH. The mechanism of action might be attributed to the preservation of eNOS activity, enhancement of eNOS phosphorylation, downregulation of iNOS activity and its antioxidative/nitrative effect.

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

    Science.gov (United States)

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

    2016-10-01

    Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Adhesion of Epstein–Barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines

    Science.gov (United States)

    Kanno, H; Watabe, D; Shimizu, N; Sawai, T

    2008-01-01

    Chronic active Epstein–Barr virus (EBV) infection (CAEBV) is characterized by chronic recurrent infectious mononucleosis-like symptoms. Approximately one-fourth of CAEBV patients develop vascular lesions with infiltration of EBV-positive lymphoid cells. Furthermore, EBV-positive natural killer (NK)/T cell lymphomas often exhibit angiocentric or angiodestructive lesions. These suggest an affinity of EBV-positive NK/T cells to vascular components. In this study, we evaluated the expression of adhesion molecules and cytokines in EBV-positive NK lymphoma cell lines, SNK1 and SNK6, and examined the role of cytokines in the interaction between NK cell lines and endothelial cells. SNKs expressed intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) at much higher levels than those in EBV-negative T cell lines. SNKs produced the larger amount of tumour necrosis factor (TNF)-α, which caused increased expression of ICAM-1 and VCAM-1 in cultured human endothelial cells, than that from EBV-negative T cell lines. Furthermore, SNKs exhibited increased adhesion to cultured endothelial cells stimulated with TNF-α or interleukin (IL)-1β, and the pretreatment of cytokine-stimulated endothelial cells with anti-VCAM-1-antibodies reduced cell adhesion. These indicate that the up-regulated expression of VCAM-1 on cytokine-stimulated endothelial cells would be important for the adhesion of EBV-positive NK cells and might initiate the vascular lesions. PMID:18190605

  4. The effect of chronic heart failure and type 2 diabetes on insulin-stimulated endothelial function is similar and additive

    DEFF Research Database (Denmark)

    Falskov, Britt; Hermann, Thomas Steffen; Rask-Madsen, Christian

    2011-01-01

    plethysmography. Insulin-stimulated endothelial function was assessed after intra-arterial infusion of insulin followed by co-infusion with serotonin in three different dosages. Forearm glucose uptake was measured during the insulin infusion. RESULTS: Patients with systolic heart failure had impaired insulin...

  5. Metoprolol compared to carvedilol deteriorates insulin-stimulated endothelial function in patients with type 2 diabetes - a randomized study

    DEFF Research Database (Denmark)

    Kveiborg, Britt; Hermann, Thomas S; Major-Pedersen, Atheline

    2010-01-01

    -stimulated endothelial function in patients with type 2 diabetes. METHOD: 24 patients with type 2 diabetes were randomized to receive either 200 mg metoprolol succinate or 50 mg carvedilol daily. Endothelium-dependent vasodilation was assessed by using venous occlusion plethysmography with increasing doses of intra...

  6. Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Wang Jing

    2002-12-01

    Full Text Available Abstract Background The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1 and Flt-1 (Fms-like tyrosine kinase-1. However, to date, it has not been determined which VEGF receptor (VEGFR is involved in binding to and activating Src kinase following VEGF stimulation of the receptors. Results In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs, and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1. Conclusions Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

  7. Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors.

    Directory of Open Access Journals (Sweden)

    Devandir Antonio de Souza Junior

    Full Text Available Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7 in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization.

  8. Insulin therapy improves insulin-stimulated endothelial function in patients with type 2 diabetes and ischemic heart disease

    DEFF Research Database (Denmark)

    Rask-Madsen, C; Ihlemann, N; Krarup, T

    2001-01-01

    Blunted insulin-stimulated endothelial function may be a mechanism for the development of atherothrombotic disease in type 2 diabetes, but it is unknown whether hypoglycemic drug therapy can modulate this abnormality. We studied patients with type 2 diabetes and stable ischemic heart disease (n......, and 69 +/- 36% (P = 0.0002). In the time control group, insulin stimulation remained without effect after 8 weeks (P = 0.7). In conclusion, insulin therapy partly restores insulin-stimulated endothelial function in patients with type 2 diabetes and ischemic heart disease....... after intrabrachial infusion of insulin. Patients were restudied after 2 months of insulin therapy with four daily subcutaneous injections (treatment group, n = 19) or without hypoglycemic drug therapy (time control group, n = 9). Insulin infusion raised venous serum insulin in the forearm to high...

  9. Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis, and migration.

    OpenAIRE

    Presta, M; Moscatelli, D; Joseph-Silverstein, J; Rifkin, D B

    1986-01-01

    A 17,500-dalton protein which stimulates plasminogen activator production in cultured bovine capillary endothelial cells has been purified from a SK-Hep-1 human hepatoma cell lysate by using heparin affinity chromatography and fast protein-liquid ion exchange chromatography. The purified molecule stimulated plasminogen activator production in a dose-dependent manner between 0.01 and 1 ng/ml. It also stimulated collagenase synthesis, DNA synthesis, and motility in capillary endothelial cells i...

  10. β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia.

    Science.gov (United States)

    Karimi Galougahi, Keyvan; Liu, Chia-Chi; Garcia, Alvaro; Gentile, Carmine; Fry, Natasha A; Hamilton, Elisha J; Hawkins, Clare L; Figtree, Gemma A

    2016-02-19

    Perturbed balance between NO and O2 (•-). (ie, NO/redox imbalance) is central in the pathobiology of diabetes-induced vascular dysfunction. We examined whether stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re-establish NO/redox balance, relieve oxidative inhibition of the membrane proteins eNOS and Na(+)-K(+) (NK) pump, and improve vascular function in a new animal model of hyperglycemia. We established hyperglycemia in male White New Zealand rabbits by infusion of S961, a competitive high-affinity peptide inhibitor of the insulin receptor. Hyperglycemia impaired endothelium-dependent vasorelaxation by "uncoupling" of eNOS via glutathionylation (eNOS-GSS) that was dependent on NADPH oxidase activity. Accordingly, NO levels were lower while O2 (•-) levels were higher in hyperglycemic rabbits. Infusion of the β3 AR agonist CL316243 (CL) decreased eNOS-GSS, reduced O2 (•-), restored NO levels, and improved endothelium-dependent relaxation. CL decreased hyperglycemia-induced NADPH oxidase activation as suggested by co-immunoprecipitation experiments, and it increased eNOS co-immunoprecipitation with glutaredoxin-1, which may reflect promotion of eNOS de-glutathionylation by CL. Moreover, CL reversed hyperglycemia-induced glutathionylation of the β1 NK pump subunit that causes NK pump inhibition, and improved K(+)-induced vasorelaxation that reflects enhancement in NK pump activity. Lastly, eNOS-GSS was higher in vessels of diabetic patients and was reduced by CL, suggesting potential significance of the experimental findings in human diabetes. β3 AR activation restored NO/redox balance and improved endothelial function in hyperglycemia. β3 AR agonists may confer protection against diabetes-induced vascular dysfunction. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

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

    Science.gov (United States)

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

    2013-01-01

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

  12. Peroxisome proliferator-activated receptor (PPAR) α and δ activators induce ICAM-1 expression in quiescent non stimulated endothelial cells.

    Science.gov (United States)

    Naidenow, Julia; Hrgovic, Igor; Doll, Monika; Hailemariam-Jahn, Tsige; Lang, Victoria; Kleemann, Johannes; Kippenberger, Stefan; Kaufmann, Roland; Zöller, Nadja; Meissner, Markus

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are implicated in the regulation of lipid and glucose homeostasis. PPAR agonists have been shown to control inflammatory processes, in part by inhibiting the expression of distinct proinflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1), IL-8, and intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is an important endothelial membrane receptor that facilitates the transmigration of leukocytes across the endothelium. To date, the influence of PPARα and δ activators on the expression of ICAM-1 in non-induced, quiescent endothelial cells has been unclear. Therefore, we examined the effects of various PPARα and δ agonists on the expression of ICAM-1 in non-stimulated primary human endothelial cells. We found that PPARα and PPARδ agonists significantly induced ICAM-1 surface, intracellular protein, and mRNA expression in a time and concentration-dependent manner. The PPARδ induced ICAM-1 expression could be paralleled with a significantly increased T-cell adherence to the endothelial cells whereas PPARα failed to do so. Transcriptional activity studies using an ICAM-1 reporter gene constructs revealed that PPARδ, but not PPARα agonists induced gene expression by stimulating ICAM-1 promoter activity via an Sp1 transcription factor binding site and inhibit the binding of the transcription factors Sp1 and Sp3. Furthermore, we performed mRNA stability assays and found that PPARα and PPARδ agonists increased ICAM-1 mRNA stability. Therefore, our data provide the first evidence that PPARα and PPARδ agonists induce ICAM-1 expression in non-stimulated endothelial cells via transcriptional and posttranscriptional mechanisms.

  13. Docosahexaenoic acid inhibits the adhesion of flowing neutrophils to cytokine stimulated human umbilical vein endothelial cells.

    Science.gov (United States)

    Yates, Clara M; Tull, Samantha P; Madden, Jackie; Calder, Philip C; Grimble, Robert F; Nash, Gerard B; Rainger, G Ed

    2011-07-01

    The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

  14. Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells.

    Science.gov (United States)

    Wang, Ying; Cao, Ying; Mangalam, Ashutosh K; Guo, Yong; LaFrance-Corey, Reghann G; Gamez, Jeffrey D; Atanga, Pascal Aliihnui; Clarkson, Benjamin D; Zhang, Yuebo; Wang, Enfeng; Angom, Ramcharan Singh; Dutta, Kirthica; Ji, Baoan; Pirko, Istvan; Lucchinetti, Claudia F; Howe, Charles L; Mukhopadhyay, Debabrata

    2016-10-15

    Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1-CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1-IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases. © 2016. Published by The Company of Biologists Ltd.

  15. Non-contact high-frequency ultrasound microbeam stimulation for studying mechanotransduction in human umbilical vein endothelial cells.

    Science.gov (United States)

    Hwang, Jae Youn; Lim, Hae Gyun; Yoon, Chi Woo; Lam, Kwok Ho; Yoon, Sangpil; Lee, Changyang; Chiu, Chi Tat; Kang, Bong Jin; Kim, Hyung Ham; Shung, K Kirk

    2014-09-01

    We describe how contactless high-frequency ultrasound microbeam stimulation (HFUMS) is capable of eliciting cytoplasmic calcium (Ca(2+)) elevation in human umbilical vein endothelial cells. The cellular mechanotransduction process, which includes cell sensing and adaptation to the mechanical micro-environment, has been studied extensively in recent years. A variety of tools for mechanical stimulation have been developed to produce cellular responses. We developed a novel tool, a highly focused ultrasound microbeam, for non-contact cell stimulation at a microscale. This tool, at 200 MHz, was applied to human umbilical vein endothelial cells to investigate its potential to elicit an elevation in cytoplasmic Ca(2+) levels. It was found that the response was dose dependent, and moreover, extracellular Ca(2+) and cytoplasmic Ca(2+) stores were involved in the Ca(2+) elevation. These results suggest that high-frequency ultrasound microbeam stimulation is potentially a novel non-contact tool for studying cellular mechanotransduction if the acoustic pressures at such high frequencies can be quantified. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

  16. Stimulation of G protein-coupled bile acid receptor enhances vascular endothelial barrier function via activation of protein kinase A and Rac1.

    Science.gov (United States)

    Kida, Taiki; Omori, Keisuke; Hori, Masatoshi; Ozaki, Hiroshi; Murata, Takahisa

    2014-01-01

    Bile acids are end products of cholesterol metabolism, and they constantly exist at high concentrations in the blood. Since vascular endothelial cells express G protein-coupled bile acid receptor (GPBAR), bile acids potentially modulate endothelial function. Here, we investigated whether and how GPBAR agonism affects endothelial barrier function. In bovine aortic endothelial cells (BAECs), treatment with a GPBAR agonist, taurolithocholic acid (TLCA) increased the transendothelial electrical resistance. In addition, TLCA suppressed the thrombin-induced dextran infiltration through the endothelial monolayer. Knockdown of GPBAR abolished the inhibitory effect of TLCA on hyperpermeability. These results indicate that stimulation of GPBAR enhances endothelial barrier function. TLCA increased intracellular cAMP production in BAECs. Inhibition of protein kinase A (PKA) or Rac1 significantly attenuated the TLCA-induced endothelial barrier protection. TLCA induced cortical actin polymerization, which was attenuated by a Rac1 inhibitor. In vivo, local administration of TLCA into the mouse ear significantly inhibited vascular leakage and edema formation induced by croton oil or vascular endothelial growth factor. These results indicate that stimulation of GPBAR enhances endothelial barrier function by cAMP/PKA/Rac1-dependent cytoskeletal rearrangement.

  17. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Ok-Nam [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Eun-Sun [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Jeong, Tae Cheon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Chun, Young-Jin [College of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Ai-Young, E-mail: leeay@duih.org [Department of Dermatology, Dongguk University Ilsan Hospital, Goyang 410-773 (Korea, Republic of); Noh, Minsoo, E-mail: minsoo@alum.mit.edu [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  18. Topical negative pressure stimulates endothelial migration and proliferation: a suggested mechanism for improved integration of Integra.

    Science.gov (United States)

    Baldwin, Christopher; Potter, Matthew; Clayton, Elizabeth; Irvine, Laurie; Dye, Julian

    2009-01-01

    Topical negative pressure is an effective technique to promote wound healing and the integration of skin graft and synthetic dermal equivalents. We describe an in vitro model to investigate the effect of negative pressure on angiogenesis, a pivotal step. Dermal fibroblasts or human microvascular endothelial cells were cultured on Integra and subjected to intermittent or continuous negative pressure. At fixed intervals of over 120 hours, the Integra was fixed and assessed for cell migration (microscopy), cell viability (MTS assay), and cell proliferation (Ki67 immunostaining). Under control conditions, endothelial cells formed a monolayer and failed to ingress, whereas fibroblasts migrated throughout the Integra within 24 hours. Negative pressure switches endothelial cell to a migratory and proliferative phenotype. Ingress is greatest with intermittent rather than continuous negative pressure. It has no effect on dermal fibroblast function. This study identifies an important, potential pro-angiogenic mechanism by which topical negative pressure promotes wound healing.

  19. Modulation of adhesion molecule expression on endothelial cells after induction by lipopolysaccharide-stimulated whole blood.

    NARCIS (Netherlands)

    Nooteboom, A.; Linden, C.J. van der; Hendriks, T.

    2004-01-01

    The relative contribution of the pro-inflammatory cytokines tumour necrosis factor (TNF)-alpha and interleukin (IL)-1 beta and the lipopolysaccharide (LPS)-induced pathways that result in endothelial activation during sepsis are not fully understood. We have examined the effects of plasma obtained

  20. Salidroside Stimulates Mitochondrial Biogenesis and Protects against H2O2-Induced Endothelial Dysfunction

    Directory of Open Access Journals (Sweden)

    Shasha Xing

    2014-01-01

    Full Text Available Salidroside (SAL is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2- induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙- production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS, adenosine monophosphate-activated protein kinase (AMPK, and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB. SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α, and mitochondrial transcription factor A (TFAM in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways.

  1. Salidroside Stimulates Mitochondrial Biogenesis and Protects against H2O2-Induced Endothelial Dysfunction

    Science.gov (United States)

    Xing, Shasha; Yang, Xiaoyan; Li, Wenjing; Bian, Fang; Wu, Dan; Chi, Jiangyang; Xu, Gao; Zhang, Yonghui; Jin, Si

    2014-01-01

    Salidroside (SAL) is an active component of Rhodiola rosea with documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙−) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways. PMID:24868319

  2. Enhanced adhesion and proliferation of human umbilical vein endothelial cells on conductive PANI-PCL fiber scaffold by electrical stimulation.

    Science.gov (United States)

    Li, Yumei; Li, Xiang; Zhao, Rui; Wang, Chuying; Qiu, Fangping; Sun, Bolun; Ji, He; Qiu, Ju; Wang, Ce

    2017-03-01

    Recently, electrically conductive biomaterial scaffolds have shown great potential in tissue regeneration. Herein, we reported an electrically conductive polyaniline (PANI) coated poly(ε-caprolactone) (PCL) electrospun micron-fiber scaffold for the enhanced attachment and proliferation of human umbilical vein endothelial cells (HUVECs) under electrical stimulation conditions. After the O2 plasma treatment toward PCL electrospun fiber, PANI could be polymerized onto their surfaces successfully. The obtained PANI-PCL fibers were characterized by SEM observations, FT-IR spectra, XPS analysis, and water contact angle measurement. The mechanical tests indicated that the fibers could satisfy the practical vascular scaffold requirements. The conductivity of the PANI-PCL fibers was 6.71×10-3S/cm which could provide a conductive in-vitro platform to study the effect of electrical stimulation on HUVECs proliferation. When PANI-coated PCL fibers were compared with PCL fibers, HUVECs exhibited highly enhanced adhesion and viability, especially under electrical stimulation (ES) of 200, 300, and 400mV/cm. Proliferation of HUVECs on PANI-PCL fibers was strongly dependent on electrical stimulation intensity. The results showed new insights into conductive scaffolds for vascular tissue engineering. Copyright © 2016. Published by Elsevier B.V.

  3. Pregnancy Augments VEGF-Stimulated In Vitro Angiogenesis and Vasodilator (NO and H2S) Production in Human Uterine Artery Endothelial Cells.

    Science.gov (United States)

    Zhang, Hong-Hai; Chen, Jennifer C; Sheibani, Lili; Lechuga, Thomas J; Chen, Dong-Bao

    2017-07-01

    Augmented uterine artery (UA) production of vasodilators, including nitric oxide (NO) and hydrogen sulfide (H2S), has been implicated in pregnancy-associated and agonist-stimulated rise in uterine blood flow that is rate-limiting to pregnancy health. Developing a human UA endothelial cell (hUAEC) culture model from main UAs of nonpregnant (NP) and pregnant (P) women for testing a hypothesis that pregnancy augments endothelial NO and H2S production and endothelial reactivity to vascular endothelial growth factor (VEGF). Main UAs from NP and P women were used for developing hUAEC culture models. Comparisons were made between NP- and P-hUAECs in in vitro angiogenesis, activation of cell signaling, expression of endothelial NO synthase (eNOS) and H2S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase, and NO/H2S production upon VEGF stimulation. NP- and P-hUAECs displayed a typical cobblestone-like shape in culture and acetylated low-density lipoprotein uptake, stained positively for endothelial and negatively for smooth muscle markers, maintained key signaling proteins during passage, and had statistically significant greater eNOS and CBS proteins in P- vs NP-hUAECs. Treatment with VEGF stimulated in vitro angiogenesis and eNOS protein and NO production only in P-hUEACs and more robust cell signaling in P- vs NP-hUAECs. VEGF stimulated CBS protein expression, accounting for VEGF-stimulated H2S production in hUAECs. Comparisons between NP- and P-hUAECs reveal that pregnancy augments VEGF-stimulated in vitro angiogenesis and NO/H2S production in hUAECs, showing that the newly established hUAEC model provides a critical in vitro tool for understanding human uterine hemodynamics.

  4. Dying endothelial cells stimulate proliferation of malignant glioma cells via a caspase 3-mediated pathway

    OpenAIRE

    Mao, Ping; Smith, Luke; XIE, WANFU; Wang, Maode

    2013-01-01

    Emerging evidence has indicated that apoptotic cells have a compensatory effect on the proliferation of neighboring cells. However, the potential role of dying vascular endothelial cells (ECs) in glioma tumor proliferation remains unclear. In the present study, three glioma cell lines were cocultured with dying ECs under various conditions to evaluate the effect of dying ECs on tumor proliferation using alamarBlue and trypan blue assays to assess cell proliferation and viability, respectively...

  5. Gas6 stimulates angiogenesis of human retinal endothelial cells and of zebrafish embryos via ERK1/2 signaling.

    Directory of Open Access Journals (Sweden)

    Young Sook Kim

    Full Text Available AIM: To determine if growth arrest-specific 6 (Gas6 plays an important role in the regulation of angiogenesis in human retinal microvascular endothelial cells (HRMECs and in vessel development of zebrafish. METHODS: Proliferation, wound-healing cell migration, and tube formation were measured in HRMECs treated with recombinant human Gas6 (rhGas6. Sprague-Dawley rat aortas in Matrigels were treated with rhGas6, and microvessel sprouting emanating from arterial rings was analyzed. Transgenic zebrafish embryos (flk:GFP were microinjected with rhGas6 at 50 hours post-fertilization (hpf, and ectopic sprouting of subintestinal vessels (SIVs was observed under a confocal microscope. Morpholino oligonucleotides (MOs were microinjected to knockdown gas6 in zebrafish embryos, and intersegmental vessel impairment was observed. The effect of the extracellular signal-regulated kinase (ERK1/2 inhibitor on the migration of HRMECs and on vessel development in zebrafish embryos was tested. RESULTS: rhGas6 stimulated proliferation, migration, and tube formation in HRMECs in a dose-dependent manner. In rat aortas, rhGas6 induced vessel outgrowth, and the sprouting length was longer than that of controls. The rhGas6-microinjected zebrafish embryos had significantly increased vessel outgrowth in the SIVs. Recombinant human vascular endothelial growth factor (rhVEGF served as a positive control. Knockdown of gas6 inhibited angiogenesis in the developing vessels of zebrafish. The ERK1/2 inhibitor inhibited HRMEC migration and intersegmental vessel formation in zebrafish embryos. CONCLUSIONS/INTERPRETATIONS: These data suggest that Gas6 plays a pivotal role in proliferation, migration, and sprouting of angiogenic endothelial cells in the retina and in zebrafish embryos. Furthermore, Gas6 induced angiogenic processes are induced via phosphorylation of ERK1/2.

  6. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells

    OpenAIRE

    Krieg, Carsten; Létourneau, Sven; Pantaleo, Giuseppe; Boyman, Onur

    2010-01-01

    IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2–induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high leve...

  7. Ginkgolide B reduces LOX-1 expression by inhibiting Akt phosphorylation and increasing Sirt1 expression in oxidized LDL-stimulated human umbilical vein endothelial cells.

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    Lina Ma

    Full Text Available Oxidized low-density lipoprotein (ox-LDL is an important risk factor in the development of atherosclerosis. LOX-1, a lectin-like receptor for ox-LDL, is present primarily on endothelial cells and upregulated by ox-LDL, tumor necrosis factor a, shear stress, and cytokines in atherosclerosis. Recent studies demonstrated that ginkgolide B, a platelet-activating factor receptor antagonist, has antiinflammatory and antioxidant effects on endothelial and nerve cells. The present study investigated the effects of ginkgolide B on LOX-1 expression and the possible mechanism of action. Our results showed that ginkgolide B inhibited LOX-1 and intercellular cell adhesion molecule-1 (ICAM-1 expression in ox-LDL-stimulated endothelial cells through a mechanism associated with the attenuation of Akt activation. Similar data were obtained by silencing Akt and LY294002. We also evaluated Sirt1 and nuclear factor erythroid 2-related factor 2 (Nrf2 expression. These molecules play a protective role in endothelial cell injury. The results showed that ginkgolide B increased Sirt1 expression in ox-LDL-treated cells. The inhibitory effects of ginkgolide B on LOX-1 and ICAM-1 expression were reduced in Sirt1 siRNA-transfected cells. Nrf2 expression was increased in ox-LDL-treated cells, and ginkgolide B downregulated Nrf2 expression. These results suggest that ginkgolide B reduces Nrf2 expression by inhibiting LOX-1 expression, consequently reducing oxidative stress injury in ox-LDL-stimulated cells. Altogether, these results indicate that the protective effect of ginkgolide B on endothelial cells may be attributable to a decrease in LOX-1 expression and an increase in Sirt1 expression in ox-LDL-stimulated endothelial cells, the mechanism of which is linked to the inhibition of Akt activation. Ginkgolide B may be a multiple-target drug that exerts protective effects in ox-LDL-treated human umbilical vein endothelial cells.

  8. Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation

    NARCIS (Netherlands)

    Arts, J.; Lansink, T.; Grimbergen, J.; Toet, K.H.; Kooistra, T.

    1995-01-01

    We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4

  9. Minimal Homozygous Endothelial Deletion of Eng with VEGF Stimulation is Sufficient to Cause Cerebrovascular Dysplasia in the Adult Mouse

    Science.gov (United States)

    Choi, Eun-Jung; Walker, Espen J.; Shen, Fanxia; Oh, S. Paul; Arthur, Helen M.; Young, William L.; Su, Hua

    2013-01-01

    Background Brain arteriovenous malformations (bAVMs) represent a high risk for hemorrhagic stroke, leading to significant neurological morbidity and mortality in young adults. The etiopathogenesis of bAVM remains unclear. Research progress has been hampered by the lack of animal models. Hereditary Hemorrhagic Telangiectasia (HHT) patients with haploinsufficiency of endoglin (ENG, HHT1) or activin receptor-like kinase 1 (ALK1, HHT2) have a higher incidence of bAVM than the general population. We previously induced cerebrovascular dysplasia in the adult mouse brain that resembles human bAVM through Alk1 deletion plus vascular endothelial growth factor (VEGF) stimulation. We hypothesized that Eng deletion plus VEGF stimulation would induce a similar degree of cerebrovascular dysplasia as the Alk1-deleted brain. Methods Ad-Cre (an adenoviral vector expressing Cre recombinase) and AAV-VEGF (an adeno-associated viral vector expressing VEGF) were co-injected into the basal ganglia of 8–10 week old Eng2f/2f (exons 5–6 flanked by loxP sites), Alk12f/2f (exons 4–6 flanked by loxP sites) and wild-type (WT) mice. Vascular density, dysplasia index and gene deletion efficiency were analyzed 8 weeks later. Results AAV-VEGF induced a similar degree of angiogenesis in the brain with or without Alk1- or Eng-deletion. Abnormally patterned and dilated dysplastic vessels were found in the viral vector-injected region of Alk12f/2f and Eng2f/2f brain sections, but not in WT. Alk12f/2f mice had about 1.8-fold higher dysplasia index than Eng2f/2f mice (4.6 ± 1.9 vs. 2.5 ± 1.1, p dysplasia index with the gene deletion efficiency (Alk12f/2f: 16% and Eng2f/2f: 1%), we found that about 8-fold higher dysplasia was induced per copy of Eng deletion (2.5) than that of Alk1 deletion (0.3). ENG-negative endothelial cells were detected in the Ad-Cre-treated brain of Eng2f/2f mice, suggesting homozygous deletion of Eng in the cells. VEGF induced more severe vascular dysplasia in the Ad

  10. Extracellular S100A4(mts1) stimulates invasive growth of mouse endothelial cells and modulates MMP-13 matrix metalloproteinase activity

    DEFF Research Database (Denmark)

    Schmidt-Hansen, Birgitte; Ornås, Dorte; Grigorian, Mariam

    2004-01-01

    S100A4(mts1) protein expression has been strongly associated with metastatic tumor progression. It has been suggested as a prognostic marker for a number of human cancers. It is proposed that extracellular S100A4 accelerates cancer progression by stimulating the motility of endothelial cells...... with the transcriptional modulation of genes involved in the proteolytic degradation of extracellular matrix (ECM). Treatment of SVEC 4-10 with the S100A4 protein leads to the transcriptional activation of collagenase 3 (MMP-13) mRNA followed by subsequent release of the protein from the cells. Beta-casein zymography...... demonstrates enhancement of proteolytic activity associated with MMP-13. This observation indicates that extracellular S100A4 stimulates the production of ECM degrading enzymes from endothelial cells, thereby stimulating the remodeling of ECM. This could explain the angiogenic and metastasis...

  11. Vascular Endothelial Growth Factor from Embryonic Status to Cardiovascular Pathology

    Directory of Open Access Journals (Sweden)

    Mohsen Azimi-Nezhad

    2014-05-01

    Full Text Available Vascular endothelial growth factor (VEGF is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, endothelial cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, 145, 165, 183, 189, or 206 amino acids. VEGF121, VEGF145, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF145 has a key role during the vascularization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of vascular pathologies including cardiovascular diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardiovascular diseases.

  12. Influence of vascular endothelial growth factor stimulation and serum deprivation on gene activation patterns of human adipose tissue-derived stromal cells

    DEFF Research Database (Denmark)

    Tratwal, Josefine; Mathiasen, Anders Bruun; Juhl, Morten

    2015-01-01

    INTRODUCTION: Stimulation of mesenchymal stromal cells and adipose tissue-derived stromal cells (ASCs) with vascular endothelial growth factor (VEGF) has been used in multiple animal studies and clinical trials for regenerative purposes. VEGF stimulation is believed to promote angiogenesis and VEGF...... stimulation is usually performed under serum deprivation. Potential regenerative molecular mechanisms are numerous and the role of contributing factors is uncertain. The aim of the current study was to investigate the effect of in vitro serum deprivation and VEGF stimulation on gene expression patterns...... of ASCs. METHODS: Gene expressions of ASCs cultured in complete medium, ASCs cultured in serum-deprived medium and ASCs stimulated with VEGF in serum-deprived medium were compared. ASC characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow...

  13. In vitro stimulation of vascular endothelial growth factor by borate-based glass fibers under dynamic flow conditions.

    Science.gov (United States)

    Chen, Sisi; Yang, Qingbo; Brow, Richard K; Liu, Kun; Brow, Katherine A; Ma, Yinfa; Shi, Honglan

    2017-04-01

    Bioactive borate glass has been recognized to have both hard and soft tissue repair and regeneration capabilities through stimulating both osteogenesis and angiogenesis. However, the underlying biochemical and cellular mechanisms remain unclear. In this study, dynamic flow culturing modules were designed to simulate the micro-environment near the vascular depletion and hyperplasia area in wound-healing regions, thus to better investigate the mechanisms underlying the biocompatibility and functionality of borate-based glass materials. Glass fibers were dosed either upstream or in contact with the pre-seeded cells in the dynamic flow module. Two types of borate glasses, doped with (1605) or without (13-93B3) CuO and ZnO, were studied along with the silicate-based glass, 45S5. Substantial fiber dissolution in cell culture medium was observed, leading to the release of ions (boron, sodium and potassium) and the deposition of a calcium phosphate phase. Different levels of vascular endothelial growth factor secretion were observed from cells exposed to these three glass fibers, and the copper/zinc containing borate 1605 fibers exhibited the most positive influence. These results indicate that dynamic studies of in vitro bioactivity provide useful information to understand the in vivo response to bioactive borate glasses. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. LPS, Oleuropein and Blueberry extracts affect the survival, morphology and Phosphoinositide signalling in stimulated human endothelial cells.

    Science.gov (United States)

    Lo Vasco, Vincenza Rita; Leopizzi, Martina; Di Maio, Valeria; Di Raimo, Tania; Cesa, Stefania; Masci, Alessandra; Rocca, Carlo Della

    2017-12-01

    Endothelial cells (EC) act as leading actors in angiogenesis. Understanding the complex network of signal transduction pathways which regulate angiogenesis might offer insights in the regulation of normal and pathological events, including tumours, vascular, inflammatory and immune diseases. The effects of olive oil and of Blueberry extracts upon the phosphoinositide (PI)-specific phospholipase C (PLC) enzymes were evaluated both in quiescent and inflammatory stimulated human umbilical vein EC (HUVEC) using molecular biology (multiliquid bioanalysis) and immunofluorescence techniques. Oleuropein significantly increased the number of surviving HUVEC compared to untreated controls, suggesting that it favours the survival and proliferation of EC. Our results suggest that Oleuropein might be useful to induce EC proliferation, an important event during angiogenesis, with special regard to wound healing. Blueberry extracts increased the number of surviving HUVEC, although the comparison to untreated controls did not result statistically significant. Lipopolysaccharide (LPS) administration significantly reduced the number of live HUVEC. LPS can also modify the expression of selected PLC genes. Adding Blueberry extracts to LPS treated HUVEC cultures did not significantly modify the variations of PLC expression induced by LPS. Oleuropein increased or reduced the expression of PLC genes, and statistically significant results were identified for selected PLC isoforms. Oleuropein also modified the effects of LPS upon PLC genes' expression. Thus, our results corroborate the hypothesis that Oleuropein owns anti-inflammatory activity. The intracellular localization of PLC enzymes was modified by the different treatments we used. Podosome-like structures were observed in differently LPS treated HUVEC.

  15. Expression and Purification of Functional Human Vascular Endothelial Growth Factor-A121; the Most Important Angiogenesis Factor

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    Fatemeh Kazemi-Lomedasht

    2014-12-01

    Full Text Available Purpose: Angiogenesis or formation of new blood vessels is an essential process for tumor growth, invasion and metastasis. Vascular Endothelial Growth Factor (VEGF and its receptors play an important role in angiogenesis-dependent tumors. VEGF-A is the most important factor in angiogenesis process. Human VEGF-A gene consists of eight exons that undergoes alternative exon splicing and produce five different proteins consisting of 121, 145, 165, 189 and 206 amino acids (named VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206. Methods: In this study, VEGF121 gene synthesized and cloned into the pET-26b plasmid. The recombinant plasmid was transferred into appropriate expression strain of BL-21. Expression of VEGF121 induced by IPTG (Isopropyl β-D-1-thiogalactopyranoside and confirmed by SDS-PAGE and Western-Blotting. Recombinant VEGF121 was purified by nickel affinity chromatography. HUVECs (Human Umbilical Vein Endothelia Cells cells were isolated from umbilical vein and the effect of VEGF121 on tube formation of endothelial cells was investigated. Results: SDS-PAGE and Western-Blotting results verified the purification of VEGF121. The final yield of recombinant protein was about 5mg per liter. Endothelial cell tube formation assay results showed that VEGF121 leads to tube formation of endothelial cell on matrix and induces angiogenesis in vitro. Conclusion: Recombinant VEGF121 is important factor in tube formation of endothelial cell, so it could be used in different cancer researches and angiogenesis assay.

  16. Proinflammatory Stimulation of Toll-Like Receptor 9 with High Dose CpG ODN 1826 Impairs Endothelial Regeneration and Promotes Atherosclerosis in Mice.

    Directory of Open Access Journals (Sweden)

    Alexander O Krogmann

    Full Text Available Toll-like receptors (TLR of the innate immune system have been closely linked with the development of atherosclerotic lesions. TLR9 is activated by unmethylated CpG motifs within ssDNA, but also by CpG motifs in nucleic acids released during vascular apoptosis and necrosis. The role of TLR9 in vascular disease remains controversial and we sought to investigate the effects of a proinflammatory TLR9 stimulation in mice.TLR9-stimulation with high dose CpG ODN at concentrations between 6.25 nM to 30 nM induced a significant proinflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid and increased numbers of circulating endothelial microparticles, as a marker for amplified endothelial damage. Chronic TLR9 agonism in apolipoprotein E-deficient (ApoE-/- mice fed a cholesterol-rich diet increased aortic production of reactive oxygen species, the number of circulating endothelial microparticles, circulating sca-1/flk-1 positive cells, and most importantly augmented atherosclerotic plaque formation when compared to vehicle treated animals. Importantly, high concentrations of CpG ODN are required for these proatherogenic effects.Systemic stimulation of TLR9 with high dose CpG ODN impaired reendothelialization upon acute vascular injury and increased atherosclerotic plaque development in ApoE-/- mice. Further studies are necessary to fully decipher the contradictory finding of TLR9 agonism in vascular biology.

  17. Mediation of endothelial cell damage by serine proteases, but not superoxide, released from antineutrophil cytoplasmic antibody-stimulated neutrophils.

    Science.gov (United States)

    Lu, X; Garfield, A; Rainger, G E; Savage, C O S; Nash, G B

    2006-05-01

    To evaluate potential mediators of endothelial cell injury in systemic vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs), we investigated the factors controlling the neutrophil respiratory burst and endothelial release of von Willebrand factor (vWF) during neutrophil-endothelial cell interactions. Superoxide release from neutrophils binding to purified P-selectin or to tumor necrosis factor-activated endothelial cells was measured under flow or static conditions using the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c. Neutrophils were activated with fMLP, normal IgG, or ANCA IgG. Enzyme-linked immunosorbent assay was used to measure vWF. Serine protease activity was measured enzymatically. ANCA IgG or fMLP induced superoxide release when perfused over neutrophils that were rolling over P-selectin, but not those that were binding to endothelial cells. In static assays, endothelial cells inhibited superoxide production by neutrophils. Adenosine inhibited the respiratory burst, and, in cocultures, adenosine deaminase overcame the inhibitory effects of endothelial cells. Serine proteases were released during activated neutrophil-endothelial cell coculture. There was enhanced release of vWF during activated neutrophil-endothelial cell coculture; this was not inhibited by diphenyleneiodonium or by SOD plus catalase, but was inhibited by diisopropylfluorophosphate. Endothelial cells inhibit superoxide generation by fMLP and ANCA-activated neutrophils. The release of vWF occurs during coculture and is sensitive to serine protease, but not NADPH oxidase inhibition. Serine proteases may play a more important role than reactive oxygen species as mediators of endothelial injury during ANCA-associated systemic vasculitis.

  18. Phosphatidic Acid Produced by RalA-activated PLD2 Stimulates Caveolae-mediated Endocytosis and Trafficking in Endothelial Cells.

    Science.gov (United States)

    Jiang, Ying; Sverdlov, Maria S; Toth, Peter T; Huang, Long Shuang; Du, Guangwei; Liu, Yiyao; Natarajan, Viswanathan; Minshall, Richard D

    2016-09-23

    Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. M3 receptor is involved in the effect of penehyclidine hydrochloride reduced endothelial injury in LPS-stimulated human pulmonary microvascular endothelial cell.

    Science.gov (United States)

    Yuan, Qinghong; Xiao, Fei; Liu, Qiangsheng; Zheng, Fei; Shen, Shiwen; He, Qianwen; Chen, Kai; Wang, Yanlin; Zhang, Zongze; Zhan, Jia

    2017-11-17

    LPS has been recently shown to induce muscarinic acetylcholine 3 receptor (M3 receptor) expression and penehyclidine hydrochloride (PHC) is an anticholinergic drug which could block the expression of M3 receptor. PHC has been demonstrated to perform protective effect on cell injury. This study is to investigate whether the effect of PHC on microvascular endothelial injury is related to its inhibition of M3 receptor or not. HPMVECs were treated with specific M3 receptor shRNA or PBS, and randomly divided into LPS group (A group), LPS+PHC group (B group), LPS + M3 shRNA group (C group) and LPS + PHC + M3 shRNA group (D group). Cells were collected at 60 min after LPS treatment to measure levels of LDH, endothelial permeability, TNF-α and IL-6 levels, NF-κB p65 activation, I-κB protein expression, p38MAPK, and ERK1/2 activations as well as M3 mRNA expression. PHC could decrease LDH levels, cell permeability, TNF-α and IL-6 levels, p38 MAPK, ERK1/2, NF-κB p65 activations and M3 mRNA expressions compared with LPS group. When M3 receptor was silence, the changes of these indices were much more obvious. These findings suggest that M3 receptor plays an important role in LPS-induced pulmonary microvascular endothelial injury, which is regulated through NF-κB p65 and MAPK activation. And knockout of M3 receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. Regulative effects of PHC on pulmonary microvascular permeability and NF-κB p65 as well as MAPK activations are including but not limited to inhibition of M3 receptor. Copyright © 2017. Published by Elsevier Ltd.

  20. Rhein inhibits the expression of vascular cell adhesion molecule 1 in human umbilical vein endothelial cells with or without lipopolysaccharide stimulation.

    Science.gov (United States)

    Hu, Gang; Liu, Jiang; Zhen, Yong-Zhan; Wei, Jie; Qiao, Yue; Lin, Ya-Jun; Tu, Ping

    2013-01-01

    Reducing the expression of endothelial cell adhesion molecules (ECAMs) is known to decrease inflammation-induced vascular complications. In this study, we explored whether rhein can reduce the inflammation-induced expression of ECAMs in human umbilical vein endothelial cells (HUVECs) with or without lipopolysaccharide (LPS) stimulation. HUVECs were treated with different concentrations of rhein with or without 2.5 μg/ml LPS stimulation. Cell viability was assayed using the MTT method. Real-time PCR and Western blot analysis were used to measure the transcription and expression levels of ECAMs, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-SELECTIN and related signaling proteins. The results indicated that rhein (0-20 μmol/L) and LPS (0-10 μg/ml) had no effect on the viability of HUVECs. LPS could promote the expression of VCAM-1, ICAM-1 and E-SELECTIN. Rhein appeared to target VCAM-1, ICAM-1 and E-SELECTIN, with the transcription and expression of all three factors being reduced by the rhein treatment (10 and 20 μmol/L). The transcription and expression of VCAM-1 were also reduced by treatment with rhein (10 and 20 μmol/L) in the presence of LPS stimulation. In conclusion, rhein treatment reduced the expression of VCAM-1 in HUVECs via a p38-dependent pathway.

  1. Stimulation of endothelial adenosine A1 receptors enhances adhesion of neutrophils in the intact guinea pig coronary system.

    Science.gov (United States)

    Zahler, S; Becker, B F; Raschke, P; Gerlach, E

    1994-09-01

    The primary aim was to determine the action of pathophysiologically relevant adenosine concentrations (0.1-1 microM) on adhesion of neutrophils to coronary endothelium. Further aims were to evaluate the nature and localisation of the adenosine receptor involved, and to assess the effect of endogenous adenosine. Adhesion was studied in isolated perfused guinea pig hearts by determining the number of cells emerging in the coronary effluent after intracoronary bolus injections of 600,000 neutrophils prepared from guinea pig or human blood. The system was characterised by the use of the proadhesive stimulus thrombin. A 5 min infusion of adenosine (0.1-0.3 microM) or the A1 receptor agonist N6-cyclopentyladenosine (CPA, 0.01 microM) significantly increased adhesion from about 20% (control) to 30%. This effect was prevented by the A1 receptor antagonist dipropyl-8-cyclopentylxanthine (DPCPX, 0.1 microM). It was not diminished by cessation of adenosine infusion 90 s prior to neutrophil injection. At a higher concentration of adenosine (1 microM), adhesion did not seem to be enhanced. However, coinfusion of the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 microM) with 1 microM adenosine unmasked the A1 action, adhesion rising to 39%. Adenosine had a quantitatively identical effect on adhesion of human neutrophils. Total ischaemia of 15 min duration raised adhesion of subsequently applied neutrophils to 35%. This effect was completely blocked by DPCPX, as well as by ischaemic preconditioning (3 x 3 min). Preconditioning raised initial postischaemic coronary effluent adenosine from about 0.8 microM to 1.5 microM. The findings suggest a bimodal participation of adenosine in the development of postischaemic dysfunction by an endothelium dependent modulation of neutrophil adhesion. Stimulation occurs via endothelial A1 receptors at submicromolar adenosine levels, whereas cardioprotection by adenosine may in part relate to the use of pharmacologically high

  2. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  3. FoxP1 stimulates angiogenesis by repressing the inhibitory guidance protein semaphorin 5B in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Grundmann

    Full Text Available Forkhead box (Fox transcription factors are important regulators of cardiovascular development and several Fox-proteins have recently been shown to modulate embryonic and post-natal angiogenesis. However, the role of the FoxP subfamily, which is highly expressed in cardiovascular tissue, has not been investigated so far. Here, we show that the transcription factor FoxP1 is the highest expressed FoxP-protein in endothelial cells and that it is upregulated at the site of neovascularization during hindlimb ischemia in mice. Silencing of FoxP1 results in a strong inhibition of proliferation, tube formation and migration of cultured endothelial cells. Accordingly, knockdown of FoxP1 in zebrafish was followed by a disruption of intersomitic vascular formation. Using gene expression profiling, we show that FoxP1 induces a specific change of the endothelial transcriptome and functions as a suppressor of semaphorin 5B, which has previously been described as a neuronal inhibitory factor. Our findings now demonstrate that semaphorin 5B also acts as a FoxP1- dependent suppressor of endothelial cell proliferation, migration and sprouting, mediating the effects of FoxP1. In summary, our data indicate that the transcription factor FoxP1 is essential for the angiogenic function of endothelial cells and functions as a suppressor of the inhibitory guidance cue semaphorin 5B, suggesting an important function of FoxP1 in the regulation of neovascularization.

  4. Both the sera of patients with Behçet's disease and Streptococcus sanguis stimulate membrane expression of hnRNP A2/B1 in endothelial cells.

    Science.gov (United States)

    Cho, S B; Zheng, Z; Cho, S; Ahn, K J; Choi, M J; Kim, D-Y; Lee, K H; Bang, D

    2013-01-01

    Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 has been identified as a target antigen of anti-endothelial cell immunglobulin (Ig)A antibodies in patients with Behçet's disease (BD). The aim was to investigate the effects of the sera from BD patients and Streptococcus sanguis on the subcellular expression of hnRNP A2/B1 in human dermal microvascular endothelial cells (HDMECs). The sera of BD patients and healthy controls (HC) as well as cultured S. sanguis were used to stimulate HDMECs. Subcellular fractions were obtained from stimulated HDMECs and were subjected to immunoblot analyses. The distribution of hnRNP A2/B1 was investigated by immunocytochemistry and direct immunofluorescence study was performed in biopsy specimens of mucosal ulcers from BD patients. BD patients' sera increased the membrane expression of hnRNP A2/B1 in HDMECs after 12 and 24 h of incubation compared with HDMECs incubated with endothelial cell culture media and HC sera. S. sanguis also increased hnRNP A2/B1 in the cellular membrane. hnRNP A2/B1 mRNA level was also significantly upregulated in HDMECs incubated with BD patients' sera and S. sanguis. Immunocytochemistry demonstrated marked expression of hnRNP A2/B1 in the cytoplasm and cellular membrane of HDMECs incubated with BD patients' sera or S. sanguis. In addition, direct immunofluorescence experiments revealed the co-localization of serum IgA antibodies and monoclonal antibodies (mAbs) against hnRNP A2/B1 in tissue sections from ulcers of BD patients. Our data indicate that both the sera of BD patients with active disease and S. sanguis infection are inflammatory stimuli that can induce membranous hnRNP A2/B1 expression in HDMECs.

  5. Activation of sonic hedgehog signaling attenuates oxidized low-density lipoprotein-stimulated brain microvascular endothelial cells dysfunction in vitro.

    Science.gov (United States)

    Jiang, Xiu-Long; Chen, Ting; Zhang, Xu

    2015-01-01

    The study was performed to investigate the role of sonic hedgehog (SHH) in the oxidized low-density lipoprotein (oxLDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to oxLDL. The results indicated that treatment of MBMECs with oxLDL decreased the cell viability, and oxidative stress was involved in oxLDL-induce MBMECs dysfunction with increasing intracellular ROS and MDA formation as well as decreasing NO release and eNOS mRNA expression. In addition, SHH signaling components, such as SHH, Smo and Gli1, mRNA and protein levels were significantly decreased after incubation with increasing concentrations of oxLDL. Treatment with oxLDL alone or SHH loss-of-function significantly increased the permeability of MBMECs, and overexpression of SHH attenuated oxLDL-induced elevation of permeability in MBMECs. Furthermore, SHH gain-of-function could reverse oxLDL-induced apoptosis through inhibition caspase3 and caspase8 levels in MBMECs. Taken together, these results demonstrated that the suppression of SHH in MBMECs might contribute to the oxLDL-induced disruption of endothelial barrier. However, the overexpression of SHH could reverse oxLDL-induced endothelial cells dysfunction in vitro.

  6. Genistein ameliorated endothelial nitric oxidase synthase uncoupling by stimulating sirtuin-1 pathway in ox-LDL-injured HUVECs.

    Science.gov (United States)

    Zhang, Hua-ping; Zhao, Jia-hui; Yu, Hai-xia; Guo, Dong-xing

    2016-03-01

    Endothelial nitric oxidase synthase (eNOS) uncoupling plays a causal role in endothelial dysfunction in atherosclerosis. Genistein consumption has been associated with the prevention of atherosclerosis. However, the effect of genistein on eNOS uncoupling has not been reported. A model of oxidized low-density lipoprotein (ox-LDL)-induced injury on human umbilical vein endothelial cells (HUVECs) was established to evaluate the effect of genistein on eNOS uncoupling. We investigated the effect of genistein on NADPH oxidase-dependent superoxide production, NOX4 expression, BH4 synthesis and oxidation, the expression of GTP cyclohydrolase 1 (GCH1) and dihydrofolate reductase (DHFR). The results showed that genistein decreased superoxide production and NOX4 expression, enhanced the ratio of BH4/BH2, augmented the expressions of GCH1 and DHFR. Accompanied with genistein ameliorating eNOS uncoupling, genistein elevated the expression of sirtuin-1; furthermore, the effects of genistein on eNOS uncoupling were blunted with sirtuin-1 siRNA. The present study indicated that genistein ameliorated eNOS uncoupling was concerned with sirtuin-1 pathway in ox-LDL-injured HUVECs. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    Science.gov (United States)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  8. Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion

    Science.gov (United States)

    Hwang, Jinah; Saha, Aniket; Boo, Yong Chool; Sorescu, George P.; McNally, J. Scott; Holland, Steven M.; Dikalov, Sergei; Giddens, Don P.; Griendling, Kathy K.; Harrison, David G.; hide

    2003-01-01

    Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of O2- from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. O2- production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (+/- 5 dynes/cm2) increased O2- (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and O2- compared with static conditions. In contrast, neither LS nor OS were able to induce O2- production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced O2- production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates O2- and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

  9. Reduction of Monocyte Chemoattractant Protein-1 and Interleukin-8 Levels by Ticlopidine in TNF-α Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Chaur-Jong Hu

    2009-01-01

    Full Text Available Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1 is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8, a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-α-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1 in human umbilical vein endothelial cells (HUVECs. Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-α stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-α induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

  10. Bioactive Copper-Doped Glass Scaffolds Can Stimulate Endothelial Cells in Co-Culture in Combination with Mesenchymal Stem Cells

    Science.gov (United States)

    Rath, Subha N.; Brandl, Andreas; Hiller, Daniel; Hoppe, Alexander; Gbureck, Uwe; Horch, Raymund E.; Boccaccini, Aldo R.; Kneser, Ulrich

    2014-01-01

    Bioactive glass (BG) scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells)-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC). In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture) or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture). Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP) assay or by PCR, there was increased vascular endothelial growth factor (VEGF) expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL) uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering approaches. PMID

  11. Bioactive copper-doped glass scaffolds can stimulate endothelial cells in co-culture in combination with mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Subha N Rath

    Full Text Available Bioactive glass (BG scaffolds are being investigated for bone tissue engineering applications because of their osteoconductive and angiogenic nature. However, to increase the in vivo performance of the scaffold, including enhancing the angiogenetic growth into the scaffolds, some researchers use different modifications of the scaffold including addition of inorganic ionic components to the basic BG composition. In this study, we investigated the in vitro biocompatibility and bioactivity of Cu2+-doped BG derived scaffolds in either BMSC (bone-marrow derived mesenchymal stem cells-only culture or co-culture of BMSC and human dermal microvascular endothelial cells (HDMEC. In BMSC-only culture, cells were seeded either directly on the scaffolds (3D or direct culture or were exposed to ionic dissolution products of the BG scaffolds, kept in permeable cell culture inserts (2D or indirect culture. Though we did not observe any direct osteoinduction of BMSCs by alkaline phosphatase (ALP assay or by PCR, there was increased vascular endothelial growth factor (VEGF expression, observed by PCR and ELISA assays. Additionally, the scaffolds showed no toxicity to BMSCs and there were healthy live cells found throughout the scaffold. To analyze further the reasons behind the increased VEGF expression and to exploit the benefits of the finding, we used the indirect method with HDMECs in culture plastic and Cu2+-doped BG scaffolds with or without BMSCs in cell culture inserts. There was clear observation of increased endothelial markers by both FACS analysis and acetylated LDL (acLDL uptake assay. Only in presence of Cu2+-doped BG scaffolds with BMSCs, a high VEGF secretion was demonstrated by ELISA; and typical tubular structures were observed in culture plastics. We conclude that Cu2+-doped BG scaffolds release Cu2+, which in turn act on BMSCs to secrete VEGF. This result is of significance for the application of BG scaffolds in bone tissue engineering

  12. Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells

    Science.gov (United States)

    Povero, Davide; Eguchi, Akiko; Niesman, Ingrid R.; Andronikou, Nektaria; de Mollerat du Jeu, Xavier; Mulya, Anny; Berk, Michael; Lazic, Milos; Thapaliya, Samjana; Parola, Maurizio; Patel, Hemal H.; Feldstein, Ariel E.

    2014-01-01

    Angiogenesis is a key pathological feature of experimental and human steatohepatitis, a common chronic liver disease that is associated with obesity. We demonstrated that hepatocytes generated a type of membrane-bound vesicle, microparticles, in response to conditions that mimicked the lipid accumulation that occurs in the liver in some forms of steatohepatitis and that these microparticles promoted angiogenesis. When applied to an endothelial cell line, medium conditioned by murine hepatocytes or a human hepatocyte cell line exposed to saturated free fatty acids induced migration and tube formation, two processes required for angiogenesis. Medium from hepatocytes in which caspase 3 was inhibited or medium in which the microparticles were removed by ultracentrifugation lacked proangiogenic activity. Isolated hepatocyte-derived microparticles induced migration and tube formation of an endothelial cell line in vitro and angiogenesis in mice, processes that depended on internalization of microparticles. Microparticle internalization required the interaction of the ectoenzyme Vanin-1 (VNN1), an abundant surface protein on the microparticles, with lipid raft domains of endothelial cells. Large quantities of hepatocyte-derived microparticles were detected in the blood of mice with diet-induced steatohepatitis, and microparticle quantity correlated with disease severity. Genetic ablation of caspase 3 or RNA interference directed against VNN1 protected mice from steatohepatitis-induced pathological angiogenesis in the liver and resulted in a loss of the proangiogenic effects of microparticles. Our data identify hepatocyte-derived microparticles as critical signals that contribute to angiogenesis and liver damage in steatohepatitis and suggest a therapeutic target for this condition. PMID:24106341

  13. Interleukin-1 and tumour necrosis factor alpha induced polymorphonuclear leukocyte-endothelial cell adhesion and transendothelial migration in vitro: the effect of apical versus basal monolayer stimulation.

    Science.gov (United States)

    Morzycki, W; Sadowska, J; Issekutz, A C

    1990-09-01

    The cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) enhance polymorphonuclear leukocyte (PMNL) adhesion to vascular endothelium by an endothelial cell dependent mechanism in vitro and induce PMNL infiltration in vivo In this study, we employed human umbilical vein endothelium (HUVE) cultured on microporous membrane filters to form a monolayer, a system in which PMNL adherence and PMNL transendothelial migration could be measured using 51Cr-labelled human PMNL. In this system, it was found that PMNL adhesion and migration were dependent on prior treatment of the HUVE monolayer with IL-1 or TNF alpha for at least 2 h and that cytokine could be removed prior to the addition of PMNL without any effect on the response. PMNL adherence to the HUVE was maximal by 30 min and was followed by progressive migration of PMNL across the monolayer and the membrane filter into the lower chamber. The effect of apical surface versus basal surface exposure of the HUVE monolayer to IL-1 alpha and TNF alpha on subsequent PMNL interaction with the HUVE monolayer in the absence of cytokine was examined. Apical or basal stimulation induced comparable PMNL adherence at 30 min following addition of PMNL (35.5% and 43.1%). However, basal (i.e., abluminal) exposure to IL-1 or TNF alpha of the HUVE induced significantly greater PMNL transendothelial migration (e.g., 27.8% vs. 15.4%; P less than 0.01). The expression of endothelial-leukocyte adhesion molecules ELAM-1 and ICAM-1 following apical versus basal stimulation was determined by ELISA on viable cells. These adhesion molecules were upregulated to a similar extent under both conditions. These observations suggest that spacial localization or orientation of adhesion molecules may be influenced by basal versus apical cytokine stimulation or that other mechanisms are responsible for the preferential PMNL migration with basal stimulation. These findings may have implications for the in vivo interactions of PMNL with

  14. Inflammation stimulates thrombopoietin (Tpo) expression in rat brain-derived microvascular endothelial cells, but suppresses Tpo in astrocytes and microglia.

    Science.gov (United States)

    Zhang, Juan; Freyer, Dorette; Rung, Olga; Im, Ae-Rie; Hoffmann, Olaf; Dame, Christof

    2010-07-01

    Thrombopoietin (Tpo) and its receptor (c-Mpl; TpoR), which primary regulate megakaryopoiesis and platelet production, are also expressed in the central nervous system (CNS). Increased Tpo concentrations are present in the cerebrospinal fluid (CSF) of some patients with bacterial or viral meningitis. Since previous data implicated a proapoptotic role of Tpo on newly generated neuronal cells, we herein elucidated the regulation of Tpo in primary rat neurons (e17), astrocytes, and microglia (p0-p3), as well as in brain-derived vascular endothelial cells of 3-week-old rats after exposure to bacterial lipopolysaccharide (LPS). LPS inhibited Tpo gene expression in astrocytes and microglia, but not in neurons, most likely due to absence of Toll-like receptor 4 in neurons. While Tpo mRNA expression recovered in astrocytes after 24 h, it remained suppressed in microglia. Furthermore, we detected Tpo mRNA expression in primary brain-derived vascular endothelial cells, which also express the TpoR. In these cells, LPS significantly up-regulated Tpo mRNA expression. TpoR mRNA and protein expression remained constitutive in all cell types. Thus, our data provide evidence for a cell-type-specific modulation of Tpo mRNA expression by inflammation in brain-derived cells. Transient down-regulation of Tpo expression in astrocytes and microglia may limit Tpo-induced neuronal cell death in inflammatory brain disorders.

  15. [The stimulation of human pulmonary artery endothelial cells by cigarette smoke extract contributed to cell senescence and induced human pulmonary artery smooth cell migration].

    Science.gov (United States)

    Cai, L; Zhu, P C; Wang, Y E; Gao, Y T; Ao, Q L

    2017-06-12

    Objective: To observe the senescent effect of human pulmonary arterial endothelial cells (HPAEC) stimulated by cigarette smoke extract (CSE) and the effect of secretion of senescent cells on human pulmonary arterial smooth muscles cell (HPASMC) proliferation and migration. Methods: HPAEC was treated with different concentrations of CSE in vitro and cell proliferation was determined by CCK8, senescence cells analyzed by detecting the β-gal activity, and the senescent proteins of cells measured by Western blot. The concentration of senescence-associated secretory phenotype (SASP) was detected by ELISA and the expression of MCP-1 and TGF-β1 was measured by Real-time PCR. The number of the proliferated cells was measured by Transwell assay and immunoflurescence. Results: The HPAEC was aging with the stimulation concentration of CSE increasing and the stimulation time prolonging (Pcells increased as the exposure time prolonged. Compared with the control group, cell viability of 48 h group(1.8±0.1) and 72 h group (1.8±0.1) decreased significantly. The flow cytometry showed a significant difference between the CSE group(14.1±1.2) and the control group(28.5±1.8) in S phase(Pcell cycle arrest. The SASP was increasing as the CSE-exposure prolonged. Compared with the control group(177±39), the 48 h group(460±43) and the 72 h group(609±64) showed a marked increase in MCP-1(Pcells could secrete SASP which induced HPASMC proliferation. After different times of conditioned medium stimulation, HPASMC proliferated especially at 72 h(P<0.05) . The immnoflorescence and Transwell assay confirmed this finding. Conclusion: CSE could induce senescence of HPAEC and SASP production which improved HPASMC proliferation and migration.

  16. Impact of obesity control on circulating level of endothelial progenitor cells and angiogenesis in response to ischemic stimulation

    Directory of Open Access Journals (Sweden)

    Chen Yung-Lung

    2012-07-01

    Full Text Available Abstract Background and aim We tested the hypothesis that obesity reduced circulating number of endothelial progenitor cells (EPCs, angiogenic ability, and blood flow in ischemic tissue that could be reversed after obesity control. Methods 8-week-old C57BL/6J mice (n = 27 were equally divided into group 1 (fed with 22-week control diet, group 2 (22-week high fat diet, and group 3 (14-week high fat diet, followed by 8-week control diet. Critical limb ischemia (CLI was induced at week 20 in groups 2 and 3. The animals were sacrificed at the end of 22 weeks. Results Heart weight, body weight, abdominal fat weight, serum total cholesterol level, and fasting blood sugar were highest in group 2 (all p  Conclusion Obesity suppressed abilities of angiogenesis and recovery from CLI that were reversed by obesity control.

  17. Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction.

    Science.gov (United States)

    Lansink, M; Kooistra, T

    1996-07-15

    We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.

  18. Function and Regulation of the Vascular Cell Adhesion Molecule-l in TNF-Stimulated Endothelial Cells

    NARCIS (Netherlands)

    A. Pietersma (Anna)

    1999-01-01

    textabstractThe accumulation of circulating monocytes in the vessel wall is an important aspect of the pathogenesis of atherosclerosis and allied problems such as restenosis after percutaneous transluminal angioplasty (PTA). Three distinct steps can be distinguished in the stimulated accumulation of

  19. Angiogenic potential of endothelial and tumor cells seeded on gelatin-based hydrogels in response to electrical stimulations.

    Science.gov (United States)

    Tzoneva, Rumiana; Uzunova, Veselina; Apostolova, Sonia; Krüger-Genge, Anne; Neffe, Axel T; Jung, Friedrich; Lendlein, Andreas

    2016-01-01

    Angiogenesis is one of the key processes during development, wound healing and tumor formation. Prerequisite for its existence is the presence of endogenous electrical fields (EFs) generated by active ion transport across polarized epithelia and endothelia, and appearance of the transcellular potentials. During angiogenesis cellular factor as endothelial growth factor (VEGF), synthesis of adhesive proteins and membrane metalloproteinases (MMPs) govern the angiogenic response to different external stimuli as biomaterials interactions and/or exogenous EF. Gelatin-based hydrogels with elasticities comparable to human tissues have shown to influence cell behavior as well as cell attachment, protein synthesis, VEGF and MMP's production after the application of EF. Gelatin-based matrices with 3 (G10_LNCO3), 5 (G10_LNCO5), and 8 (G10_LNCO8) fold excess of isocyanate groups per mol of amine groups present in gelatin were used. Human umbilical endothelial cells (HUVEC) (Lonza Basel, Switzerland) and highly invasive breast cancer MDA-MB-231 cells (ATCC®HTB-26TM) were used. For an estimation of the amount of VEGF released from cells a commercially available VEGF ELISA (Thermo Fisher Scientific, Germany) kit was used. Fibronectin (FN) enzyme immunoassay (EIA) was used to analyze the secreted amount of FN by cells seeded on the materials. Secreted MMPs were analyzed by zymography. Gelatin-based hydrogels attracted HUVEC adhesion and diminished the adhesion of MDA-MB-231 cells. The applied direct current (DC) EF induced an almost 5-fold increase in VEGF production by HUVEC seeded on gelatin-based hydrogels, while in contrast, the applied EF decreased the production of VEGF by cancer cells. FN synthesis was elevated in HUVEC cells seeded on gelatin-based materials in comparison to FN synthesis by cancer cells. HUVEC seeded on gelatin hydrogels showed an expression mainly of MMP-2. The application of EF increased the production of MMP-2 in HUVEC seeded on gelatin materials. In

  20. Involvement of retinoic acid receptor alpha in the stimulation of tissue-type plasminogen-activator gene expression in human endothelial cells.

    Science.gov (United States)

    Kooistra, T; Lansink, M; Arts, J; Sitter, T; Toet, K

    1995-09-01

    Retinoids stimulate tissue-type plasminogen-activator (t-PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern-blot analysis of total or poly(A)-rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR-alpha (3.6 kb and 2.8 kb), and low levels of transcripts for RAR-beta (3.4 kb and 3.2 kb) and RAR-gamma (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR-alpha (4.8 kb) and, at a much lower concentration, RXR-beta (2.4 kb); no evidence for the presence of RXR-gamma was found. Furthermore, HUVEC express cellular retinol-binding protein I (CRBP-I) and cellular retinoic-acid-binding protein I (CRABP-I) mRNA. Exposure of HUVEC to 1 microM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR-beta mRNAs, RXR-alpha mRNA and CRBP-I mRNA, whereas the expression of the other receptor and CRABP-I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR-alpha is involved in the retinoid-induced t-PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR-alpha with a specific RAR-alpha antagonist, Ro 41-5253, was demonstrated to suppress the induction of t-PA by retinoids.

  1. Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Z.H. [Division of Cardiology, Shanghai Sixth People' s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, Division of Cardiology, Shanghai Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Yancheng People' s First Hospital, Division of Cardiology, Yancheng, Jiangsu, China, Division of Cardiology, Yancheng People’s First Hospital, Yancheng, Jiangsu (China); Zhu, W.; Tao, J.P.; Zhang, Q.Y.; Wei, M. [Division of Cardiology, Shanghai Sixth People' s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, Division of Cardiology, Shanghai Sixth People’s Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China)

    2013-10-22

    Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

  2. Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

    Science.gov (United States)

    Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

    2016-01-01

    Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from

  3. Endothelial precursor cells stimulate pericyte-like coverage of bone marrow-derived mesenchymal stem cells through platelet-derived growth factor-BB induction, which is enhanced by substance P.

    Science.gov (United States)

    Zhang, Mingzi; Ahn, Woosung; Kim, Sumin; Hong, Hyun Sook; Quan, Chengshi; Son, Youngsook

    2017-11-01

    The aim of this study was to evaluate the angiogenicity of a combination of BM-EPCs and BM-MSCs in vitro in the presence of SP and its working mechanism. BM-MSCs and BM-EPCs were cocultured with or without SP. ELISA and RT-PCR were performed to detect angiogenic factors such as VEGF and PDGF-BB. N-cadherin was detected by Western blot analysis. The tubular network-forming ability was evaluated by a Matrigel tube-forming assay. BM-EPCs coculture with BM-MSCs strongly stimulated the recruitment of BM-MSCs onto the BM-EPC-generated endothelial tubular network. Upon SP treatment, endothelial branching point, tubule length, and tubular recruitment of BM-MSCs were further increased and stabilized. The coculture of BM-EPCs and BM-MSCs synergistically stimulated expression of VEGF, VEGF receptor, N-cadherin, and PDGF-BB, all of which were further enhanced by SP treatment. Blockade of PDGF-BB by its functional blocking antibodies markedly reduced the BM-MSC incorporation into the endothelial tubules. SP-pretreated BM-MSCs were preferentially incorporated into the preformed BM-EPC tubular network. BM-EPCs along with SP promote the pericyte-like coverage of BM-MSCs on endothelial tubules possibly through the induction of PDGF-BB. © 2017 John Wiley & Sons Ltd.

  4. Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation.

    Science.gov (United States)

    Arts, J; Lansink, M; Grimbergen, J; Toet, K H; Kooistra, T

    1995-08-15

    We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.

  5. Effective Mobilization of Very Small Embryonic-Like Stem Cells and Hematopoietic Stem/Progenitor Cells but Not Endothelial Progenitor Cells by Follicle-Stimulating Hormone Therapy

    Directory of Open Access Journals (Sweden)

    Monika Zbucka-Kretowska

    2016-01-01

    Full Text Available Recently, murine hematopoietic progenitor stem cells (HSCs and very small embryonic-like stem cells (VSELs were demonstrated to express receptors for sex hormones including follicle-stimulating hormone (FSH. This raised the question of whether FSH therapy at clinically applied doses can mobilize stem/progenitor cells in humans. Here we assessed frequencies of VSELs (referred to as Lin−CD235a−CD45−CD133+ cells, HSPCs (referred to as Lin−CD235a−CD45+CD133+ cells, and endothelial progenitor cells (EPCs, identified as CD34+CD144+, CD34+CD133+, and CD34+CD309+CD133+ cells in fifteen female patients subjected to the FSH therapy. We demonstrated that FSH therapy resulted in statistically significant enhancement in peripheral blood (PB number of both VSELs and HSPCs. In contrast, the pattern of responses of EPCs delineated by different cell phenotypes was not uniform and we did not observe any significant changes in EPC numbers following hormone therapy. Our data indicate that FSH therapy mobilizes VSELs and HSPCs into peripheral blood that on one hand supports their developmental origin from germ lineage, and on the other hand FSH can become a promising candidate tool for mobilizing HSCs and stem cells with VSEL phenotype in clinical settings.

  6. Diagnostic Power of Vascular Endothelial Growth Factor and Macrophage Colony-Stimulating Factor in Breast Cancer Patients Based on ROC Analysis

    Directory of Open Access Journals (Sweden)

    Monika Zajkowska

    2016-01-01

    Full Text Available Breast cancer (BC is the most common malignancy in women. Vascular endothelial growth factor (VEGF has been described as an important regulator of angiogenesis which plays a vital role in the progression of tumor. Macrophage colony-stimulating factor (M-CSF is a cytokine whose functions include regulation of hematopoietic lineages cells growth, proliferation, and differentiation. We investigated the diagnostic significance of these parameters in comparison to CA15-3 in BC patients and in relation to the control group (benign breast tumor and healthy women. Plasma levels of the tested parameters were determined by ELISA and CA15-3 was determined by CMIA. VEGF was shown to be comparable to CA15-3 values of sensitivity in BC group and, what is more important, higher values in early stages of BC. VEGF was also the only parameter which has statistically significant AUC in all stages of cancer. M-CSF has been shown to be comparable to CA15-3 and VEGF, specificity, and AUC values only in stages III and IV of BC. These results indicate the usefulness and high diagnostic power of VEGF in the detection of BC. Also, it occurred to be the best candidate for cancer diagnostics in stages I and II of BC and in the differentiation between BC and benign cases.

  7. Propolis Reduces Phosphatidylcholine-Specific Phospholipase C Activity and Increases Annexin a7 Level in Oxidized-LDL-Stimulated Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hongzhuan Xuan

    2014-01-01

    Full Text Available To understand the mechanisms underlying the regulating dyslipidemia action of Chinese propolis and Brazilian green propolis, we investigated their effects on phosphatidylcholine-specific phospholipase C (PC-PLC activity and annexin a7 (ANXA7 level which play crucial roles in the control of the progress of atherosclerosis. Furthermore, active oxygen species (ROS levels, nuclear factor-KappaB p65 (NF-κB p65, and mitochondrial membrane potential (MMP were also investigated in oxidized-LDL- (ox-LDL- stimulated human umbilical vein endothelial cells (HUVECs. Our data indicated that the treatment of both types of propolis 12.5 μg/mL significantly increased cell viability and attenuated apoptosis rate, increased ANXA7 level, and decreased PC-PLC activity. Both types of propolis also inhibited ROS generation as well as the subsequent MMP collapse, and NF-κB p65 activation induced by ox-LDL in HUVECs. Our results also indicated that Chinese propolis and Brazilian green propolis had similar biological activities and prevented ox-LDL induced cellular dysfunction in HUVECs.

  8. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

    Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  9. Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

    Science.gov (United States)

    Thebault, Stéphanie; González, Carmen; García, Celina; Zamarripa, David Arredondo; Nava, Gabriel; Vaca, Luis; López-Casillas, Fernando; de la Escalera, Gonzalo Martínez; Clapp, Carmen

    2011-01-01

    Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK). Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS), as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i) upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC) channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

  10. Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

    Directory of Open Access Journals (Sweden)

    Carmen Clapp

    2011-07-01

    Full Text Available Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK. Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS, as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

  11. Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway.

    Science.gov (United States)

    Yang, Yi; Chen, Qi-Hong; Liu, Ai-Ran; Xu, Xiu-Ping; Han, Ji-Bin; Qiu, Hai-Bo

    2015-12-16

    Mesenchymal stem cells (MSCs) stabilise endothelial barrier function in acute lung injury via paracrine hepatocyte growth factor (HGF). Vascular endothelial growth factor (VEGF), which is secreted by MSCs, is another key regulator of endothelial permeability; however, its role in adjusting permeability remains controversial. In addition, whether an interaction occurs between HGF and VEGF, which are secreted by MSCs, is not completely understood. We introduced a co-cultured model of human pulmonary microvascular endothelial cells (HPMECs) and MSC conditioned medium (CM) collected from MSCs after 24 h of hypoxic culture. The presence of VEGF and HGF in the MSC-CM was neutralised by anti-VEGF and anti-HGF antibodies, respectively. To determine the roles and mechanisms of MSC-secreted HGF and VEGF, we employed recombinant humanised HGF and recombinant humanised VEGF to co-culture with HPMECs. Additionally, we employed the RhoA inhibitor C3 transferase and the Rac1 inhibitor NSC23766 to inhibit the activities of RhoA and Rac1 in HPMECs treated with MSC-CM or VEGF/HGF with the same dosage as in the MSC-CM. Then, endothelial paracellular and transcellular permeability was detected. VE-cadherin, occludin and caveolin-1 protein expression in HPMECs was measured by western blot. Adherens junction proteins, including F-actin and VE-cadherin, were detected by immunofluorescence. MSC-CM treatment significantly decreased lipopolysaccharide-induced endothelial paracellular and transcellular permeability, which was significantly inhibited by pretreatment with HGF antibody or with both VEGF and HGF antibodies. Furthermore, MSC-CM treatment increased the expression of the endothelial intercellular adherence junction proteins VE-cadherin and occludin and decreased the expression of caveolin-1 protein. MSC-CM treatment also decreased endothelial apoptosis and induced endothelial cell proliferation; however, the effects of MSC-CM treatment were inhibited by pretreatment with HGF

  12. Granulocyte-macrophage colony-stimulating factor increases tumor growth and angiogenesis directly by promoting endothelial cell function and indirectly by enhancing the mobilization and recruitment of proangiogenic granulocytes.

    Science.gov (United States)

    Zheng, Qiaowei; Li, Xueqian; Cheng, Xiaoliang; Cui, Ting; Zhuo, Yingcheng; Ma, Wenbin; Zhao, Xue; Zhao, Peipei; Liu, Xuanlin; Feng, Weiyi

    2017-02-01

    Granulocyte-macrophage colony-stimulating factor has been widely used as an adjuvant therapy for cancer patients exhibiting myelosuppression induced by chemotherapy or radiotherapy. However, the effects of granulocyte-macrophage colony-stimulating factor on tumor growth, as well as its precise mechanism, are still controversial due to inconsistent evidence. This study investigated the effect of exogenous granulocyte-macrophage colony-stimulating factor on the growth of B16 melanoma, S180 sarcoma, and U14 cervical carcinoma in mice. The angiogenesis and recruitment of bone-marrow-derived cells were analyzed in tumor tissues. Interactions among granulocyte-macrophage colony-stimulating factor, bone-marrow-derived cells, and B16 tumor cells were investigated in vitro. Proangiogenic types of bone-marrow-derived cells in blood were assessed both in vivo and in vitro. The results showed that granulocyte-macrophage colony-stimulating factor markedly facilitated the growth of B16 and S180 tumors, but not U14 tumors. Granulocyte-macrophage colony-stimulating factor increased the densities of blood vessels and the number of bone-marrow-derived cells in B16 tumor tissues. The granulocyte-macrophage colony-stimulating factor-induced enhancement of tumor cell proliferation was mediated by bone-marrow-derived cells in vitro. Meanwhile, a distinct synergistic effect on endothelial cell function between granulocyte-macrophage colony-stimulating factor and bone-marrow-derived cells was observed. After separating two types of bone-marrow-derived cells, granulocyte-macrophage colony-stimulating factor-induced enhancement of tumor growth and angiogenesis in vivo was mediated by proangiogenic cells in granulocytes, but not monocytes, with CD11b+, vascular endothelial growth factor receptor 2, and C-X-C chemokine receptor 4 granulocytes possibly involved. These data suggest that granulocyte-macrophage colony-stimulating factor contributes to the growth and angiogenesis of certain types

  13. Endothelial RIG-I activation impairs endothelial function.

    Science.gov (United States)

    Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2012-03-30

    Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5'end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Receptor for advanced glycation end products - membrane type1 matrix metalloproteinase axis regulates tissue factor expression via RhoA and Rac1 activation in high-mobility group box-1 stimulated endothelial cells.

    Directory of Open Access Journals (Sweden)

    Koichi Sugimoto

    Full Text Available BACKGROUND: Atherosclerosis is understood to be a blood vessel inflammation. High-mobility group box-1 (HMGB-1 plays a key role in the systemic inflammation. Tissue factor (TF is known to lead to inflammation which promotes thrombus formation. Membrane type1 matrix metalloprotease (MT1-MMP associates with advanced glycation endproducts (AGE triggered-TF protein expression and phosphorylation of NF-κB. However, it is still unclear about the correlation of MT1-MMP and HMBG-1-mediated TF expression. In this study, we investigated the molecular mechanisms of TF expression in response to HMGB-1 stimulation and the involvement of MT1-MMP in endothelial cells. METHODS AND RESULTS: Pull-down assays and Western blotting revealed that HMGB-1 induced RhoA/Rac1 activation and NF-kB phosphorylation in cultured human aortic endothelial cells. HMGB-1 increased the activity of MT1-MMP, and inhibition of RAGE or MT1-MMP by siRNA suppressed HMGB-1-induced TF upregulation as well as HMGB-1-triggered RhoA/Rac1 activation and NF-kB phosphorylation. CONCLUSIONS: The present study showed that RAGE/MT1-MMP axis modified HMBG-1-mediated TF expression through RhoA and Rac1 activation and NF-κB phosphorylation in endothelial cells. These results suggested that MT1-MMP was involved in vascular inflammation and might be a good target for treating atherosclerosis.

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

    Science.gov (United States)

    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.

  16. Positive Feedback Regulation of Agonist-Stimulated Endothelial Ca2+ Dynamics by KCa3.1 Channels in Mouse Mesenteric Arteries

    DEFF Research Database (Denmark)

    Qian, Xun; Francis, Michael; Köhler, Ralf

    2014-01-01

    Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca(2+) signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca(2+) transients from internal ...... stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1(-/-)) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca(2+) dynamics....

  17. Water extract of Korean red ginseng stimulates angiogenesis by activating the PI3K/Akt-dependent ERK1/2 and eNOS pathways in human umbilical vein endothelial cells.

    Science.gov (United States)

    Kim, Young-Mi; Namkoong, Seung; Yun, Young-Gab; Hong, Hee-Do; Lee, Young-Chul; Ha, Kwon-Soo; Lee, Hansoo; Kwon, Ho Jeong; Kwon, Young-Guen; Kim, Young-Myeong

    2007-09-01

    Angiogenesis is important for promoting cardiovascular disease, wound healing, and tissue regeneration. We investigated the effects of Korean red ginseng water extract (KRGE) on angiogenesis and its underlying signal mechanism. KRGE increased in vitro proliferation, migration, and tube formation of human umbilical vein endothelial cells, as well as stimulated in vivo angiogenesis without increasing VEGF expression. KRGE-induced angiogenesis was accompanied by phosphorylation of ERK1/2, phosphatidylinositol 3-kinase (Akt), and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. Inhibition of PI3K activity by wortmannin completely inhibited KRGE-induced angiogenesis and phosphorylation of Akt, ERK1/2, and eNOS, indicating that PI3K/Akt activation is an upstream event of the KRGE-mediated angiogenic pathway. The MEK inhibitor PD98059 blocked KRGE-induced ERK1/2 phosphorylation without affecting Akt and eNOS activation. However, the eNOS inhibitor N(G)-monomethyl-L-arginine effectively inhibited tube formation, but partially blocked proliferation and migration as well as ERK phosphorylation, without altering Akt and eNOS activation, revealing that the eNOS/NO pathway is partially involved in ERK1/2 activation. This study demonstrated that KRGE stimulates in vitro and in vivo angiogenesis through the activation of the PI3K/Akt-dependent ERK1/2 and eNOS signal pathways and their cross talk.

  18. Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor β2 induction

    NARCIS (Netherlands)

    Lansink, M.; Kooistra, T.

    1996-01-01

    We previously showed the involvement of retinoic acid receptor α (RARα) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of

  19. Vascular Endothelial Growth Factor A Isoform mRNA Expression in Pediatric Acute Myeloid Leukemia

    NARCIS (Netherlands)

    Kruizinga, R. C.; de Jonge, H. J. M.; Kampen, K. R.; Walenkamp, A. M. E.; de Bont, E. S. J. M.

    In AML high VEGFA protein expression correlates with poor overall and relapse-free survival (OS/RFS). To date, the relevance of the various VEGFA isoforms is unclear. We determined VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, and VEGF189 mRNA expression in pediatric AML samples and investigated the

  20. Resveratrol: A Multifunctional Compound Improving Endothelial Function

    OpenAIRE

    Li, Huige; F?rstermann, Ulrich

    2009-01-01

    The red wine polyphenol resveratrol boosts endothelium-dependent and -independent vasorelaxations. The improvement of endothelial function by resveratrol is largely attributable to nitric oxide (NO) derived from endothelial NO synthase (eNOS). By stimulating eNOS expression, eNOS phosphorylation and eNOS deacetylation, resveratrol enhances endothelial NO production. By upregulating antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and suppressing the expression a...

  1. Evaluation of Endothelial Dysfunction In Vivo.

    Science.gov (United States)

    Todiras, Mihail; Alenina, Natalia; Bader, Michael

    2017-01-01

    Vascular endothelial cells play a major role in maintaining cardiovascular homeostasis. Impairment of physiological properties of the endothelium, such as the promotion of vasodilation and anti-aggregation, leads to a condition called endothelial dysfunction. Endothelial dysfunction is an important early event in the pathogenesis of atherosclerosis and has been shown to have prognostic value in predicting vascular events including stroke and myocardial infarction.Endothelial-dependent vasodilation is one of the most widely used methods for assessment of endothelial function in rodents. It includes pharmacological stimulation (for example by acetylcholine) of endothelial release of NO and other vasoactive compounds in comparison with vascular response to endothelium-independent dilators such as sodium nitroprusside. However, usually this technique is performed in anesthetized animals. Here we describe a method which allows evaluation of endothelial dysfunction in conscious, freely moving mice and rats.

  2. Telmisartan attenuates hyperglycemia-exacerbated VCAM-1 expression and monocytes adhesion in TNFα-stimulated endothelial cells by inhibiting IKKβ expression.

    Science.gov (United States)

    Song, Kee-Ho; Park, Jung-Hyun; Jo, Inho; Park, Joong-Yeol; Seo, Jungwon; Kim, Soon Ae; Cho, Du-Hyong

    2016-03-01

    Uncontrolled hyperglycemia accelerates endothelial damage and vascular inflammation caused by proinflammatory cytokines including tumor necrosis factor α (TNFα), which leads to arteriosclerotic cardiovascular diseases such as myocardial infarction. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is prescribed for treatment of hypertensive patients with concurrent diabetes mellitus (DM). Although a few clinical trials have suggested that telmisartan decreases cardiovascular complications in diabetic patients, the molecular mechanism for the beneficial effects remains elusive. Here, we investigated a molecular mechanism and effects of telmisartan on the expression of vascular cell adhesion molecule-1 (VCAM-1) and attachment of monocytes onto endothelial cells induced by TNFα in hyperglycemia-treated bovine aortic endothelial cells (BAEC). Telmisartan dose-dependently decreased hyperglycemia-aggravated IκB kinase β (IKKβ) expression and nuclear factor-κB (NF-κB) p65-Ser(536) phosphorylation, which accompanied a decrease in VCAM-1 expression and THP-1 monocytes adhesion. Among ARBs, including losartan and fimasartan, only telmisartan showed the inhibitory effects on expression of VCAM-1 and IKKβ, and phosphorylation of NF-κB p65-Ser(536). The telmisartan's beneficial effects were not changed by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, although GW9662 clearly inhibited rosiglitazone-induced CD36 expression. Finally, ectopic expression of wild type (WT)-IKKβ significantly restored telmisartan-attenuated VCAM-1 expression, NF-κB p65-Ser(536) phosphorylation, and THP-1 monocytes adhesion. Taken together, our findings demonstrate that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by decreasing expression of IKKβ and VCAM-1 independently of PPARγ. Telmisartan may be useful for the treatment of DM-associated vascular

  3. PDGF receptor-α does not promote HCMV entry into epithelial and endothelial cells but increased quantities stimulate entry by an abnormal pathway.

    Directory of Open Access Journals (Sweden)

    Adam L Vanarsdall

    2012-09-01

    Full Text Available Epidermal growth factor receptor (EGFR and platelet-derived growth factor receptor-α (PDGFRα were reported to mediate entry of HCMV, including HCMV lab strain AD169. AD169 cannot assemble gH/gL/UL128-131, a glycoprotein complex that is essential for HCMV entry into biologically important epithelial cells, endothelial cells, and monocyte-macrophages. Given this, it appeared incongruous that EGFR and PDGFRα play widespread roles in HCMV entry. Thus, we investigated whether PDGFRα and EGFR could promote entry of wild type HCMV strain TR. EGFR did not promote HCMV entry into any cell type. PDGFRα-transduction of epithelial and endothelial cells and several non-permissive cells markedly enhanced HCMV TR entry and surprisingly, promoted entry of HCMV mutants lacking gH/gL/UL128-131 into epithelial and endothelial cells. Entry of HCMV was not blocked by a panel of PDGFRα antibodies or the PDGFR ligand in fibroblasts, epithelial, or endothelial cells or by shRNA silencing of PDGFRα in epithelial cells. Moreover, HCMV glycoprotein induced cell-cell fusion was not increased when PDGFRα was expressed in cells. Together these results suggested that HCMV does not interact directly with PDGFRα. Instead, the enhanced entry produced by PDGFRα resulted from a novel entry pathway involving clathrin-independent, dynamin-dependent endocytosis of HCMV followed by low pH-independent fusion. When PDGFRα was expressed in cells, an HCMV lab strain escaped endosomes and tegument proteins reached the nucleus, but without PDGFRα virions were degraded. By contrast, wild type HCMV uses another pathway to enter epithelial cells involving macropinocytosis and low pH-dependent fusion, a pathway that lab strains (lacking gH/gL/UL128-131 cannot follow. Thus, PDGFRα does not act as a receptor for HCMV but increased PDGFRα alters cells, facilitating virus entry by an abnormal pathway. Given that PDGFRα increased infection of some cells to 90%, PDGFRα may be very

  4. Stimulation of Transforming Growth Factor-β1-Induced Endothelial-To-Mesenchymal Transition and Tissue Fibrosis by Endothelin-1 (ET-1: A Novel Profibrotic Effect of ET-1.

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    Peter J Wermuth

    Full Text Available TGF-β-induced endothelial-to-mesenchymal transition (EndoMT is a newly recognized source of profibrotic activated myofibroblasts and has been suggested to play a role in the pathogenesis of various fibrotic processes. Endothelin-1 (ET-1 has been implicated in the development of tissue fibrosis but its participation in TGF-β-induced EndoMT has not been studied. Here we evaluated the role of ET-1 on TGF-β1-induced EndoMT in immunopurified CD31+/CD102+ murine lung microvascular endothelial cells. The expression levels of α-smooth muscle actin (α-SMA, of relevant profibrotic genes, and of various transcription factors involved in the EndoMT process were assessed employing quantitative RT-PCR, immunofluorescence histology and Western blot analysis. TGF-β1 caused potent induction of EndoMT whereas ET-1 alone had a minimal effect. However, ET-1 potentiated TGF-β1-induced EndoMT and TGF-β1-stimulated expression of mesenchymal cell specific and profibrotic genes and proteins. ET-1 also induced expression of the TGF-β receptor 1 and 2 genes, suggesting a plausible autocrine mechanism to potentiate TGF-β-mediated EndoMT and fibrosis. Stimulation of TGF-β1-induced skin and lung fibrosis by ET-1 was confirmed in vivo in an animal model of TGF-β1-induced tissue fibrosis. These results suggest a novel role for ET-1 in the establishment and progression of tissue fibrosis.

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

    Science.gov (United States)

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

    2016-02-01

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

  6. Oxidative stress induced pulmonary endothelial cell proliferation is ...

    African Journals Online (AJOL)

    Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

  7. Activation of endothelial toll-like receptor 3 impairs endothelial function.

    Science.gov (United States)

    Zimmer, Sebastian; Steinmetz, Martin; Asdonk, Tobias; Motz, Inga; Coch, Christoph; Hartmann, Evelyn; Barchet, Winfried; Wassmann, Sven; Hartmann, Gunther; Nickenig, Georg

    2011-05-27

    Endothelial dysfunction and atherosclerosis are chronic inflammatory diseases characterized by activation of the innate and acquired immune system. Specialized protein receptors of the innate immune system recognize products of microorganisms and endogenous ligands such as nucleic acids. Toll-like receptor 3 (TLR3), for example, detects long double-stranded RNA and is abundantly expressed in endothelial cells. Whether innate immunity contributes to atherogenic mechanisms in endothelial cells is poorly understood. We sought to determine the effects of TLR3 activation in endothelial cells. We first investigated whether stimulation of TLR3 influences endothelial biology in mice. Intravenous injection of polyinosine polycytidylic acid, a synthetic double-stranded RNA analog and TLR3 ligand, impaired endothelium-dependent vasodilation, increased vascular production of reactive oxygen species, and reduced reendothelialization after carotid artery injury in wild-type mice compared with controls but had no effect in TLR3(-/-) animals. TLR3 stimulation not only induced endothelial dysfunction but also enhanced the formation of atherosclerotic plaques in apolipoprotein E-deficient mice. In vitro incubation of endothelial cells with polyinosine polycytidylic acid induced production of the proinflammatory cytokines interleukin-8 and interferon-γ-induced protein 10, increased formation of reactive oxygen species, diminished proliferation, and increased apoptosis, which suggests that endothelial cells are able to directly detect and respond to TLR3 ligands. Neutralization of interleukin-8 and interferon-γ-induced protein 10 antagonizes the observed negative effects of polyinosine polycytidylic acid. We found elevated levels of circulating endothelial progenitor cells in polyinosine polycytidylic acid-treated mice, although they displayed increased endothelial dysfunction. Stimulation of TLR3 in cultured endothelial progenitor cells, however, led to increased formation of

  8. Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

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    Annie O Smith

    Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

  9. Hematopoietic Stem Cell Cytokines and Fibroblast Growth factor-2 Stimulate Human Endothelial Cell-Pericyte Tube Co-Assembly in 3D Fibrin Matrices under Serum-Free Defined Conditions

    Science.gov (United States)

    Smith, Annie O.; Bowers, Stephanie L. K.; Stratman, Amber N.; Davis, George E.

    2013-01-01

    We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC) tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF)-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay) to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices) that regulates angiogenic events in postnatal life. PMID:24391990

  10. Endothelial function in vasovagal syncope.

    Science.gov (United States)

    Pietrucha, Artur Z

    2014-12-01

    Vasovagal syncope (VVS) is a common form of fainting. The pathophysiology of VVS is complex and involves changes in the autonomic and vascular tone, resulting in reflex bradycardia with marked hypotension. Paradoxical peripheral vasodilation caused by endothelial dysfunction may also play a key role in inappropriate hypotension during VVS. Endothelial hyperactivity due to up regulation of nitric oxide synthase leads to profound vasodilation, much stronger than vasodilation caused by adrenergic stimulation in response to orthostatic stress alone. Studies have reported significantly higher flow-mediated dilation and higher plasma nitric oxide concentration in people with vasovagal syndrome. Patients with VVS showed decreased vasoconstrictive agent endothelin-1 levels during orthostatic stress. Coagulation and fibrinolysis activity also play important roles in endothelial function in syncopal patients. The response of the endothelium to orthostatic stress is similar to the reaction to haemorrhagic stress and is likely to be a remnant from the evolutionary adaptation of primates.

  11. Bubble-Induced Endothelial Microparticles Promote Endothelial Dysfunction.

    Science.gov (United States)

    Yu, Xuhua; Xu, Jiajun; Huang, Guoyang; Zhang, Kun; Qing, Long; Liu, Wenwu; Xu, Weigang

    2017-01-01

    Decompression sickness is a systemic pathophysiological process caused by bubbles and endothelial microparticles (EMPs) are established markers reflecting competency of endothelial function and vascular biology. Here, we investigated the effects of bubble-induced EMPs on endothelial cells in vitro and vivo. Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and stimulated by bubbles and bubble-induced EMPs were collected and incubated with normal PMVECs in vitro. Cell viability and apoptosis were detected using Cell Counting Kit-8 assay and Annexin V FITC/PI double staining, respectively. Cell permeability and pro-inflammatory cytokines were determined by electric cell substrate impedance sensing and enzyme-linked immunosorbent assay, respectively. Intracellular nitric oxide and reactive oxygen species production were analyzed microscopically. In vivo study, bubble-induced EMPs were intravenously injected to the rats and soluble thrombomodulin, intercellular adhesion molecule 1, and vascullar adhesion molecule 1 were involved in evaluating endothelial dysfunction. In our study, bubble stimulus resulted in a significant increase of EMPs release by 3 fold. Bubble-induced EMPs significantly decreased cell viability and increased cell apoptosis. Moreover, bubble-induced EMPs induced abnormal increase of cell permeability and over-expression of pro-inflammatory cytokines. Intracellular ROS production increased while NO production decreased. These negative effects caused by bubble-induced EMPs were remarkably suppressed when EMPs pretreated with surfactant FSN-100. Finally, intravenous injection of bubble-induced EMPs caused elevations of soluble thrombomodulin and pro-inflammatory cytokines in the circulation. Altogether, our results demonstrated that bubble-induced EMPs can mediate endothelial dysfunction in vitro and vivo, which can be attenuated by EMPs abatement strategy. These data expanded our horizon of the detrimental effects of bubble

  12. Bubble-Induced Endothelial Microparticles Promote Endothelial Dysfunction.

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    Xuhua Yu

    Full Text Available Decompression sickness is a systemic pathophysiological process caused by bubbles and endothelial microparticles (EMPs are established markers reflecting competency of endothelial function and vascular biology. Here, we investigated the effects of bubble-induced EMPs on endothelial cells in vitro and vivo. Rat pulmonary microvascular endothelial cells (PMVECs were isolated and stimulated by bubbles and bubble-induced EMPs were collected and incubated with normal PMVECs in vitro. Cell viability and apoptosis were detected using Cell Counting Kit-8 assay and Annexin V FITC/PI double staining, respectively. Cell permeability and pro-inflammatory cytokines were determined by electric cell substrate impedance sensing and enzyme-linked immunosorbent assay, respectively. Intracellular nitric oxide and reactive oxygen species production were analyzed microscopically. In vivo study, bubble-induced EMPs were intravenously injected to the rats and soluble thrombomodulin, intercellular adhesion molecule 1, and vascullar adhesion molecule 1 were involved in evaluating endothelial dysfunction. In our study, bubble stimulus resulted in a significant increase of EMPs release by 3 fold. Bubble-induced EMPs significantly decreased cell viability and increased cell apoptosis. Moreover, bubble-induced EMPs induced abnormal increase of cell permeability and over-expression of pro-inflammatory cytokines. Intracellular ROS production increased while NO production decreased. These negative effects caused by bubble-induced EMPs were remarkably suppressed when EMPs pretreated with surfactant FSN-100. Finally, intravenous injection of bubble-induced EMPs caused elevations of soluble thrombomodulin and pro-inflammatory cytokines in the circulation. Altogether, our results demonstrated that bubble-induced EMPs can mediate endothelial dysfunction in vitro and vivo, which can be attenuated by EMPs abatement strategy. These data expanded our horizon of the detrimental effects

  13. Estetrol modulates endothelial nitric oxide synthesis in human endothelial cells

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    Maria Magdalena eMontt-Guevara

    2015-07-01

    Full Text Available Estetrol (E4 is a natural human estrogen that is present at high concentrations during pregnancy. E4 has been reported to act as an endogenous estrogen receptor modulator, exerting estrogenic actions on the endometrium or the central nervous system but presenting antagonistic effects on the breast. Due to these characteristics, E4 is currently being developed for a number of clinical applications, including contraception and menopausal hormone therapy. Endothelial nitric oxide (NO is a key player for vascular function and disease during pregnancy and throughout ageing in women. Endothelial NO is an established target of estrogens that enhance its formation in human endothelial cells. We here addressed the effects of E4 on the activity and expression of the endothelial nitric oxide synthase (eNOS in cultured human umbilical vein endothelial cells (HUVEC. E4 stimulated the activation of eNOS and NO secretion in HUVEC. E4 was significantly less effective compared to E2 and a peculiar concentration-dependent effect was found, with higher amounts of E4 being less effective than lower concentrations. When E2 was combined with E4, an interesting pattern was noted. E4 antagonized NO synthesis induced by pregnancy-like E2 concentrations. However, E4 did not impede the modest induction of NO synthesis associated with postmenopausal-like E2 levels. These results support the hypothesis that E4 may be a regulator of NO synthesis in endothelial cells and raise questions on its peculiar signaling in this context. Our results may be useful to interpret the role of E4 during human pregnancy and possibly to help develop this interesting steroid for clinical use.

  14. Plasma vascular endothelial growth factor B levels are increased in patients with newly diagnosed type 2 diabetes mellitus and associated with the first phase of glucose-stimulated insulin secretion function of β-cell.

    Science.gov (United States)

    Wu, J; Wei, H; Qu, H; Feng, Z; Long, J; Ge, Q; Deng, H

    2017-11-01

    To detect plasma vascular endothelial growth factor B (VEGF-B) in individuals with different glucose tolerance and investigate the relationship between plasma VEGF-B levels and the first phase of glucose-stimulated insulin secretion. A cross-sectional study was conducted involving 45 patients with newly diagnosed type 2 diabetes mellitus (T2DM), 37 patients with impaired glucose regulation (IGR), and 39 Normal glucose tolerance (NGT) subjects, all of whom underwent intravenous glucose tolerance test. Plasma VEGF-B levels were assayed by ELISA. The first phase of insulin secretion was evaluated by acute insulin response (AIR), the area under the curve of the first-phase (0-10 min) insulin secretion (AUC) and glucose disposition index (GDI). The T2DM and IGR groups had higher plasma VEGF-B levels than the NGT group (P B levels were negatively correlated with AIR, AUC, GDI, HOMA-β (P B levels [145.59-180.07 pg/ml, OR 3.55 (95% CI 1.05-12.02) and >180.07 pg/ml, OR 3.64 (95% CI 1.16-11.42)] were related to a greater probability of β-cell hypofunction, compared with low VEGF-B levels (B levels and β-cell hypofunction disappeared (P > 0.05). Our study provides evidence that plasma VEGF-B levels were higher in patients with newly diagnosed T2DM, and were strongly associated with glucose and lipid metabolism and the first-phase insulin secretion function of β-cells. VEGF-B may be involved in the mechanism of β-cell dysfunction in T2DM.

  15. AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro.

    Science.gov (United States)

    Szczesny, Bartosz; Módis, Katalin; Yanagi, Kazunori; Coletta, Ciro; Le Trionnaire, Sophie; Perry, Alexis; Wood, Mark E; Whiteman, Matthew; Szabo, Csaba

    2014-09-15

    The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. Addition of AP39 (30-300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30-100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage. Copyright © 2014 Elsevier Inc. All

  16. Hydrogen Sulfide Stimulates Ischemic Vascular Remodeling Through Nitric Oxide Synthase and Nitrite Reduction Activity Regulating Hypoxia‐Inducible Factor‐1α and Vascular Endothelial Growth Factor–Dependent Angiogenesis

    Science.gov (United States)

    Bir, Shyamal C.; Kolluru, Gopi K.; McCarthy, Paul; Shen, Xinggui; Pardue, Sibile; Pattillo, Christopher B.; Kevil, Christopher G.

    2012-01-01

    Background Hydrogen sulfide (H2S) therapy is recognized as a modulator of vascular function during tissue ischemia with the notion of potential interactions of nitric oxide (NO) metabolism. However, little is known about specific biochemical mechanisms or the importance of H2S activation of NO metabolism during ischemic tissue vascular remodeling. The goal of this study was to determine the effect of H2S on NO metabolism during chronic tissue ischemia and subsequent effects on ischemic vascular remodeling responses. Methods and Results The unilateral, permanent femoral artery ligation model of hind‐limb ischemia was performed in C57BL/6J wild‐type and endothelial NO synthase–knockout mice to evaluate exogenous H2S effects on NO bioavailability and ischemic revascularization. We found that H2S selectively restored chronic ischemic tissue function and viability by enhancing NO production involving both endothelial NO synthase and nitrite reduction mechanisms. Importantly, H2S increased ischemic tissue xanthine oxidase activity, hind‐limb blood flow, and angiogenesis, which were blunted by the xanthine oxidase inhibitor febuxostat. H2S treatment increased ischemic tissue and endothelial cell hypoxia‐inducible factor‐1α expression and activity and vascular endothelial growth factor protein expression and function in a NO‐dependent manner that was required for ischemic vascular remodeling. Conclusions These data demonstrate that H2S differentially regulates NO metabolism during chronic tissue ischemia, highlighting novel biochemical pathways to increase NO bioavailability for ischemic vascular remodeling. PMID:23316304

  17. VEGF and endothelial guidance in angiogenic sprouting.

    Science.gov (United States)

    Gerhardt, Holger

    2008-10-01

    The cellular actions of VEGF need to be coordinated to guide vascular patterning during sprouting angiogenesis. Individual endothelial tip cells lead and guide the blood vessel sprout, while neighbouring stalk cells proliferate and form the vascular lumen. Recent studies illustrate how endothelial DLL4/NOTCH signalling, stimulated by VEGF, regulates the sprouting response by limiting tip cell formation in the stalk. The spatial distribution of VEGF, in turn, regulates the shape of the ensuing sprout by directing tip cell migration and determining stalk cell proliferation.

  18. Resveratrol and Endothelial Nitric Oxide

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    Ning Xia

    2014-10-01

    Full Text Available Nitric oxide (NO derived from the endothelial NO synthase (eNOS has antihypertensive, antithrombotic, anti-atherosclerotic and antiobesogenic properties. Resveratrol is a polyphenol phytoalexin with multiple cardiovascular and metabolic effects. Part of the beneficial effects of resveratrol are mediated by eNOS. Resveratrol stimulates NO production from eNOS by a number of mechanisms, including upregulation of eNOS expression, stimulation of eNOS enzymatic activity and reversal of eNOS uncoupling. In addition, by reducing oxidative stress, resveratrol prevents oxidative NO inactivation by superoxide thereby enhancing NO bioavailability. Molecular pathways underlying these effects of resveratrol involve SIRT1, AMPK, Nrf2 and estrogen receptors.

  19. Endothelial dysfunction: a comprehensive appraisal

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    Vilariño Jorge O

    2006-02-01

    Full Text Available Abstract The endothelium is a thin monocelular layer that covers all the inner surface of the blood vessels, separating the circulating blood from the tissues. It is not an inactive organ, quite the opposite. It works as a receptor-efector organ and responds to each physical or chemical stimulus with the release of the correct substance with which it may maintain vasomotor balance and vascular-tissue homeostasis. It has the property of producing, independently, both agonistic and antagonistic substances that help to keep homeostasis and its function is not only autocrine, but also paracrine and endocrine. In this way it modulates the vascular smooth muscle cells producing relaxation or contraction, and therefore vasodilatation or vasoconstriction. The endothelium regulating homeostasis by controlling the production of prothrombotic and antithrombotic components, and fibrynolitics and antifibrynolitics. Also intervenes in cell proliferation and migration, in leukocyte adhesion and activation and in immunological and inflammatory processes. Cardiovascular risk factors cause oxidative stress that alters the endothelial cells capacity and leads to the so called endothelial "dysfunction" reducing its capacity to maintain homeostasis and leads to the development of pathological inflammatory processes and vascular disease. There are different techniques to evaluate the endothelium functional capacity, that depend on the amount of NO produced and the vasodilatation effect. The percentage of vasodilatation with respect to the basal value represents the endothelial functional capacity. Taking into account that shear stress is one of the most important stimulants for the synthesis and release of NO, the non-invasive technique most often used is the transient flow-modulate "endothelium-dependent" post-ischemic vasodilatation, performed on conductance arteries such as the brachial, radial or femoral arteries. This vasodilatation is compared with the

  20. Enhanced assay of endothelial exocytosis using extracellular matrix components.

    Science.gov (United States)

    LoMonaco, Michael B; Lowenstein, Charles J

    2014-05-01

    Vascular inflammation plays a key role in the pathogenesis of atherosclerosis. The first step in vascular inflammation is endothelial exocytosis, in which endothelial granules fuse with the plasma membrane, releasing prothrombotic and proinflammatory messenger molecules. The development of cell culture models to study endothelial exocytosis has been challenging because the factors that modulate exocytosis in vitro are not well understood. Here we report a method for studying endothelial exocytosis that optimizes extracellular matrix components, cell density, and duration of culture. Human umbilical vein endothelial cells plated on collagen I-coated plates and cultured in the confluent state for 7-12 days in low-serum medium showed robust secretion of von Willebrand factor when stimulated with various agonists. This exocytosis assay is rapid and applicable to high-throughput screening. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Endothelial cell oxidative stress and signal transduction

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    ROCIO FONCEA

    2000-01-01

    Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

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

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    Jason Tran

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Tumor endothelial inflammation predicts clinical outcome in diverse human cancers.

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    Sean P Pitroda

    Full Text Available Vascular endothelial cells contribute to the pathogenesis of numerous human diseases by actively regulating the stromal inflammatory response; however, little is known regarding the role of endothelial inflammation in the growth of human tumors and its influence on the prognosis of human cancers.Using an experimental model of tumor necrosis factor-alpha (TNF-α-mediated inflammation, we characterized inflammatory gene expression in immunopurified tumor-associated endothelial cells. These genes formed the basis of a multivariate molecular predictor of overall survival that was trained and validated in four types of human cancer.We report that expression of experimentally derived tumor endothelial genes distinguished pathologic tissue specimens from normal controls in several human diseases associated with chronic inflammation. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. This endothelial-derived signature predicted outcome independently of, but cooperatively with, standard clinical and pathological prognostic factors. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells.This study provides the first prognostic cancer gene signature derived from an experimental model of tumor-associated endothelial inflammation. These findings support the notion that activation of inflammatory pathways in non-malignant tumor-infiltrating endothelial cells contributes to tumor growth and progression in multiple human cancers. Importantly, these results identify endothelial-derived factors that could serve as potential targets for therapy in diverse human cancers.

  5. Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients.

    Science.gov (United States)

    Haller, H; Ziegler, E M; Homuth, V; Drab, M; Eichhorn, J; Nagy, Z; Busjahn, A; Vetter, K; Luft, F C

    1997-01-01

    Endothelial cell activation is important in the pathogenesis of preeclampsia; however, the nature of the activation is unknown. We investigated 22 patients with preeclampsia. 29 normotensive pregnancies, and 18 nonpregnant women to test the hypothesis that serum from preeclamptic patients induces expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) and stimulates intracellular free calcium concentrations [Ca2+]i in cultured endothelial cells. We then asked whether the corresponding integrin adhesive counter receptors lymphocyte function-associated antigen-1 (CD11a/CD18), macrophage-1 antigen (CD11b/CD18), p150,95 (CD11c/CD18), and very late activation antigen-4 (CD49/CD29) are increased in patients with preeclampsia. In the pregnant women, the measurements were conducted both before and after delivery. Integrin expression was measured by fluorescent antibody cell sorting analysis using monoclonal antibodies. ICAM-1 and VCAM-1 were analyzed on endothelial cells by enzyme-linked immunosorbent assay. [Ca2+]i was measured with fura 2. Serum from preeclamptic patients increased endothelial cell ICAM-1 expression but not VCAM-1 expression. Preeclamptic patients' serum also increased [Ca2+]i in endothelial cells compared with serum from normal nonpregnant or normal pregnant women. Endothelial cell [Ca2+]i concentrations were correlated with the ICAM-1 expression in preeclamptic patients (r = .80, P preclampsia and normal pregnancy compared with the nonpregnant state. The expression decreased significantly after delivery in both groups. Our results demonstrate that serum from preeclamptic women induces increased ICAM-1 surface expression on endothelial cells, while the expression of the integrin counterreceptors was not different. The effect on endothelial cells may be related to an increase in [Ca2+]i. The effect on cultured endothelial cells and the rapid decrease after delivery suggests the presence of a circulating serum

  6. G Protein-Coupled Receptors Sense Fluid Shear Stress in Endothelial Cells

    National Research Council Canada - National Science Library

    Mirianas Chachisvilis; Yan-Liang Zhang; John A. Frangos

    2006-01-01

    ...). By using time-resolved fluorescence microscopy and GPCR conformation-sensitive FRET we found that stimulation of endothelial cells with fluid shear stress, hypotonic stress, or membrane fluidizing...

  7. Basic fibroblast growth factor/vascular endothelial growth factor in the serum from severe burn patients stimulates the proliferation of cultured human umbilical cord mesenchymal stem cells via activation of Notch signaling pathways.

    Science.gov (United States)

    Liu, Ling-Ying; Hou, Yu-Sen; Chai, Jia-Ke; Hu, Quan; Duan, Hong-Jie; Yu, Yong-Hui; Yin, Hui-Nan; Hao, Dai-Feng; Feng, Guang; Li, Tao; Du, Jun-Dong

    2013-11-01

    Mesenchymal stem cells (MSCs) are the leading cellular constituents used in regenerative medicine. MSCs repair and reconstruct wounds of acute traumata and radiation-induced burns through proliferation, differentiation, and trophic activity. However, repair effect of MSCs on severe burn wounds remain to be clarified because severe burns are much more complex traumata than radiation-induced burns. Survival and proliferation of MSCs in microenvironments affected by severe burns are very important for improving wound repair/regeneration. This study aimed to elucidate the survival and proliferation effects and the potential proliferation mechanism of serum from severe burn patients (BPS) on human umbilical cord MSCs (hUCMSCs) in vitro. The hUCMSCs were isolated, cultured, and identified. Next, we evaluated the effects of BPS on cell numbers, cell cycle progression, cyclin D expression, and key proteins and genes of the Notch signaling pathway. Putative mechanisms underlying the proliferation of hUCMSCs were investigated. BPS markedly increased the number of hUCMSCs, and the results of the cell cycle studies indicated that BPS induced cell cycle progression into the M phase. Cyclin D expression was higher with BPS than in the control group. Moreover, Notch-1, a key determinant of hUCMSC activation and proliferation, and its target gene Hes-1 were overexpressed after BPS treatment. Proliferation numbers of hUCMSC, rate of proliferation period (G2/M+S), and the expression of cyclin D, Notch-1, and Hes-1 were markedly decreased by Notch signaling inhibitors (DAPT/GSI). In the case of BPS, basic fibroblast growth factor and vascular endothelial growth factor were the key factors that promoted hUCMSC proliferation. This study provides novel evidence for the role of BPS in the survival and rapid proliferation of hUCMSCs and suggests that these cells could be used for cell therapy-based clinical applications for treating severe burns. Furthermore, hUCMSC proliferation was

  8. Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.

    Science.gov (United States)

    Olszewska-Pazdrak, Barbara; Hein, Travis W; Olszewska, Paulina; Carney, Darrell H

    2009-05-01

    Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

  9. The endothelial border to health

    DEFF Research Database (Denmark)

    Hansen, Nina Wærling; Hansen, Anker Jon; Sams, Anette

    2017-01-01

    by hyperglycemic events because the endothelium transduces “high glucose” signaling into significant pathophysiological phenomena leading to reduced endothelial barrier function, compromised vascular tone regulation and inflammation (e.g., cytokine secretion and RAGE activation). In addition, endothelial...

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

    Science.gov (United States)

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

    2015-06-01

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

  11. Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes.

    Science.gov (United States)

    Edwards, Sarah; Lalor, Patricia F; Nash, Gerard B; Rainger, G Ed; Adams, David H

    2005-03-01

    Crosstalk between hepatic sinusoidal ECs and closely juxtaposed hepatocytes via vascular endothelial growth factor is essential for the maintenance of sinusoidal endothelial growth and differentiation. We propose that paracrine interactions between endothelial cells and hepatocytes also may be responsible for the unique complement of adhesion receptors expressed on sinusoidal endothelium that regulate the recruitment of lymphocytes into the liver. To address this hypothesis, we developed an in vitro model of the hepatic sinusoid in which flowing lymphocytes could interact with hepatic endothelium conditioned by the presence of hepatocytes. Human hepatic sinusoidal endothelial cells cocultured with hepatocytes were activated so that they supported the adhesion of lymphocytes at levels equivalent to those seen on endothelium stimulated with the inflammatory cytokine tumour necrosis factor-beta. Lymphocyte adhesion was supported by intracellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin, with an additional contribution from the novel adhesion receptor VAP-1. In conclusion, we show that interactions between hepatocytes and endothelial cells amplify leukocyte recruitment through the sinusoids by regulating the expression and function of endothelial adhesion molecules. These paracrine interactions may be responsible for the induction of the adhesion molecules that support constitutive lymphocyte recruitment to the liver as well as contributing significantly to the patterns of leukocyte adhesion seen during episodes of hepatic inflammation.

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

    Directory of Open Access Journals (Sweden)

    Shanshan W Howland

    2015-06-01

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

  13. Activated Brain Endothelial Cells Cross-Present Malaria Antigen

    Science.gov (United States)

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

    2015-01-01

    In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8+ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8+ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention. PMID:26046849

  14. Mechanisms of Endothelial Dysfunction in Hypertensive Pregnancy and Preeclampsia

    Science.gov (United States)

    Possomato-Vieira, José S.; Khalil, Raouf A.

    2016-01-01

    Preeclampsia is a pregnancy-related disorder characterized by hypertension, and could lead to maternal and fetal morbidity and mortality. Although the causative factors and pathophysiological mechanisms are unclear, endothelial dysfunction is a major hallmark of preeclampsia. Clinical tests and experimental research have suggested that generalized endotheliosis in the systemic, renal, cerebral and hepatic circulation could decrease endothelium-derived vasodilators such as nitric oxide, prostacyclin and hyperpolarization factor and increase vasoconstrictors such as endothelin-1 and thromboxane A2, leading to increased vasoconstriction, hypertension and other manifestation of preeclampsia. In search for the upstream mechanisms that could cause endothelial dysfunction, certain genetic, demographic and environmental risk factors have been suggested to cause abnormal expression of uteroplacental integrins, cytokines and matrix metalloproteinases, leading to decreased maternal tolerance, apoptosis of invasive trophoblast cells, inadequate spiral arteries remodeling, reduced uterine perfusion pressure (RUPP), and placental ischemia/hypoxia. RUPP may cause imbalance between the anti-angiogenic factors soluble fms-like tyrosine kinase-1 and soluble endoglin and the pro-angiogenic factors vascular endothelial growth factor and placental growth factor, or stimulate the release of other circulating bioactive factors such as inflammatory cytokines, hypoxia-inducible factor-1, reactive oxygen species, and angiotensin AT1 receptor agonistic autoantibodies. These circulating factors could then target endothelial cells and cause generalized endothelial dysfunction. Therapeutic options are currently limited, but understanding the factors involved in endothelial dysfunction could help design new approaches for prediction and management of preeclampsia. PMID:27451103

  15. Nanomedicines for Endothelial Disorders

    OpenAIRE

    Chung, Bomy Lee; Toth, Michael J.; Kamaly, Nazila; Sei, Yoshitaka J.; Becraft, Jacob; Mulder, Willem J. M.; Fayad, Zahi A; Farokhzad, Omid C.; Kim, YongTae; Langer, Robert

    2015-01-01

    The endothelium lines the internal surfaces of blood and lymphatic vessels and has a critical role in maintaining homeostasis. Endothelial dysfunction is involved in the pathology of many diseases and conditions, including disorders such as diabetes, cardiovascular diseases, and cancer. Given this common etiology in a range of diseases, medicines targeting an impaired endothelium can strengthen the arsenal of therapeutics. Nanomedicine – the application of nanotechnology to healthcare – prese...

  16. Construction of extracellular microenvironment to improve surface endothelialization of NiTi alloy substrate

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Peng, E-mail: liupeng79@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200433 (China); Zhao, Yongchun; Yan, Ying; Hu, Yan; Yang, Weihu [Key Laboratory of Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Cai, Kaiyong, E-mail: kaiyong_cai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology of Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2015-10-01

    To mimic extracellular microenvironment of endothelial cell, a bioactive multilayered structure of gelatin/chitosan pair, embedding with vascular endothelial growth factor (VEGF), was constructed onto NiTi alloy substrate surface via a layer-by-layer assembly technique. The successful fabrication of the multilayered structure was demonstrated by scanning electron microscopy, atomic force microscopy, contact angle measurement, attenuated total reflection-fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The growth behaviors of endothelial cells on various NiTi alloy substrates were investigated in vitro. Cytoskeleton observation, MTT assay, and wound healing assay proved that the VEGF-embedded multilayer structure positively stimulated adhesion, proliferation and motogenic responses of endothelial cells. More importantly, the present system promoted the nitric oxide production of endothelial cells. The approach affords an alternative to construct extracellular microenvironment for improving surface endothelialization of a cardiovascular implant. - Highlights: • Biofunctional multilayer films mimicking extracellular microenvironment were successfully fabricated. • Multilayered structure stimulated the biological responses of endothelial cells. • The approach affords an efficient approach for surface endothelialization of stent implant.

  17. Proinflammatory mediators stimulate neutrophil-directed angiogenesis.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Vascular endothelial growth factor (VEGF; vascular permeability factor) is one of the most potent proangiogenic cytokines, and it plays a central role in mediating the process of angiogenesis or new blood vessel formation. Neutrophils (PMNs) recently have been shown to produce VEGF. HYPOTHESIS: The acute inflammatory response is a potent stimulus for PMN-directed angiogenesis. METHODS: Neutrophils were isolated from healthy volunteers and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and anti-human Fas monoclonal antibody. Culture supernatants were assayed for VEGF using enzyme-linked immunosorbent assays. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs were then added to human umbilical vein endothelial cells and human microvessel endothelial cells and assessed for endothelial cell proliferation using 5-bromodeoxyuridine labeling. Tubule formation was also assessed on MATRIGEL basement membrane matrix. Neutrophils were lysed to measure total VEGF release, and VEGF expression was detected using Western blot analysis. RESULTS: Lipopolysaccharide and TNF-alpha stimulation resulted in significantly increased release of PMN VEGF (532+\\/-49 and 484+\\/-80 pg\\/mL, respectively; for all, presented as mean +\\/- SEM) compared with control experiments (32+\\/-4 pg\\/mL). Interleukin 6 and Fas had no effect. Culture supernatants from LPS- and TNF-alpha-stimulated PMNs also resulted in significant increases (P<.005) in macrovascular and microvascular endothelial cell proliferation and tubule formation. Adding anti-human VEGF-neutralizing polyclonal antibody to stimulated PMN supernatant inhibited these effects. Total VEGF release following cell lysis and Western blot analysis suggests that the VEGF is released from an intracellular store. CONCLUSION: Activated human PMNs are directly angiogenic by releasing VEGF, and this has important implications for inflammation, capillary leak syndrome

  18. Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes.

    Science.gov (United States)

    Zapolska-Downar, Danuta; Zapolski-Downar, Andrzej; Naruszewicz, Marek; Siennicka, Aldona; Krasnodebska, Barbara; Kołdziej, Blanka

    2002-11-01

    It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFalpha and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2', 7'-dichlorofluorescin (DCFH) to 2', 7'-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (partichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes.

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

    Directory of Open Access Journals (Sweden)

    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  20. Effects of Parietaria judaica pollen extract on human microvascular endothelial cells.

    Science.gov (United States)

    Taverna, Simona; Flugy, Anna; Colomba, Paolo; Barranca, Marilisa; De Leo, Giacomo; Alessandro, Riccardo

    2008-08-08

    Pollinosis from Parietaria judaica is one of the main causes of allergy in the Mediterranean area. The present study is designed to assess if P. judaica pollens contain bioactive compounds able to elicit a functional response in endothelial cells. We have demonstrated that addition of pollen extract to human lung microvascular endothelial cells (HMVEC-L) induces a modification of cell morphology, actin cytoskeletal rearrangements and an increase in endothelial cell permeability. We further showed that the treatment of endothelial cells with pollen extract causes an increase of E-selectin and VCAM-1 protein levels as well as an increase of IL-8 production. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of polymorphonuclear cells (PMNs) to HMVEC-L monolayer. Our results suggest for the first time that pollen affect directly endothelial cells (EC) modulating critical functions related to the inflammatory response.

  1. Reversibility of endothelial dysfunction in diabetes: role of polyphenols.

    Science.gov (United States)

    Suganya, N; Bhakkiyalakshmi, E; Sarada, D V L; Ramkumar, K M

    2016-07-01

    The endothelium, a thin single sheet of endothelial cells, is a metabolically active layer that coats the inner surface of blood vessels and acts as an interface between the circulating blood and the vessel wall. The endothelium through the secretion of vasodilators and vasoconstrictors serves as a critical mediator of vascular homeostasis. During the development of the vascular system, it regulates cellular adhesion and vessel wall inflammation in addition to maintaining vasculogenesis and angiogenesis. A shift in the functions of the endothelium towards vasoconstriction, proinflammatory and prothrombic states characterise improper functioning of these cells, leading to endothelial dysfunction (ED), implicated in the pathogenesis of many diseases including diabetes. Major mechanisms of ED include the down-regulation of endothelial nitric oxide synthase levels, differential expression of vascular endothelial growth factor, endoplasmic reticulum stress, inflammatory pathways and oxidative stress. ED tends to be the initial event in macrovascular complications such as coronary artery disease, peripheral arterial disease, stroke and microvascular complications such as nephropathy, neuropathy and retinopathy. Numerous strategies have been developed to protect endothelial cells against various stimuli, of which the role of polyphenolic compounds in modulating the differentially regulated pathways and thus maintaining vascular homeostasis has been proven to be beneficial. This review addresses the factors stimulating ED in diabetes and the molecular mechanisms of natural polyphenol antioxidants in maintaining vascular homeostasis.

  2. Infection with Porphyromonas gingivalis exacerbates endothelial injury in obese mice.

    Directory of Open Access Journals (Sweden)

    Min Ao

    Full Text Available BACKGROUND: A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg, on pathogenesis of atherosclerosis in obesity. METHODS: In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD or normal chow diet (CD, as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1 were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA induced endothelial cells apoptosis and regulation of cytokine gene expression. RESULTS: Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. CONCLUSIONS: Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.

  3. Endothelial lipase is a major determinant of HDL level

    Energy Technology Data Exchange (ETDEWEB)

    Ishida, Tatsuro; Choi, Sungshin; Kundu, Ramendra K.; Hirata, Ken-Ichi; Rubin, Edward M.; Cooper, Allen D.; Quertermous, Thomas

    2003-01-30

    For the past three decades, epidemiologic studies have consistently demonstrated an inverse relationship between plasma HDL cholesterol (HDL-C) concentrations and coronary heart disease (CHD). Population-based studies have provided compelling evidence that low HDL-C levels are a risk factor for CHD, and several clinical interventions that increased plasma levels of HDL-C were associated with a reduction in CHD risk. These findings have stimulated extensive investigation into the determinants of plasma HDL-C levels. Turnover studies using radiolabeled apolipoprotein A-I, the major protein component of HDL, suggest that plasma HDL-C concentrations are highly correlated with the rate of clearance of apolipoprotein AI. However, the metabolic mechanisms by which HDL are catabolized have not been fully defined. Previous studies in humans with genetic deficiency of cholesteryl ester transfer protein, and in mice lacking the scavenger receptor BI (SR-BI), have demonstrated that these proteins participate in the removal of cholesterol from HDL, while observations in individuals with mutations in hepatic lipase indicate that this enzyme hydrolyzes HDL triglycerides. In this issue of the JCI, reports from laboratories of Tom Quertermous and Dan Rader now indicate that endothelial lipase (LIPG), a newly identified member of the lipase family, catalyzes the hydrolysis of HDL phospholipids and facilitates the clearance of HDL from the circulation. Endothelial lipase was initially cloned by both of these laboratories using entirely different strategies. Quertermous and his colleagues identified endothelial lipase as a transcript that was upregulated in cultured human umbilical vein endothelial cells undergoing tube formation, whereas the Rader group cloned endothelial lipase as a transcript that was upregulated in the human macrophage-like cell line THP-1 exposed to oxidized LDL. Database searches revealed that endothelial lipase shows strong sequence similarity to lipoprotein

  4. Interaction of human endothelial cells and nickel-titanium materials modified with silicon ions

    Energy Technology Data Exchange (ETDEWEB)

    Lotkov, Aleksandr I., E-mail: lotkov@ispms.tsc.ru; Kashin, Oleg A., E-mail: okashin@ispms.tsc.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); Kudryavtseva, Yuliya A., E-mail: yulia-k1970@mail.ru; Antonova, Larisa V., E-mail: antonova.la@mail.ru; Matveeva, Vera G., E-mail: matveeva-vg@mail.ru; Sergeeva, Evgeniya A., E-mail: sergeewa.ew@yandex.ru [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation); Kudryashov, Andrey N., E-mail: kudryashov@angioline.ru [Angioline Interventional Device Ltd, Novosibirsk, 630090 (Russian Federation)

    2015-10-27

    The paper studies the influence of chemical and phase compositions of NiTi surface layers modified with Si ions by plasma immersion implantation on their interaction with endothelial cells. It is shown that certain technological modes of Si ion implantation enhance the adhesion, proliferation, and viability of endothelial cells. It is found that the Si-modified NiTi surface is capable of stimulating the formation of capillary-like structures in the cell culture.

  5. Vascular endothelial growth factor in children with cyanotic and acyanotic and congenital heart disease

    OpenAIRE

    Baghdady, Yasser; Hussein, Yasser; Shehata, Mohamed

    2010-01-01

    Introduction Vascular endothelial growth factor is a potent stimulator of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis resulting in increased morbidity and mortality. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor compared t...

  6. Fibroblast growth factor signaling pathway in endothelial cells is activated by BMPER to promote angiogenesis.

    Science.gov (United States)

    Esser, Jennifer S; Rahner, Susanne; Deckler, Meike; Bode, Christoph; Patterson, Cam; Moser, Martin

    2015-02-01

    Previously, we have identified bone morphogenetic protein endothelial cell precursor-derived regulator (BMPER) to increase the angiogenic activity of endothelial cells in a concentration-dependent manner. In this project, we now investigate how BMPER acts in concert with key molecules of angiogenesis to promote blood vessel formation. To assess the effect of BMPER on angiogenesis-related signaling pathways, we performed an angiogenesis antibody array with BMPER-stimulated endothelial cells. We detected increased basic fibroblast growth factor (bFGF/FGF-2) expression after BMPER stimulation and decreased expression of thrombospondin-1. Additionally, FGF receptor-1 expression, phosphorylation, FGF signaling pathway activity, and cell survival were increased. Consistently, silencing of BMPER by small interfering RNA decreased bFGF and FGF receptor-1 expression and increased thrombospondin-1 expression and cell apoptosis. Next, we investigated the interaction of BMPER and the FGF signaling pathway in endothelial cell function. BMPER stimulation increased endothelial cell angiogenic activity in migration, Matrigel, and spheroid assays. To block FGF signaling, an anti-bFGF antibody was used, which effectively inhibited the proangiogenic BMPER effect. Accordingly, BMPER-silenced endothelial cells under bFGF stimulation showed decreased angiogenic activity compared with bFGF control. We confirmed these findings in vivo by subcutaneous Matrigel injections with and without bFGF in C57BL/6_Bmper(+/-) mice. Aortic ring assays of C57BL/6_Bmper(+/-) mice confirmed a specific effect for bFGF but not for vascular endothelial growth factor. Taken together, the proangiogenic BMPER effect in endothelial cells is mediated by inhibition of antiangiogenic thrombospondin-1 and enhanced expression and activation of the FGF signaling pathway that is crucial in the promotion of angiogenesis. © 2014 American Heart Association, Inc.

  7. [Medical significance of endothelial glycocalyx].

    Science.gov (United States)

    Frati-Munari, Alberto C

    2013-01-01

    Endothelial glycocalyx is a layer composed by glycosaminoglycans, proteoglycans and glycoproteins attached to the vascular endothelial luminal surface. It has several physiological roles: shear stress mechanotransduction to the endothelial cells, regulation of fluids and macromolecules vascular permeability, of coagulation cascade activation and fibrinolysis, and protects the endothelium from platelets and leukocytes adhesion. In general, glycocalyx protects vascular wall against pathogenic insults. The glycocalyx may be damaged by abnormal shear stress, reactive oxygen species, hypernatremia, hyperglycemia, hypercholesterolemia and inflammatory molecules, resulting in endothelial dysfunction, enhanced vascular permeability, lipoproteins leakage to subendothelial space, activation of plasma coagulation, and increased adherence of platelets and leukocytes to the endothelial cells. Shredding of glycocalyx appears as an important initial step in the pathophysiology of vascular diseases. Copyright © 2012 Instituto Nacional de Cardiología Ignacio Chávez. Published by Masson Doyma México S.A. All rights reserved.

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

    Science.gov (United States)

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

    2016-04-01

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

  9. Histamine Induces Vascular Hyperpermeability by Increasing Blood Flow and Endothelial Barrier Disruption In Vivo

    Science.gov (United States)

    Ashina, Kohei; Tsubosaka, Yoshiki; Nakamura, Tatsuro; Omori, Keisuke; Kobayashi, Koji; Hori, Masatoshi; Ozaki, Hiroshi; Murata, Takahisa

    2015-01-01

    Histamine is a mediator of allergic inflammation released mainly from mast cells. Although histamine strongly increases vascular permeability, its precise mechanism under in vivo situation remains unknown. We here attempted to reveal how histamine induces vascular hyperpermeability focusing on the key regulators of vascular permeability, blood flow and endothelial barrier. Degranulation of mast cells by antigen-stimulation or histamine treatment induced vascular hyperpermeability and tissue swelling in mouse ears. These were abolished by histamine H1 receptor antagonism. Intravital imaging showed that histamine dilated vasculature, increased blood flow, while it induced hyperpermeability in venula. Whole-mount staining showed that histamine disrupted endothelial barrier formation of venula indicated by changes in vascular endothelial cadherin (VE-cadherin) localization at endothelial cell junction. Inhibition of nitric oxide synthesis (NOS) by L-NAME or vasoconstriction by phenylephrine strongly inhibited the histamine-induced blood flow increase and hyperpermeability without changing the VE-cadherin localization. In vitro, measurements of trans-endothelial electrical resistance of human dermal microvascular endothelial cells (HDMECs) showed that histamine disrupted endothelial barrier. Inhibition of protein kinase C (PKC) or Rho-associated protein kinase (ROCK), NOS attenuated the histamine-induced barrier disruption. These observations suggested that histamine increases vascular permeability mainly by nitric oxide (NO)-dependent vascular dilation and subsequent blood flow increase and maybe partially by PKC/ROCK/NO-dependent endothelial barrier disruption. PMID:26158531

  10. Endothelial plasticity governs the site-specific leukocyte recruitment in hepatocellular cancer.

    Science.gov (United States)

    Salnikova, Olga; Breuhahn, Kai; Hartmann, Natalie; Schmidt, Jan; Ryschich, Eduard

    2013-11-15

    The correct programming of the endothelial cell phenotype is crucial for efficient leukocyte recruitment to tumor tissue. It has been previously described that T cells infiltrated hepatocellular cancer (HCC) tissue mainly in peritumoral, stromal and tumor border areas. In the current study, phenotype features of tumor endothelial cells and their potential impact on leukocyte recruitment were analyzed in murine tissue of HCC. In the murine model, proinflammatory stimulation with IL-1β induced leukocyte recruitment in the blood vessels of peripheral tumor areas and in nonmalignant liver tissue, but not in deeper tumor blood vessels. Furthermore, peripheral tumor endothelium, but not deeper tumor blood vessels exhibited a "normalized" hepatic sinusoidal endothelial cell (HSEC)-like phenotype with regard to the expression of adhesion molecules and liver sinusoidal endothelial markers. When tumor endothelial cells were isolated and incubated in vitro, their phenotype rapidly changed and became almost identical to normal hepatic endothelial cells. Interestingly, cytokine production in HCC was strongly dysregulated as compared to normal liver, with IL-1RN exhibiting the most prominent elevation. Experiments with isolated hepatic endothelial cells showed that IL-1RN effectively antagonized the activating action of IL-1β on the expression of adhesion molecules and T cell attachment. These novel insights indicate that tumor endothelium of HCC represents a plastic system that is susceptible to microenvironmental changes. The peritumoral and tumor border areas have distinct endothelial cell phenotype, which promotes leukocyte recruitment to HCC tissue. Copyright © 2013 UICC.

  11. Expression of an insulin-regulatable glucose carrier in muscle and fat endothelial cells

    Science.gov (United States)

    Vilaró, Senen; Palacín, Manuel; Pilch, Paul F.; Testar, Xavier; Zorzano, Antonio

    1989-12-01

    INSULIN rapidly stimulates glucose use in the major target tissues, muscle and fat, by modulating a tissue-specific glucose transporter isoform1-6. Access of glucose to the target tissue is restricted by endothelial cells which line the walls of nonfenestrated capillaries of fat and muscle7. Thus, we examined whether the capillary endothelial cells are actively involved in the modulation of glucose availability by these tissues. We report here the abundant expression of the muscle/fat glucose transporter isoform in endothelial cells, using an immunocytochemical analysis with a monoclonal antibody specific for this isoform1. This expression is restricted to endothelial cells from the major insulin target tissues, and it is not detected in brain and liver where insulin does not activate glucose transport. The expression of the muscle/fat transporter isoform in endothelial cells is significantly greater than in the neighbouring muscle and fat cells. Following administration of insulin to animals in vivo, there occurs a rapid increase in the number of muscle/fat transporters present in the lumenal plasma membrane of the capillary endothelial cells. These results document that insulin promotes the translocation of the muscle/fat glucose transporter in endothelial cells. It is therefore likely that endothelial cells play an important role in the regulation of glucose use by the major insulin target tissues in normal and diseased states.

  12. Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells.

    Science.gov (United States)

    Unger, R E; Peters, K; Wolf, M; Motta, A; Migliaresi, C; Kirkpatrick, C J

    2004-09-01

    We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps between individual fibers. Higher attachment and growth coverage was obtained if nets were first coated with gelatin, fibronectin or collagen type I. Proinflammatory markers of endothelial cells on the fibers exhibited a non-activated state and LPS-stimulated cells exhibited activation of these markers. Furthermore, a typical PECAM-1 localization at cell-cell contacts was observed. Scanning electron microscopic examination of fibroin nets after removal of cells did not demonstrate any changes to the fibroin structure. HUVEC and HDMEC on fibroin nets embedded in collagen type I gels formed microvessel-like structures. Thus, silk fibroin nets are a highly endothelial cell-compatible scaffolding material that support the growth, normal and inducible cell functions and angiogenesis potential of human endothelial cells in vitro similar to that observed in vivo.

  13. Chlorine gas exposure causes systemic endothelial dysfunction by inhibiting endothelial nitric oxide synthase-dependent signaling.

    Science.gov (United States)

    Honavar, Jaideep; Samal, Andrey A; Bradley, Kelley M; Brandon, Angela; Balanay, Joann; Squadrito, Giuseppe L; MohanKumar, Krishnan; Maheshwari, Akhil; Postlethwait, Edward M; Matalon, Sadis; Patel, Rakesh P

    2011-08-01

    Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

  14. Chlorine Gas Exposure Causes Systemic Endothelial Dysfunction by Inhibiting Endothelial Nitric Oxide Synthase–Dependent Signaling

    Science.gov (United States)

    Honavar, Jaideep; Samal, Andrey A.; Bradley, Kelley M.; Brandon, Angela; Balanay, Joann; Squadrito, Giuseppe L.; MohanKumar, Krishnan; Maheshwari, Akhil; Postlethwait, Edward M.; Matalon, Sadis; Patel, Rakesh P.

    2011-01-01

    Chlorine gas (Cl2) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl2 exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl2 promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl2 for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl2 dose (0–400 ppm) and time after exposure (0–48 h) were determined. Exposure to Cl2 (250–400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl2–exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl2 exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl2–exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl2 exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms. PMID:21131444

  15. growth stimulant

    African Journals Online (AJOL)

    Effects of timing and duration of supplementation of LIVFIT VET ® (growth stimulant) as substitute for fish meal on the growth performance, haematology and clinical enzymes concentration of growing pigs.

  16. SIRT1 regulates endothelial Notch signaling in lung cancer.

    Directory of Open Access Journals (Sweden)

    Mian Xie

    Full Text Available BACKGROUND: Sirtuin 1 (SIRT1 acts as a key regulator of vascular endothelial homeostasis, angiogenesis, and endothelial dysfunction. However, the underlying mechanism for SIRT1-mediated lung carcinoma angiogenesis remains unknown. Herein, we report that the nicotinamide adenine dinucleotide 1 (NAD1-dependent deacetylase SIRT1 can function as an intrinsic negative modulator of Delta-like ligand 4 (DLL4/Notch signaling in Lewis lung carcinoma (LLC xenograft-derived vascular endothelial cells (lung cancer-derived ECs. PRINCIPAL FINDINGS: SIRT1 negatively regulates Notch1 intracellular domain (N1IC and Notch1 target genes HEY1 and HEY2 in response to Delta-like ligand 4 (DLL4 stimulation. Furthermore, SIRT1 deacetylated and repressed N1IC expression. Quantitative chromatin immunoprecipitation (qChIP analysis and gene reporter assay demonstrated that SIRT1 bound to one highly conserved region, which was located at approximately -500 bp upstream of the transcriptional start site of Notch1,and repressed Notch1 transcription. Inhibition of endothelial cell growth and sprouting angiogenesis by DLL4/Notch signaling was enhanced in SIRT1-silenced lung cancer-derived EC and rescued by Notch inhibitor DAPT. In vivo, an increase in proangiogenic activity was observed in Matrigel plugs from endothelial-specific SIRT1 knock-in mice. SIRT1 also enhanced tumor neovascularization and tumor growth of LLC xenografts. CONCLUSIONS: Our results show that SIRT1 facilitates endothelial cell branching and proliferation to increase vessel density and promote lung tumor growth through down-regulation of DLL4/Notch signaling and deacetylation of N1IC. Thus, targeting SIRT1 activity or/and gene expression may represent a novel mechanism in the treatment of lung cancer.

  17. Lifestyle factors and endothelial function.

    Science.gov (United States)

    Papageorgiou, Nikolaos; Tousoulis, Dimitris; Androulakis, Emmanuel; Giotakis, Aris; Siasos, Gerasimos; Latsios, George; Stefanadis, Christodoulos

    2012-01-01

    Atherosclerotic disease remains a major health problem around the world. The central role of endothelium and inflammation in all stages of the atherosclerotic process is advocated by significant data. Moreover, clinical evidence supports the prognostic potential of endothelial dysfunction for the development of ischemic events and for adverse outcome after acute coronary syndromes. Interestingly, suboptimal lifestyle choices are implicated in the development and deterioration of this endothelial dysfunction, a fact with significant impact, considering the contribution of endothelial dysfunction in atherosclerosis and its complications. Many epidemiological research studies, using a variety of strategies, provide encouraging evidence suggesting that lifestyle modifications may have significant impact regarding the improvement of endothelial function. However, little is known about how individual's genetic background interacts with environmental influences on vascular health, thereby making the interpretation of the relative importance of lifestyle interventions more complicated.

  18. Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro

    NARCIS (Netherlands)

    Bakker, A.D.; Huesa, C.; Hughes, A.; Aspden, R.M.; van 't Hof, R.J.; Klein-Nulend, J.; Helfrich, M.H.

    2013-01-01

    Endothelial nitric oxide synthase (eNOS) has long been held responsible for NO production by mechanically stimulated osteoblasts, but this has recently been disputed. We investigated whether one of the three known NOS isoforms is essential for NO production by mechanically stimulated osteoblasts in

  19. Store-operated calcium entry and increased endothelial cell permeability.

    Science.gov (United States)

    Norwood, N; Moore, T M; Dean, D A; Bhattacharjee, R; Li, M; Stevens, T

    2000-11-01

    We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.

  20. Epithelial: Endothelial cross-talk regulates exocrine differentiation in developing pancreas.

    Science.gov (United States)

    Pierreux, Christophe E; Cordi, Sabine; Hick, Anne-Christine; Achouri, Younes; Ruiz de Almodovar, Carmen; Prévot, Pierre-Paul; Courtoy, Pierre J; Carmeliet, Peter; Lemaigre, Frédéric P

    2010-11-01

    Endothelial cells are required to initiate pancreas development from the endoderm. They also control the function of endocrine islets after birth. Here we investigate in developing pancreas how the endothelial cells become organized during branching morphogenesis and how their development affects pancreatic cell differentiation. We show that endothelial cells closely surround the epithelial bud at the onset of pancreas morphogenesis. During branching morphogenesis, the endothelial cells become preferentially located near the central (trunk) epithelial cells and remain at a distance from the branch tips where acinar cells differentiate. This correlates with predominant expression of the angiogenic factor vascular endothelial growth factor-A (VEGF-A) in trunk cells. In vivo ablation of VEGF-A expression by pancreas-specific inactivation of floxed Vegfa alleles results in reduced endothelial development and in excessive acinar differentiation. On the contrary, acinar differentiation is repressed when endothelial cells are recruited around tip cells that overexpress VEGF-A. Treatment of embryonic day 12.5 explants with VEGF-A or with VEGF receptor antagonists confirms that acinar development is tightly controlled by endothelial cells. We also provide evidence that endothelial cells repress the expression of Ptf1a, a transcription factor essential for acinar differentiation, and stimulate the expression of Hey-1 and Hey-2, two repressors of Ptf1a activity. In explants, we provide evidence that VEGF-A signaling is required, but not sufficient, to induce endocrine differentiation. In conclusion, our data suggest that, in developing pancreas, epithelial production of VEGF-A determines the spatial organization of endothelial cells which, in turn, limit acinar differentiation of the epithelium. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Endothelial-regenerating cells: an expanding universe.

    Science.gov (United States)

    Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

    2010-03-01

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

  2. Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

    Directory of Open Access Journals (Sweden)

    Gina A. Smith

    2017-10-01

    Full Text Available Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A and vascular endothelial growth factor receptor 2 (VEGFR2 regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response.

  3. Forkhead box O-1 modulation improves endothelial insulin resistance in human obesity.

    Science.gov (United States)

    Karki, Shakun; Farb, Melissa G; Ngo, Doan T M; Myers, Samantha; Puri, Vishwajeet; Hamburg, Naomi M; Carmine, Brian; Hess, Donald T; Gokce, Noyan

    2015-06-01

    Increased visceral adiposity has been closely linked to insulin resistance, endothelial dysfunction, and cardiometabolic disease in obesity, but pathophysiological mechanisms are poorly understood. We sought to investigate mechanisms of vascular insulin resistance by characterizing depot-specific insulin responses and gain evidence that altered functionality of transcription factor forkhead box O-1 (FOXO-1) may play an important role in obesity-related endothelial dysfunction. We intraoperatively collected paired subcutaneous and visceral adipose tissue samples from 56 severely obese (body mass index, 43 ± 7 kg/m(2)) and 14 nonobese subjects during planned surgical operations, and characterized depot-specific insulin-mediated responses using Western blot and quantitative immunofluorescence techniques. Insulin signaling via phosphorylation of FOXO-1 and consequent endothelial nitric oxide synthase stimulation was selectively impaired in the visceral compared with subcutaneous adipose tissue and endothelial cells of obese subjects. In contrast, tissue actions of insulin were preserved in nonobese individuals. Pharmacological antagonism with AS1842856 and biological silencing using small interfering RNA-mediated FOXO-1 knockdown reversed insulin resistance and restored endothelial nitric oxide synthase activation in the obese. We observed profound endothelial insulin resistance in the visceral adipose tissue of obese humans which improved with FOXO-1 inhibition. FOXO-1 modulation may represent a novel therapeutic target to diminish vascular insulin resistance. In addition, characterization of endothelial insulin resistance in the adipose microenvironment may provide clues to mechanisms of systemic disease in human obesity. © 2015 American Heart Association, Inc.

  4. Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

    Science.gov (United States)

    Medici, Damian; Olsen, Bjorn R.

    2012-01-01

    Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas. PMID:22900063

  5. Rapamycin inhibits proliferation of hemangioma endothelial cells by reducing HIF-1-dependent expression of VEGF.

    Directory of Open Access Journals (Sweden)

    Damian Medici

    Full Text Available Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF signaling through VEGF receptor 2 (VEGFR2. Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1. VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.

  6. Endothelial cells, fibroblasts and vasculitis.

    Science.gov (United States)

    Buckley, Christopher D; Rainger, G Ed; Nash, Gerard B; Raza, Karim

    2005-07-01

    One of the most important questions in vasculitis research is not why inflammation of blood vessels occurs but why it persists, often in a site-specific manner. In this review we illustrate how stromal cells, such as fibroblasts and pericytes, might play an important role in regulating the site at which vasculitis occurs. Smooth muscle cells and fibroblasts directly influence the behaviour of overlying vascular cells, amplifying the response of the endothelium to proinflammatory agents such as TNF-alpha and allowing enhanced and inappropriate leucocyte recruitment. An abnormal local vascular stromal environment can therefore influence local endothelial function and drive the persistence of local vascular inflammation. However, such local vascular inflammation can have distant effects on the systemic vascular system, leading to widespread endothelial cell dysfunction. Vascular endothelial dysfunction is common in a range of immune-mediated inflammatory diseases, is seen in multiple vascular beds, and is reversible following the induction of disease remission. The mechanisms that drive such systemic vascular endothelial dysfunction are unclear but factors such as TNF-alpha and CRP may play a role. Persistence of such widespread endothelial dysfunction in systemic vasculitis appears to have long-term consequences, leading to the acceleration of atherosclerosis and premature ischaemic heart disease. It may also underlie the accelerated atherosclerosis seen in other immune-mediated rheumatic diseases, such as rheumatoid arthritis.

  7. The role of vascular endothelial growth factor in inflammatory processes

    Directory of Open Access Journals (Sweden)

    Ewa Koczy-Baron

    2014-01-01

    Full Text Available The vascular endothelial growth factor (VEGF is produced by different types of cells and has a major role in both, physiological and pathological angiogenesis. On the one hand VEGF is a strong mitotic and chemotactic factor for the endothelial cells, stimulating thus formation of new vessels, while on the other, it enhances the vascular endothelium permeability of the existing blood vessels which contributes to development and persistence of the inflammatory conditions. In the latter its activity is by 50 000 times higher than that of histamine. VEGF facilitates formation of oedema and leukocyte migration from the circulation to the site of inflammation. VEGF is also important in remodeling of the extracellular matrix. Moreover, it has an important significance in regulation of the immunological response, therefore plays a role in autoaggressive phenomena as well as immediate- and delayed-type hypersensitivity. Its role in the pathogenesis of immunological and inflammatory diseases, including allergy, asthma and different skin disorders has been indicated.

  8. Brain Stimulation Therapies

    Science.gov (United States)

    ... Magnetic Seizure Therapy Deep Brain Stimulation Additional Resources Brain Stimulation Therapies Overview Brain stimulation therapies can play ... for a shorter recovery time than ECT Deep Brain Stimulation Deep brain stimulation (DBS) was first developed ...

  9. Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

    NARCIS (Netherlands)

    Wiersma, Harmen; Gatti, Alberto; Nijstad, Niels; Kuipers, Folkert; Tietge, Uwe J. F.

    High density lipoprotein cholesterol is thought to represent a preferred source of sterols secreted into bile following hepatic uptake by scavenger receptor class B type I (SR-BI). The present study aimed to determine the metabolic effects of an endothelial lipase (EL)-mediated stimulation of HDL

  10. Some Phenolic Compounds Increase the Nitric Oxide Level in Endothelial Cells in Vitro

    NARCIS (Netherlands)

    Appeldoorn, M.M.; Venema, D.P.; Peters, T.H.F.; Koenen, M.E.; Arts, I.C.W.; Vincken, J.P.; Gruppen, H.; Keijer, J.; Hollman, P.C.H.

    2009-01-01

    The vasorelaxing properties of chocolate and wine might relate to the presence of phenolic compounds. One of the potential mechanisms involved is stimulation of endothelial nitric oxide (NO) production, as NO is a major regulator of vasodilatation. This study aimed to develop an in vitro assay using

  11. Some phenolic compounds increase the nitric oxide level in endothelial cells in vitro

    NARCIS (Netherlands)

    Appeldoorn, M.M.; Venema, D.P.; Peters, T.H.F.; Koenen, M.E.; Arts, I.C.W.; Vincken, J.-P.; Gruppen, H.; Keuer, J.; Hollman, P.C.H.

    2009-01-01

    The vasorelaxing properties of chocolate and wine might relate to the presence of phenolic compounds. One of the potential mechanisms involved is stimulation of endothelial nitric oxide (NO) production, as NO is a major regulator of vasodilatation. This study aimed to develop an in vitro assay using

  12. IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation

    DEFF Research Database (Denmark)

    Hammer, Troels; Tritsaris, Katerina; Hübschmann, Martin V

    2009-01-01

    IL-20 is an arteriogenic cytokine that remodels collateral networks in vivo, and plays a role in cellular organization. Here, we investigate its role in lymphangiogenesis using a lymphatic endothelial cell line, hTERT-HDLEC, which expresses the lymphatic markers LYVE-1 and podoplanin. Upon stimul...

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

    Science.gov (United States)

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

    2012-08-01

    The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Small GTP-Binding Protein Rac Is an Essential Mediator of Vascular Endothelial Growth Factor-Induced Endothelial Fenestrations and Vascular Permeability

    DEFF Research Database (Denmark)

    Eriksson, A.; Cao, R.; Tritsaris, K.

    2003-01-01

    ), endothelial nitric oxide synthase (eNOS), and extracellular regulated kinase (Erk1/2). We further found that phosphatidylinositol-3-OH kinase (PI3K) acted upstream of Rac and Akt-eNOS in VEGF/VEGFR-2 signaling. Conclusions- Our findings indicate that the small GTP-binding protein Rac is a key component...... and vascular permeability but only partially inhibited angiogenesis. Studies on endothelial cell cultures further revealed that VEGF stimulated phosphorylation of VEGF receptor-2 (VEGFR-2), leading to activation of Rac as well as increased phosphorylation of phospholipase C (PLC ), protein kinase B (Akt...

  15. Endothelial keratoplasty: evolution and horizons

    Directory of Open Access Journals (Sweden)

    Gustavo Teixeira Grottone

    2012-12-01

    Full Text Available Endothelial keratoplasty has been adopted by corneal surgeons worldwide as an alternative to penetrating keratoplasty (PK in the treatment of corneal endothelial disorders. Since the first surgeries in 1998, different surgical techniques have been used to replace the diseased endothelium. Compared with penetrating keratoplasty, all these techniques may provide faster and better visual rehabilitation with minimal change in refractive power of the transplanted cornea, minimal induced astigmatism, elimination of suture-induced complications and late wound dehiscence, and a reduced demand for postoperative care. Translational research involving cell-based therapy is the next step in work on endothelial keratoplasty. The present review updates information on comparisons among different techniques and predicts the direction of future treatment.

  16. Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

    Science.gov (United States)

    Parker, William H.; Qu, Zhi-chao; May, James M.

    2015-01-01

    Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729

  17. Selenium Inhibits Homocysteine-Induced Endothelial Dysfunction and Apoptosis via Activation of AKT

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    Hui Ren

    2016-02-01

    Full Text Available Background/Aims: Endothelial cells are crucial in vascular homeostasis. Dysfunction of endothelial cells is involved in the development of cardiovascular diseases (CVD. High plasma homocysteine (Hcy correlates with CVD while selenium supplementation counteracts development of CVD. However, the underlying mechanism remained unclear. Here, we investigated the effects of selenium on homocysteine-induced endothelial dysfunction. Methods: An animal model of Hcy-induced endothelial dysfunction was established by intragastric administration of L-methionine. Plasma NO and von Willebrand factor (vWF were quantified using NO assay and ELISA kit respectively. Relaxation was measured in thoracic aortic ring assays. Cell viability and migration were detected by Cell Counting Kit-8 and Bio-Coat cell migration chambers respectively. Cellular apoptosis was determined by Annexin V-FITC apoptosis kit. Results: Selenium prevented homocysteine-induced endothelial injury and impairment of endothelium-dependent relaxation. Selenium reversed the impaired viability and migration of endothelial cells induced by homocysteine in a dose-dependent manner. Selenium inhibited the apoptosis of endothelial cells induced by homocysteine, through downregulating of Caspase-3 activity and expression of Caspase-3 and Bax, and by stimulating Bcl-2 expression. Selenium reversed the homocysteine-induced reduction of NO release, and increased the expression and phosphoylation of endothelial nitric oxide synthetase (eNOS in a dose-dependent manner. Moreover, selenium enhanced AKT phosphorylation, and selenium-induced phosphorylation and expression of eNOS were inhibited by AKT inhibition. NO production, cell viability and migration rescued by selenium were inhibited, while cell apoptosis was reversed by AKT inhibition. Conclusion: Selenium protected against homocysteine-induced dysfunction and apoptosis of endothelial cells through AKT pathway. The observations may provide novel

  18. Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells.

    Science.gov (United States)

    LeClaire, R D; Kell, W M; Sadik, R A; Downs, M B; Parker, G W

    1995-01-01

    The effect of staphylococcal enterotoxin B (SEB)-elicited inducible nitric oxide synthase (iNOS) in mouse endothelial cells was investigated. Results showed that SEB stimulated the same level of NO production in gamma interferon (IFN-gamma)-primed cells as did trichloroacetic acid-extracted lipopolysaccharide. The kinetics of induced NO production and expression of mRNA for iNOS differed markedly in endothelial and macrophage cells. Induced endothelial nitrite production was transient and was 15 to 20% of that generated by macrophage cells; mRNA levels peaked by 2 h and then steadily declined, whereas macrophage message levels continually increased. The ability of endothelial cells to produce SEB-induced NO depended on priming with IFN-gamma, although detectable mRNA could be elicited by SEB alone. Induction of endothelial iNOS mRNA was inhibited by cycloheximide, which indicated a requirement for de novo protein synthesis. Niacinamide and interleukin-10 significantly reduced SEB-induced endothelial NO production. Both are reported to affect IFN-gamma-induced class II major histocompatibility complex (MHC) expression on antigen-presenting cells. Niacinamide reduced iNOS mRNA levels and markedly reduced IFN-gamma induction of endothelial class II MHC surface antigen. Interleukin-10 did not consistently reduce iNOS mRNA expression and had no effect on IFN-gamma induction of endothelial class II MHC surface antigen. These results suggest that SEB interacts with IFN-gamma-primed endothelial cells to elicit induced NO and that this induction can be effectively modulated at the receptor or transcriptional level. PMID:7529748

  19. The role of caveolin1 and sprouty1 in genistein's regulation of vascular smooth muscle cell and endothelial cell proliferation.

    Science.gov (United States)

    Xiang, QiuLing; Lin, GuiPing; Xu, JinWen; Zheng, ShuHui; Chen, SiJuan; Zhou, KeWen; Wang, TingHuai

    2010-12-01

    Genistein prevents atherosclerosis by exerting protective effects on blood vessels. The aim of this study is to investigate the role of caveolin1 and sprouty1 in the regulation of proliferation of vascular smooth muscle cell (VSMC) and endothelial cell by genistein. Using thiazolyl blue tetrazolium bromide(MTT) and [3H]-TdR assay, we found genistein inhibited angiotensin II-induced proliferation in primary cultured VSMC while it stimulated proliferation of quiescent endothelial cells. The effects were attenuated by caveolin1 or sprouty1 siRNA. Western blot analysis indicated that genistein attenuated the phosphorylation of extracellular regulated kinase1/2(ERK1/2) in angiotensin II-induced proliferated VSMC but stimulated the phosphorylation of ERK1/2 in quiescent endothelial cell. Double staining immunofluorescence identified caveolin1 and sprouty1 coexpressed in the cytoplasm of both VSMC and endothelial cell. Genistein increased the expression of caveolin1, p-caveolin1 and sprouty1 in VSMC, while it had opposite effects in quiescent endothelial cell. Co-immunoprecipitation suggested that genistein exerted its effects through interaction of caveolin1 and sprouty1. Our results demonstrate that the inhibition of angiotensin II-induced proliferation of VSMC and stimulation of quiescent endothelial cell by genistein are regulated by caveolin1 and sprouty1, which are implemented through Ras/MAPK pathway. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Calcification and endothelialization of thrombi in acute stroke.

    Science.gov (United States)

    Almekhlafi, Mohammed A; Hu, William Y; Hill, Michael D; Auer, Roland N

    2008-09-01

    We report chronic histopathological features in thrombi mechanically retrieved from five acute ischemic stroke patients with a median age of 68 years and a median pretreatment National Institutes of Health Stroke Scale score of 13. Early endothelialization occurred over and within the thrombus, and calcifications were seen, in addition to the usual acute laminar fibrin, intervening red blood cells, and neutrophils. The effectiveness of tissue plasminogen activator in clot dissolution might be affected by these features, if extensive. Thrombus composition could critically determine the success of chemical thrombolysis. Our results should stimulate the development of imaging modalities to determine thrombus composition.

  1. Role of endothelial cell metabolism in vessel sprouting.

    Science.gov (United States)

    De Bock, Katrien; Georgiadou, Maria; Carmeliet, Peter

    2013-11-05

    Endothelial cells (ECs) are quiescent for years but can plastically switch to angiogenesis. Vascular sprouting relies on the coordinated activity of migrating tip cells at the forefront and proliferating stalk cells that elongate the sprout. Past studies have identified genetic signals that control vascular branching. Prominent are VEGF, activating tip cells, and Notch, which stimulates stalk cells. After the branch is formed and perfused, ECs become quiescent phalanx cells. Now, emerging evidence has accumulated indicating that ECs not only adapt their metabolism when switching from quiescence to sprouting but also that metabolism regulates vascular sprouting in parallel to the control by genetic signals. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Endothelial keratoplasty versus penetrating keratoplasty for Fuchs endothelial dystrophy.

    Science.gov (United States)

    Nanavaty, Mayank A; Wang, Xue; Shortt, Alex J

    2014-02-14

    Fuchs endothelial dystrophy (FED) is a condition in which there is premature degeneration of corneal endothelial cells. When the number of endothelial cells is reduced to a significant degree, fluid begins to accumulate within the cornea. As a result, the cornea loses its transparency and the individual suffers a reduction in vision. The only successful surgical treatment for this condition is replacement of part or all of the cornea with healthy tissue from a donor. The established procedure, penetrating keratoplasty (PKP), has been used for many years and its safety and efficacy are well known. Endothelial keratoplasty (EK) techniques are relatively new surgical procedures and their safety and efficacy relative to PKP are uncertain. The objective of this review was to compare the benefits and complications related to two surgical methods (EK and PKP) of replacing the diseased endothelial layer of the cornea with a healthy layer in people with FED. We searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2014, Issue 1), MEDLINE (January 1950 to January 2014), EMBASE (January 1980 to January 2014), Latin American and Caribbean Health Sciences Literature Database (LILACS) (January 1982 to January 2014), the metaRegister of Controlled Trials (mRCT) (www.controlled-trials.com) and ClinicalTrials.gov (www.clinicaltrials.gov). There were no date or language restrictions in the electronic searches for trials. The electronic databases were last searched on 27 January 2014. We included all randomised controlled trials (RCTs) comparing EK versus PKP for people (of any age and gender) who had been clinically diagnosed with FED. Two authors independently screened the search results, assessed trial quality and extracted data using the standard methodological procedures expected by The Cochrane Collaboration. We included three RCTs that enrolled a total of 139 eyes of 136 participants and analysed 123 (88%) eyes. Two RCTs

  3. [Multisensory stimulation].

    Science.gov (United States)

    Etchepareborda, M C; Abad-Mas, L; Pina, J

    2003-02-01

    Diagnosis in early care (EC) involves a global study that covers the child's development, their personal history, family and surroundings. The specific aims of an intervention programme in EC could be summed up in four areas: the prevention of deficits or difficulties, the detection of problems linked with a socio-family deficiency or shortages, the stimulation of development, and help and assistance for families. Multisensory stimulation (MSS) of small children is essential for their future existence. The presentation of stimuli must follow a strict schedule; indeed, this observation is so important that if the critical moment for incorporating a stimulus is missed, providing the stimulus at another time will not have the same effect. Intellectual development during early childhood was taken into account when defining the fundamental aims of a therapeutic intervention programme in EC. To develop suitable therapy according to these concepts, an EMS (Snoezelen) room with certain special characteristics is required. This room must allow the stimuli offered in each moment and under each sensory modality to be controlled. Applying intervention programmes in a proper, specific and timely manner will enable us to accompany each child, as far as is possible in each case, in the development of his or her abilities and capabilities.

  4. Glycosaminoglycan-Mediated Downstream Signaling of CXCL8 Binding to Endothelial Cells

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    Rupert Derler

    2017-12-01

    Full Text Available The recruitment of leukocytes, mediated by endothelium bound chemokine gradients, is a vital process in inflammation. The highly negatively charged, unbranched polysaccharide family of glycosaminoglycans (GAGs, such as heparan sulfate and chondroitin sulfate mediate chemokine immobilization. Specifically the binding of CXCL8 (interleukin 8 to GAGs on endothelial cell surfaces is known to regulate neutrophil recruitment. Currently, it is not clear if binding of CXCL8 to GAGs leads to endothelial downstream signaling in addition to the typical CXCR1/CXCR2 (C-X-C motif chemokine receptor 1 and 2-mediated signaling which activates neutrophils. Here we have investigated the changes in protein expression of human microvascular endothelial cells induced by CXCL8. Tumor necrosis factor alpha (TNFα stimulation was used to mimic an inflammatory state which allowed us to identify syndecan-4 (SDC4 as the potential proteoglycan co-receptor of CXCL8 by gene array, real-time PCR and flow cytometry experiments. Enzymatic GAG depolymerization via heparinase III and chondroitinase ABC was used to emulate the effect of glycocalyx remodeling on CXCL8-induced endothelial downstream signaling. Proteomic analyses showed changes in the expression pattern of a number of endothelial proteins such as Zyxin and Caldesmon involved in cytoskeletal organization, cell adhesion and cell mobility. These results demonstrate for the first time a potential role of GAG-mediated endothelial downstream signaling in addition to the well-known CXCL8-CXCR1/CXCR2 signaling pathways in neutrophils.

  5. Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion.

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    Raghav Oberoi

    Full Text Available It is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab-which is approved for several inflammatory disorders-on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions.Phorbol myristate acetate (PMA differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1, intercellular adhesion molecule-1 (ICAM-1 and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice.Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.

  6. Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10

    Science.gov (United States)

    Obiako, Boniface; Calchary, Wendy; Xu, Ningyong; Kunstadt, Ryan; Richardson, Bianca; Nix, Jessica

    2013-01-01

    It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients. PMID:23686854

  7. Double-stranded RNA attenuates the barrier function of human pulmonary artery endothelial cells.

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    Zoltán Bálint

    Full Text Available Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs. The effect of natural and synthetic double-stranded RNA (dsRNA on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca(2+ homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca(2+ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca(2+-ATPase (SERCA which is involved in the regulation of the intracellular Ca(2+ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.

  8. Abl family kinases regulate endothelial barrier function in vitro and in mice.

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    Elizabeth M Chislock

    Full Text Available The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg, as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR, Kit, colony stimulating factor 1 receptor (CSF1R, and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca(2+ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use

  9. CXCR2 is essential for cerebral endothelial activation and leukocyte recruitment during neuroinflammation.

    Science.gov (United States)

    Wu, Fengjiao; Zhao, Yawei; Jiao, Tian; Shi, Dongyan; Zhu, Xingxing; Zhang, Mingshun; Shi, Meiqing; Zhou, Hong

    2015-05-21

    Chemokines and chemokine receptors cooperate to promote immune cell recruitment to the central nervous system (CNS). In this study, we investigated the roles of CXCR2 and CXCL1 in leukocyte recruitment to the CNS using a murine model of neuroinflammation. Wild-type (WT), CXCL1(-/-), and CXCR2(-/-) mice each received an intracerebroventricular (i.c.v.) injection of lipopolysaccharide (LPS). Esterase staining and intravital microscopy were performed to examine neutrophil recruitment to the brain. To assess endothelial activation in these mice, the expression of adhesion molecules was measured via quantitative real-time polymerase chain reaction (PCR) and Western blotting. To identify the cellular source of functional CXCR2, chimeric mice were generated by transferring bone marrow cells between the WT and CXCR2(-/-) mice. Expression levels of the chemokines CXCL1, CXCL2, and CXCL5 were significantly increased in the brain following the i.c.v. injection of LPS. CXCR2 or CXCL1 deficiency blocked neutrophil infiltration and leukocyte recruitment in the cerebral microvessels. In the CXCR2(-/-) and CXCL1(-/-) mice, the cerebral endothelial expression of adhesion molecules such as P-selectin and VCAM-1 was dramatically reduced. Furthermore, the bone marrow transfer experiments demonstrated that CXCR2 expression on CNS-residing cells is essential for cerebral endothelial activation and leukocyte recruitment. Compared with microglia, cultured astrocytes secreted a much higher level of CXCL1 in vitro. Astrocyte culture conditioned medium significantly increased the expression of VCAM-1 and ICAM-1 in cerebral endothelial cells in a CXCR2-dependent manner. Additionally, CXCR2 messenger RNA (mRNA) expression in cerebral endothelial cells but not in microglia or astrocytes was increased following tumor necrosis factor-α (TNF-α) stimulation. The intravenous injection of the CXCR2 antagonist SB225002 significantly inhibited endothelial activation and leukocyte recruitment to

  10. Inhibition of TGF-β Signaling in SHED Enhances Endothelial Differentiation.

    Science.gov (United States)

    Xu, J G; Gong, T; Wang, Y Y; Zou, T; Heng, B C; Yang, Y Q; Zhang, C F

    2017-10-01

    Low efficiency of deriving endothelial cells (ECs) from adult stem cells hampers their utilization in tissue engineering studies. The purpose of this study was to investigate whether suppression of transforming growth factor beta (TGF-β) signaling could enhance the differentiation efficiency of dental pulp-derived stem cells into ECs. We initially used vascular endothelial growth factor A (VEGF-A) to stimulate 2 dental pulp-derived stem cells (dental pulp stem cells and stem cells from human exfoliated deciduous teeth [SHED]) and compared their differentiation capacity into ECs. We further evaluated whether the vascular endothelial growth factor receptor I (VEGF-RI)-specific ligand placental growth factor-1 (PlGF-1) could mediate endothelial differentiation. Finally, we investigated whether the TGF-β signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than dental pulp stem cells. VEGFR1-specific ligand-PLGF exerted a negligible effect on SHED-ECs differentiation. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-β-SMAD2/3 signaling.

  11. Challenges in pediatric endothelial keratoplasty

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    Vikas Mittal

    2014-01-01

    Full Text Available We performed endothelial keratoplasty (EK in three eyes of two siblings (2.5 years, male and 3.5 years, female with congenital hereditary endothelial dystrophy (CHED and report the intraoperative and postoperative difficulties. Repeated iris prolapse, apprehension of crystalline lens touch due to positive vitreous pressure, and need for frequent air injections to attach the graft were intraoperative challenges in all three eyes. These were addressed by use of Sheet′s glide instead of Busin′s glide during graft insertion and suturing of main and side ports before air injection. One eye had graft dislocation on second postoperative day due to eye rubbing by the child. Graft was repositioned with air and a venting incision was created. Postoperative examination required repeated general anesthesia. Corneal edema resolved completely in all three eyes. Present case series highlights the possible intraoperative and postoperative challenges and their solutions in pediatric EK for CHED.

  12. Flow-Mediated Endothelial Mechanotransduction

    Science.gov (United States)

    Davies, Peter F.

    2011-01-01

    Mechanical forces associated with blood flow play important roles in the acute control of vascular tone, the regulation of arterial structure and remodeling, and the localization of atherosclerotic lesions. Major regulation of the blood vessel responses occurs by the action of hemodynamic shear stresses on the endothelium. The transmission of hemodynamic forces throughout the endothelium and the mechanotransduction mechanisms that lead to biophysical, biochemical, and gene regulatory responses of endothelial cells to hemodynamic shear stresses are reviewed. PMID:7624393

  13. Polyphenols in preventing endothelial dysfunction

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    Sylwia Biegańska-Hensoldt

    2017-03-01

    Full Text Available 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.

  14. Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation.

    Science.gov (United States)

    Hick, Anne-Christine; Delmarcelle, Anne-Sophie; Bouquet, Mahé; Klotz, Sabrina; Copetti, Tamara; Forez, Celine; Van Der Smissen, Patrick; Sonveaux, Pierre; Collet, Jean-François; Feron, Olivier; Courtoy, Pierre J; Pierreux, Christophe E

    2013-09-01

    The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by > 90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Endothelial microparticles prevent lipid-induced endothelial damageviaAkt/eNOS signaling and reduced oxidative stress.

    Science.gov (United States)

    Mahmoud, Ayman M; Wilkinson, Fiona L; McCarthy, Eoghan M; Moreno-Martinez, Daniel; Langford-Smith, Alexander; Romero, Miguel; Duarte, Juan; Alexander, M Yvonne

    2017-10-01

    Endothelial microparticles (EMPs) are endothelium-derived submicron vesicles that are released in response to diverse stimuli and are elevated in cardiovascular disease, which is correlated with risk factors. This study investigates the effect of EMPs on endothelial cell function and dysfunction in a model of free fatty acid (FFA) palmitate-induced oxidative stress. EMPs were generated from TNF-α-stimulated HUVECs and quantified by using flow cytometry. HUVECs were treated with and without palmitate in the presence or absence of EMPs. EMPs were found to carry functional eNOS and to protect against oxidative stress by positively regulating eNOS/Akt signaling, which restored NO production, increased superoxide dismutase and catalase, and suppressed NADPH oxidase and reactive oxygen species (ROS) production, with the involvement of NF-erythroid 2-related factor 2 and heme oxygenase-1. Conversely, under normal conditions, EMPs reduced NO release and increased ROS and redox-sensitive marker expression. In addition, functional assays using EMP-treated mouse aortic rings that were performed under homeostatic conditions demonstrated a decline in endothelium-dependent vasodilatation, but restored the functional response under lipid-induced oxidative stress. These data indicate that EMPs harbor functional eNOS and potentially play a role in the feedback loop of damage and repair during homeostasis, but are also effective in protecting against FFA-induced oxidative stress; thus, EMP function is reflected by the microenvironment.-Mahmoud, A. M., Wilkinson, F. L., McCarthy, E. M., Moreno-Martinez, D., Langford-Smith, A., Romero, M., Duarte, J., Alexander, M. Y. Endothelial microparticles prevent lipid-induced endothelial damage via Akt/eNOS signaling and reduced oxidative stress. © FASEB.

  16. Cataract surgery after Descemet stripping endothelial keratoplasty

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    Sunita Chaurasia

    2012-01-01

    Full Text Available Management of endothelial dysfunction in phakic patients is sometimes a dilemma for corneal surgeons. Phakic patients with visually significant cataract and endothelial dysfunction are preferably managed by performing combined cataract surgery with endothelial keratoplasty. However, combined surgery may be deferred in eyes with early incipient cataract, younger age and where anterior chamber is poorly visualized. As cataract formation may be accelerated after endothelial keratoplasty, these eyes may need cataract surgery subsequently. Surgical intervention in eyes with endothelial keratoplasty is of concern as this may affect the graft adversely and threaten graft survival. In this report, we describe the intraoperative surgical details and postoperative clinical course of a patient who underwent phacoemulsification with intraocular lens implantation after Descemet stripping automated endothelial keratoplasty (DSAEK.

  17. Endothelial cell loss after autologous rotational keratoplasty.

    Science.gov (United States)

    Birnbaum, Florian; Reinhard, Thomas; Böhringer, Daniel; Sundmacher, Rainer

    2005-01-01

    To investigate whether it may be possible to ascertain the influence of immunological factors on chronic endothelial cell loss by comparing chronic endothelial cell loss after autologous rotational penetrating keratoplasty and after homologous penetrating keratoplasty. For six patients who had undergone autologous rotational penetrating keratoplasty the relative annual loss of endothelial cells was calculated by means of an exponential regression analysis. The findings were compared with those in a homogeneous historical control group (53 patients undergoing homologous penetrating keratoplasty for keratoconus). After autologous rotational keratoplasty relative annual loss of endothelial cells was 1.1%+/-2.6% (mean +/- standard deviation). Relative annual loss of endothelial cells in the control-group was 16.7%+/-20.8%. The results of the study lead to the assumption that immunological influences might be the main cause for chronic endothelial cell loss after homologous penetrating keratoplasty.

  18. Herpes Simplex Virus endotheliitis following descemet‘s membrane endothelial keratoplasty

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    Siamak Zarei-Ghanavati1

    2015-01-01

    Conclusion:HSV endotheliitis may occur in the early postoperative period after DMEK and manifest as endothelial dysfunction leading to donor detachment. Anti-viral medication may help treat the acute phase and reduce the risk of recurrence.

  19. Role of Lipotoxicity in Endothelial Dysfunction

    OpenAIRE

    Kim, Jeong-a; Montagnani, Monica; Chandrasekran, Sruti; Quon, Michael J.

    2012-01-01

    Lipotoxicity, caused in large part by overnutrition, directly leads to endothelial dysfunction. Excess lipids in both the circulation and at the tissue level contribute to endothelial dysfunction that underlies much of the pathophysiology of both metabolic disease, including obesity and diabetes and their CV complications. Direct lipotoxic effects on other organs as well as secondary insults from endothelial dysfunction synergize to cause substantial morbidity and mortality. Lifestyle interve...

  20. Nitride coating enhances endothelialization on biomedical NiTi shape memory alloy

    Energy Technology Data Exchange (ETDEWEB)

    Ion, Raluca [University of Bucharest, Department of Biochemistry and Molecular Biology, 91-95 Spl. Independentei, 050095 Bucharest (Romania); Luculescu, Catalin [National Institute for Laser, Plasma and Radiation Physics, 409 Atomistilor, P.O. Box MG-36, 077125 Magurele-Bucharest (Romania); Cimpean, Anisoara, E-mail: anisoara.cimpean@bio.unibuc.ro [University of Bucharest, Department of Biochemistry and Molecular Biology, 91-95 Spl. Independentei, 050095 Bucharest (Romania); Marx, Philippe [AMF Company, Route de Quincy, 18120 Lury-sur-Arnon (France); Gordin, Doina-Margareta; Gloriant, Thierry [INSA Rennes, UMR CNRS 6226 ISCR, 20 Avenue des Buttes de Coësmes, 35708 Rennes Cedex 7 (France)

    2016-05-01

    Surface nitriding was demonstrated to be an effective process for improving the biocompatibility of implantable devices. In this study, we investigated the benefits of nitriding the NiTi shape memory alloy for vascular stent applications. Results from cell experiments indicated that, compared to untreated NiTi, a superficial gas nitriding treatment enhanced the adhesion of human umbilical vein endothelial cells (HUVECs), cell spreading and proliferation. This investigation provides data to demonstrate the possibility of improving the rate of endothelialization on NiTi by means of nitride coating. - Highlights: • Gas nitriding process of NiTi is competent to promote cell spreading. • Surface nitriding of NiTi is able to stimulate focal adhesion formation and cell proliferation. • Similar expression pattern of vWf and eNOS was exhibited by bare and nitrided NiTi. • Gas nitriding treatment of NiTi shows promise for better in vivo endothelialization.

  1. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens.

    Science.gov (United States)

    Nakamura, Junko; Shigematsu, Satoshi; Yamauchi, Keishi; Takeda, Teiji; Yamazaki, Masanori; Kakizawa, Tomoko; Hashizume, Kiyoshi

    2008-10-03

    Migration and tube formation of endothelial cells are important in angiogenesis and require a coordinated response to the extra-cellular matrix (ECM) and growth factor. Since focal adhesion kinase (FAK) integrates signals from both ECM and growth factor, we investigated its role in angiogenesis. Type I and II collagens are fibril-forming collagens and stimulate human umbilical vein endothelial cells (HUVECs) to form tube structure. Although knockdown of FAK restrained cell motility and resulted in inhibition of tube formation, FAK degradation and tube formation occurred simultaneously after incubation with fibril-forming collagens. The compensation for the FAK degradation by a calpain inhibitor or transient over-expression of FAK resulted in disturbance of tube formation. These phenomena are specific to fibril-forming collagens and mediated via alpha2beta1 integrin. In conclusion, our data indicate that FAK is functioning in cell migration, but fibril-forming collagen-induced FAK degradation is necessary for endothelial tube formation.

  2. Synchronization of endothelial Dll4-Notch dynamics switch blood vessels from branching to expansion.

    Science.gov (United States)

    Ubezio, Benedetta; Blanco, Raquel Agudo; Geudens, Ilse; Stanchi, Fabio; Mathivet, Thomas; Jones, Martin L; Ragab, Anan; Bentley, Katie; Gerhardt, Holger

    2016-04-13

    Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.

  3. ALTERED EXPRESSION OF SURFACE RECEPTORS AT EA.HY926 ENDOTHELIAL CELL LINE INDUCED WITH PLACENTAL SECRETORY FACTORS

    Directory of Open Access Journals (Sweden)

    O. I. Stepanova

    2012-01-01

    Full Text Available Abstract. Placental cell populations produce a great variety of angiogenic factors and cytokines than control angiogenesis in placenta. Functional regulation of endothelial cells proceeds via modulation of endothelial cell receptors for endogenous angiogenic and apoptotic signals. Endothelial phenotype alteration during normal pregnancy and in cases of preclampsia is not well understood. The goal of this investigation was to evaluate altered expression of angiogenic and cytokine receptors at EA.hy926 endothelial cells under the influence of placental tissue supernatants. Normal placental tissue supernatants from 1st and 3rd trimesters, and pre-eclamptic placental tissue supernatants (3rd trimester stimulated angiogenic and cytokine receptors expression by the cultured endothelial cells, as compared with their background expression. Tissue supernatants from placental samples of 3rd trimester caused a decreased expression of angiogenic and cytokine receptors by endothelial cells, thus reflecting maturation of placental vascular system at these terms. Supernatants from preeclamptic placental tissue induced an increase of CD119 expression, in comparison with normal placental supernatants from the 3rd trimester. This finding suggests that IFNγ may be a factor of endothelial activation in pre-eclampsia. The study was supported by grants ГК №02.740.11.0711, НШ-3594.2010.7., and МД-150.2011.7.

  4. Methotrexate improves perivascular adipose tissue/endothelial dysfunction via activation of AMPK/eNOS pathway.

    Science.gov (United States)

    Ma, Yanmin; Li, Li; Shao, Yating; Bai, Xiaohong; Bai, Tiao; Huang, Xinliang

    2017-04-01

    Adipose and endothelial dysfunction is associated with cardiovascular disease. Perivascular adipose tissue (PVAT) directly surrounds vessels and influences vessel function via a paracrine effect, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) modulates the metabolic pathway, thus, the present study hypothesized that activation of AMPK in PVAT may regulate endothelial function in pathological settings. The present study investigated the effect of methotrexate (MTX) on adipocytokine expression in PVAT with an emphasis on the regulation of endothelial function. The effects of MTX and the mechanisms involved were investigated using a relaxation assay and western blot analysis. Reverse transcription‑quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels. ELISA assay was used to quantify the level of TNF‑α and IL‑6. Palmitic acid (PA) stimulation induced inflammation and dysregulation of adipocytokine expression in PVAT. MTX treatment inhibited nuclear factor‑κB p65 phosphorylation and downregulated expression of pro‑inflammatory cytokines, including tumor necrosis factor‑α and interleukin-6, whereas adiponectin expression increased. MTX increased AMPK phosphorylation under basal and inflammatory conditions in PVAT, whereas knockdown of AMPK via small interfering RNA diminished its modulatory effect, indicating that MTX inhibits inflammation in an AMPK‑dependent manner. The present study prepared conditioned medium from PA‑stimulated PVAT to induce endothelial dysfunction and observed that pre‑treatment of PVAT with MTX effectively restored the loss of acetylcholine‑induced vasodilation and increased endothelial nitric oxide synthase phosphorylation in the rat aorta. The results of the present study demonstrated that MTX ameliorated inflammation-associated adipocytokine dysregulation and thus prevented endothelial dysfunction. These data provide further

  5. The regulatory mechanism of Hsp90{alpha} secretion from endothelial cells and its role in angiogenesis during wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Song, Xiaomin [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China); Luo, Yongzhang, E-mail: yluo@tsinghua.edu.cn [National Engineering Laboratory for Anti-tumor Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Beijing Key Laboratory for Protein Therapeutics, Tsinghua University, Beijing 100084 (China); Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing 100084 (China)

    2010-07-16

    Research highlights: {yields} Growth factors such as bFGF, VEGF, PDGF and SDF-1 stimulate Hsp90{alpha} secretion from endothelial cells. {yields} Secreted Hsp90{alpha} localizes on the leading edge of activated endothelial cells. {yields} Secreted Hsp90{alpha} promotes angiogenesis in wound healing. -- Abstract: Heat shock protein 90{alpha} (Hsp90{alpha}) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90{alpha} can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90{alpha} from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90{alpha} in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90{alpha} but not Hsp90{beta} is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90{alpha} localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90{alpha} neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90{alpha} localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90{alpha} can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90{alpha} as a stimulator for wound repair.

  6. Circulating Endothelial Cells and Endothelial Progenitor Cells in Pediatric Sepsis.

    Science.gov (United States)

    Zahran, Asmaa Mohamad; Elsayh, Khalid Ibrahim; Mohamad, Ismail Lotfy; Hassan, Gamal Mohamad; Abdou, Madleen Adel A

    2016-03-01

    The aim of the study was to measure the number of circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs) in pediatric patients with sepsis and correlating it with the severity of the disease and its outcome. The study included 19 children with sepsis, 26 with complicated sepsis, and 30 healthy controls. The patients were investigated within 48 hours of pediatric intensive care unit admission together with flow cytometric detection of CECs and CEPs. The levels of both CECs and CEPs were significantly higher in patient with sepsis and complicated sepsis than the controls. The levels of CECs were higher in patients with complicated sepsis, whereas the levels of CEPs were lower in patients with complicated sepsis. Comparing the survival and nonsurvival septic patients, the levels of CEPs were significantly higher in the survival than in nonsurvival patients, whereas the levels of CECs were significantly lower in the survival than in nonsurvival patients. Serum albumin was higher in survival than in nonsurvival patients. Estimation of CECs and CEPs and their correlation with other parameters such as serum albumen could add important information regarding prognosis in septic pediatric patients.

  7. Endothelial Cell-derived Extracellular Vesicles Size-dependently Exert Procoagulant Activity Detected by Thromboelastometry.

    Science.gov (United States)

    Holnthoner, Wolfgang; Bonstingl, Cornelia; Hromada, Carina; Muehleder, Severin; Zipperle, Johannes; Stojkovic, Stefan; Redl, Heinz; Wojta, Johann; Schöchl, Herbert; Grillari, Johannes; Weilner, Sylvia; Schlimp, Christoph J

    2017-06-16

    Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures.

  8. Whole blood-mediated endothelial permeability and adhesion molecule expression: a model study into the effects of bacteria and antibiotics.

    NARCIS (Netherlands)

    Nooteboom, A.; Linden, C.J. van der; Hendriks, T.

    2005-01-01

    AIM: To investigate whether the inflammatory response of cultured endothelial cells, as induced by conditioned plasma, depends on the bacterial species or type of antibiotic used for incubation with whole blood. MATERIALS AND METHODS: Blood from healthy volunteers was stimulated ex vivo with

  9. Transmission of Anaplasma phagocytophilum from endothelial cells to peripheral granulocytes in vitro under shear flow conditions.

    Science.gov (United States)

    Wang, Jinyong; Dyachenko, Viktor; Munderloh, Ulrike G; Straubinger, Reinhard K

    2015-10-01

    Anaplasma phagocytophilum (Ap) is a tick-borne pathogen, which can cause granulocytic anaplasmosis in humans and animals. In vivo this obligate intracellular pathogen is primarily located in circulating mature granulocytes, but it also infects endothelial cells. In order to study the interaction between Ap-infected endothelial cells and human granulocytes under conditions similar to those found naturally in the infected host, an in vitro model that mimics physiological flow conditions in the microvasculature was established. Cell-to-cell interactions were then visualized by microscopy, which showed that granulocytes adhered strongly to Ap-infected endothelial cells at a shear stress of 0.5 dyne/cm(2). In addition, Ap-transmission assays under flow conditions showed that the bacteria transferred from infected endothelial cells to circulating granulocytes and were able to establish infection in constantly moving granulocytes. Cell surface analysis showed that Ap induced up-regulation of the cell adhesion molecules ICAM-1 and VCAM-1 on infected endothelial cells in a dose-dependent manner. Furthermore, IL-8 secretion by endothelial cells indicated that the presence of Ap induced a pro-inflammatory response. In summary, the results of this study suggest that endothelial cells of the microvasculature (1) provide an excellent site for Ap dissemination to peripheral blood granulocytes under flow conditions and therefore may play a crucial role in the development of persistent infection, and (2) are stimulated by Ap to express surface molecules and cytokines that may lead to inflammatory responses at the site of the infection.

  10. Oxidative stress and apoptosis induced by iron oxide nanoparticles in cultured human umbilical endothelial cells.

    Science.gov (United States)

    Zhu, Mo-Tao; Wang, Yun; Feng, Wei-Yue; Wang, Bing; Wang, Meng; Ouyang, Hong; Chai, Zhi-Fang

    2010-12-01

    Recent epidemiologic researches indicate that exposure to ultrafine particles (nanoparticles) is an independent risk factor for several cardiovascular diseases. The induction of endothelial injuries is hypothesized to be an attractive mechanism involved in these cardiovascular diseases. To investigate this hypothesis, the widely used iron nanomaterials, ferric oxide (Fe2O3) and ferriferrous oxide (Fe3O4) nanoparticles were incubated with human umbilical endothelial cells (ECV304 cells) at different concentrations of 2, 20, 100 microg/mL. The cell viability, the rate of apoptosis, the apoptotic nuclear morphology and the mitochondria membrane potential were measured to estimate the cell necrosis and apoptosis caused by the nanoparticle exposure. The stimulation of superoxide anion (O2*-) and nitric oxide (NO) were examined to evaluate the stress responses of endothelial cells. Our results indicated that both the Fe2O3 and Fe3O4 nanoparticles could generate oxidative stress as well as the significant increase of nitric oxide in ECV304 cells. The loss of mitochondria membrane potential and the apoptotic chromatin condensation in the nucleus were observed as the early signs of apoptosis. It is inferred the stress response might be an important mechanism involving in endothelial cells apoptosis and death, and these injuries in endothelial cells might play a key role in downstream cardiovascular diseases such as atheroscelerosis, hypertension and myocardial infarction (MI).

  11. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Directory of Open Access Journals (Sweden)

    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  12. Vagus Nerve Stimulation

    Science.gov (United States)

    Vagus nerve stimulation Overview By Mayo Clinic Staff Vagus nerve stimulation is a procedure that involves implantation of a device that stimulates the vagus nerve with electrical impulses. There's one vagus nerve ...

  13. APPLICATION OF STEM CELLS AND PRECURSOR CELLS FOR STIMULATION OF ORGAN REVASCULARIZATION AND REGENERATION

    Directory of Open Access Journals (Sweden)

    M. V. Eremeeva

    2010-01-01

    Full Text Available Different angiogenic factors induced angiogenesis stimulation in ischemic tissues stays in the focus of scientific research for long time. The key role in ischemic angiogenesis belongs to endothelial precursor cells, plenty of which are reserved in bone marrow. Resident endothelial precursor cells are also found in some tissues and in circulation. These cells are involved in neoangiogenesis as well. Theoretically, injection of exogeneous endothelial precusor cells might contribute to restoration of circulation in the ischemic organ. Various types of cells have been approved for regeneration stimulation in a number of experimental protocols. A various degree of improvement of myocardial contractive function has been obtained as a universal result of these investigations, though the mechanisms underlying observed effect remain evasive. The paper focuses on advantages and drawbacks of embryonic, hematopoetic and mesenhimal stem cells application for angiogenesis stimulation and organs and tissues regeneration. 

  14. Effect of cytokines on tumour cell-endothelial interactions.

    Science.gov (United States)

    Cohen, M C; Bereta, M; Bereta, J

    1997-01-01

    The adherence of tumour cells to microvascular endothelium is believed to be a necessary step in their migration to sites of metastasis. It has been proposed that this process occurs when cell surface molecules on tumour cells bind to complementary sites on endothelial cells. The expression of these endothelial-derived cell adhesion molecules appears to be modulated by cytokines, a broad class of protein mediators which play important roles in immune and inflammatory reactions. It has been found by ourselves and others that exposure of endothelium to some cytokines augments the adhesion of inflammatory cells as well as tumour cells in in vitro assays. We used a murine model consisting of P815 mastocytoma cells and microvascular endothelium and found that pretreatment of endothelial monolayers with TNF-alpha, IL-1, LPS or PMA augmented the number of tumour cells that attach in a dose-dependent fashion. FACS analysis showed that the change in binding was due to an increase in the expression of VCAM-1 on the surface of the endothelial cell. Methylxanthines (caffeine and theophylline) as well as "classical" calcium-mobilizing agents (ionomycin and thapsigargin) inhibited the expression of VCAM-1 in MME. We also studied the possible mechanisms of TNF-alpha signal transduction in endothelial cells. We examined the involvement of protein kinases in the TNF-alpha effect. Although we found that inhibitors of PKC could inhibit the TNF-alpha effect, our studies suggest that the "classical" PKC pathway is not completely responsible for signaling since TNF-alpha did not cause translocation of PKC to the cell membrane and its effect could not be completely mimicked by PMA. We also studied the effect of TGF-beta on the binding of tumour cells to endothelium. Exposure of endothelium to TGF-beta led to the inhibition of both basal and TNF-alpha enhanced binding of P815 cells. Inhibitors of G-proteins do not abolish TGF-beta action, and PKC and PKA activators elicit an opposite

  15. Endothelial progenitor cell dysfunction in diabetes mellitus

    NARCIS (Netherlands)

    Loomans, Cindy Johanna Maria

    2007-01-01

    Postnatally, Endothelial Progenitor Cells are needed to maintain the integrity of the endothelium (re-endothelialization) and to augment wound healing or vascularize hypoxic areas (neovascularization). Complex networks of different signals and regulators have been identified to be involved in these

  16. Endothelial dysfunction after non-cardiac surgery

    DEFF Research Database (Denmark)

    Søndergaard, E S; Fonnes, S; Gögenur, I

    2015-01-01

    was to systematically review the literature to evaluate the association between non-cardiac surgery and non-invasive markers of endothelial function. METHODS: A systematic search was conducted in MEDLINE, EMBASE and Cochrane Library Database according to the PRISMA guidelines. Endothelial dysfunction was described only...

  17. PPAR Gamma and Angiogenesis: Endothelial Cells Perspective

    Directory of Open Access Journals (Sweden)

    Jerzy Kotlinowski

    2016-01-01

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

  18. Molecular expression in transfected corneal endothelial cells

    Science.gov (United States)

    Wang, Fan; Miao, Zhuang; Lu, Chengwei; Hao, Jilong

    2017-10-01

    To investigate the capability of human corneal endothelial cells serving as immunological cells. Expression of HLA-DP, -DQ, -DR, CD40, CD80, and CD86 was determined by immunohistochemical methods. Meanwhile, purified peripheral blood mononuclear cells were cocultured with human corneal endothelial cells which were pre-treated with and without -IFN respectively, activation of lymphocytes was determined by FACS analysis. In coculture system, T lymphocyte was activated by corneal endothelial cells, HLA-DP, -DQ, -DR and CD40 expression were increased by - IFN induction. Costimulatory molecular CD80 was shown on the endothelial cells. Human corneal endothelial cells were assumed to be involved in the corneal transplantation rejection process as potential antigen presenting cells.

  19. SNAP23 Regulates Endothelial Exocytosis of von Willebrand Factor.

    Science.gov (United States)

    Zhu, Qiuyu Martin; Zhu, Qiuyu; Yamakuchi, Munekazu; Lowenstein, Charles J

    2015-01-01

    Endothelial exocytosis regulates vascular thrombosis and inflammation. The trafficking and release of endothelial vesicles is mediated by SNARE (Soluble NSF Attachment protein REceptors) molecules, but the exact identity of endothelial SNAREs has been unclear. Three SNARE molecules form a ternary complex, including isoforms of the syntaxin (STX), vesicle-associated membrane protein (VAMP), and synaptosomal-associated protein (SNAP) families. We now identify SNAP23 as the predominant endothelial SNAP isoform that mediates endothelial exocytosis of von Willebrand Factor (VWF). SNAP23 was localized to the plasma membrane. Knockdown of SNAP23 decreased endothelial exocytosis, suggesting it is important for endothelial exocytosis. SNAP23 interacted with the endothelial exocytic machinery, and formed complexes with other known endothelial SNARE molecules. Taken together, these data suggest that SNAP23 is a key component of the endothelial SNARE machinery that mediates endothelial exocytosis.

  20. Docosapentaenoic acid (22:5, n-3) suppressed tube-forming activity in endothelial cells induced by vascular endothelial growth factor.

    Science.gov (United States)

    Tsuji, Masako; Murota, Se-itsu; Morita, Ikuo

    2003-05-01

    It is generally accepted that n-3 polyunsaturated fatty acids have beneficial effects on vascular homeostasis. Among the several functions of endothelial cells, angiogenesis contributes to tumor growth, inflammation, and microangiopathy. We have already demonstrated that eicosapentaenoic acid (EPA, 20:5, n-3) suppressed angiogenesis. In this paper, we examined the effect of docosapentaenoic acid (DPA, 22:5, n-3), an elongated metabolite of EPA, on tube-forming activity in bovine aortic endothelial cells (BAE cells) incubated between type I collagen gels. The pretreatment of BAE cells with DPA suppressed tube-forming activity induced by vascular endothelial growth factor (VEGF). The effect of DPA was stronger than those of EPA and docosahexaenoic acid (22:6, n-3). The migrating activity of endothelial cells stimulated with VEGF was also suppressed by DPA pretreatment. The treatment of BAE cells with DPA caused the suppression of VEGF receptor-2 (VEGFR-2, the kinase insert domain-containing receptor, KDR) expression in both plastic dish and collagen gel cultures. These data indicate that DPA has a potent inhibitory effect on angiogenesis through the suppression of VEGFR-2 expression.

  1. [Transplantation of corneal endothelial cells].

    Science.gov (United States)

    Amano, Shiro

    2002-12-01

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

  2. Hypothyroidism Is Associated With Coronary Endothelial Dysfunction in Women.

    Science.gov (United States)

    Sara, Jaskanwal D; Zhang, Ming; Gharib, Hossein; Lerman, Lilach O; Lerman, Amir

    2015-07-29

    Hypothyroidism is associated with an increased risk of coronary artery disease, beyond that which can be explained by its association with conventional cardiovascular risk factors. Coronary endothelial dysfunction precedes atherosclerosis, has been linked to adverse cardiovascular events, and may account for some of the increased risk in patients with hypothyroidism. The aim of this study was to determine whether there is an association between epicardial and microvascular coronary endothelial dysfunction and hypothyroidism. In 1388 patients (mean age 50.5 [12.3] years, 34% male) presenting with stable chest pain to Mayo Clinic, Rochester, MN for diagnostic coronary angiography, and who were found to have nonobstructive coronary artery disease (hypothyroidism, defined as a documented history of hypothyroidism or a thyroid-stimulating hormone (TSH) >10.0 mU/mL, n=188, and euthyroidism, defined as an absence of a history of hypothyroidism in the clinical record and/or 0.3hypothyroidism had a significantly lower % Δ CBF Ach (48.26 [80.66] versus 64.58 [128.30]) compared to patients with euthyroidism, while the % Δ CAD Ach did not vary significantly between groups. After adjusting for covariates, females with hypothyroidism still had a significantly lower % Δ CBF Ach (estimated difference in % Δ CBF Ach [SE]: -16.79 [8.18]). Hypothyroidism in women is associated with microvascular endothelial dysfunction, even after adjusting for confounders, and may explain some of the increased risk of cardiovascular disease in these patients. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  3. Dexamethasone Enhances ATP-Induced Inflammatory Responses in Endothelial Cells

    Science.gov (United States)

    Ding, Yi; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation. PMID:20826566

  4. Hypothyroidism Is Associated With Coronary Endothelial Dysfunction in Women

    Science.gov (United States)

    Sara, Jaskanwal D; Zhang, Ming; Gharib, Hossein; Lerman, Lilach O; Lerman, Amir

    2015-01-01

    Background Hypothyroidism is associated with an increased risk of coronary artery disease, beyond that which can be explained by its association with conventional cardiovascular risk factors. Coronary endothelial dysfunction precedes atherosclerosis, has been linked to adverse cardiovascular events, and may account for some of the increased risk in patients with hypothyroidism. The aim of this study was to determine whether there is an association between epicardial and microvascular coronary endothelial dysfunction and hypothyroidism. Methods and Results In 1388 patients (mean age 50.5 [12.3] years, 34% male) presenting with stable chest pain to Mayo Clinic, Rochester, MN for diagnostic coronary angiography, and who were found to have nonobstructive coronary artery disease (hypothyroidism, defined as a documented history of hypothyroidism or a thyroid-stimulating hormone (TSH) >10.0 mU/mL, n=188, and euthyroidism, defined as an absence of a history of hypothyroidism in the clinical record and/or 0.3hypothyroidism had a significantly lower % Δ CBF Ach (48.26 [80.66] versus 64.58 [128.30]) compared to patients with euthyroidism, while the % Δ CAD Ach did not vary significantly between groups. After adjusting for covariates, females with hypothyroidism still had a significantly lower % Δ CBF Ach (estimated difference in % Δ CBF Ach [SE]: −16.79 [8.18]). Conclusions Hypothyroidism in women is associated with microvascular endothelial dysfunction, even after adjusting for confounders, and may explain some of the increased risk of cardiovascular disease in these patients. PMID:26224049

  5. VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration

    Directory of Open Access Journals (Sweden)

    Gareth W. Fearnley

    2015-07-01

    Full Text Available Vascular endothelial growth factor A (VEGF-A regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

  6. Dysfunctional Endothelial Progenitor Cells in Metabolic Syndrome

    Science.gov (United States)

    Devaraj, Sridevi; Jialal, Ishwarlal

    2012-01-01

    The metabolic syndrome (MetS) is highly prevalent and confers an increased risk of diabetes and cardiovascular disease. A key early event in atherosclerosis is endothelial dysfunction. Numerous groups have reported endothelial dysfunction in MetS. However, the measurement of endothelial function is far from optimum. There has been much interest recently in a subtype of progenitor cells, termed endothelial progenitor cells (EPCs), that can circulate, proliferate, and dfferentiate into mature endothelial cells. EPCs can be characterized by the assessment of surface markers, CD34 and vascular endothelial growth factor receptor-2, VEGFR-2 (KDR). The CD34+KDR+ phenotype has been demonstrated to be an independent predictor of cardiovascular outcomes. MetS patients without diabetes or cardiovascular diseases have decreased EPC number and functionality as evidenced by decreased numbers of colony forming units, decreased adhesion and migration, and decreased tubule formation. Strategies that have been shown to upregulate and enhance EPC number and functionality include statins, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and peroxisome-proliferator-activating-receptor gamma agonists. Mechanisms by which they affect EPC number and functionality need to be studied. Thus, EPC number and/or functionality could emerge as novel cellular biomarkers of endothelial dysfunction and cardiovascular disease risk in MetS. PMID:21941528

  7. Danggui Buxue Tang, Chinese Herbal Decoction Containing Astragali Radix and Angelicae Sinensis Radix, Induces Production of Nitric Oxide in Endothelial Cells: Signaling Mediated by Phosphorylation of Endothelial Nitric Oxide Synthase.

    Science.gov (United States)

    Gong, Amy G W; Lau, K M; Zhang, Laura M L; Lin, H Q; Dong, Tina T X; Tsim, Karl W K

    2016-03-01

    Danggui Buxue Tang, an ancient Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix at the weight ratio of 5:1, is used to mitigate menopausal syndromes in women. The pharmacological properties of Danggui Buxue Tang have been illustrated in bone development, blood enhancement, and immune stimulation. Here, we extended the possible pharmacological role of Danggui Buxue Tang in cardiovascular function. In cultured human umbilical vein endothelial cells, the application of Danggui Buxue Tang induced the release of nitric oxide and the phosphorylation of endothelial nitric oxide synthase and Akt kinase in time- and dose-dependent manners. The robust activation of nitric oxide signaling, however, required the boiling of Astragali Radix and Angelicae Sinensis Radix together, i.e., as Danggui Buxue Tang instead of other herbal extracts. The Danggui Buxue Tang-induced phosphorylation of endothelial nitric oxide synthase and Akt kinase in human umbilical vein endothelial cells were fully blocked by treatment with an endothelial nitric oxide synthase inhibitor (L-NAME), a PI3K/Akt inhibitor (LY294002), and a Ca(2+) chelator (BAPTA-AM). In parallel, the blockage of endothelial nitric oxide synthase and Akt activation subsequently fully abolished the Danggui Buxue Tang-induced nitric oxide production. Georg Thieme Verlag KG Stuttgart · New York.

  8. Endothelial TLR4 and the microbiome drive cerebral cavernous malformations.

    Science.gov (United States)

    Tang, Alan T; Choi, Jaesung P; Kotzin, Jonathan J; Yang, Yiqing; Hong, Courtney C; Hobson, Nicholas; Girard, Romuald; Zeineddine, Hussein A; Lightle, Rhonda; Moore, Thomas; Cao, Ying; Shenkar, Robert; Chen, Mei; Mericko, Patricia; Yang, Jisheng; Li, Li; Tanes, Ceylan; Kobuley, Dmytro; Võsa, Urmo; Whitehead, Kevin J; Li, Dean Y; Franke, Lude; Hart, Blaine; Schwaninger, Markus; Henao-Mejia, Jorge; Morrison, Leslie; Kim, Helen; Awad, Issam A; Zheng, Xiangjian; Kahn, Mark L

    2017-05-18

    Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3-KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.

  9. Transcellular transport of CCL2 across brain microvascular endothelial cells.

    Science.gov (United States)

    Ge, Shujun; Song, Li; Serwanski, David R; Kuziel, William A; Pachter, Joel S

    2008-03-01

    The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood-brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal-to-luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous (125)I-labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin-1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double-label immuno-electronmicroscopy, which revealed co-localization of caveolin-1 with exogenous CCL2, during this chemokine's transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.

  10. Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger*

    Science.gov (United States)

    Andrikopoulos, Petros; Kieswich, Julius; Harwood, Steven M.; Baba, Akemichi; Matsuda, Toshio; Barbeau, Olivier; Jones, Keith; Eccles, Suzanne A.; Yaqoob, Muhammad M.

    2015-01-01

    Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na+/Ca2+ exchanger (NCX) operating in the Ca2+-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca2+ influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na+-K+-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving

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

    Science.gov (United States)

    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+.

  12. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    Science.gov (United States)

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

  13. Effect of high-glucose conditions on human periodontal ligament endothelial cells: in vitro analysis.

    Science.gov (United States)

    Maruyama, Kosuke; Sato, Soh

    2017-01-01

    Endothelial cells participate in key aspects of vascular biology, such as maintenance of capillary permeability and regulation of inflammation. According to previous reports, endothelial cells have revealed highly specific characteristics depending on the organs and tissues. In particular, periodontal endothelial cells have a higher permeability than vascular endothelial cells of other types of tissue. Periodontal disease is not only a chronic disease in oral, but also affect the entire body. Diabetes and periodontal disease are closely related, with periodontal disease even been referred to as the sixth complication of disease. However, no reports have investigated the pathophysiology of microvascular in periodontal tissue once diabetes has developed. Therefore, the aim of the present study is to investigate changes in the properties of human periodontal endothelial cells (HPDLECs) that were cultured under high-glucose conditions. We isolated HPDLECs from human periodontal ligament cells. HPDLECs were cultured under high-glucose (5.5, 11.0, 22.0 mM) and investigated proliferation, apoptosis, tube formation and the expression of cell adhesion molecules. A 5.5 mM (100 mg/dl) control was used in this study. HPDLECs stimulated with high glucose concentration exhibited suppression of cell proliferation and an increased percentage of apoptosis-positive cells. This results suggested that apoptosis was caused by TNF-α expression. The expression levels cell adhesion molecules increased. These results suggest that when HPDLECs are stimulated with a high glucose concentrations, PKC in the intracellular cell substrate is activated, increasing the expression of intercellular and vascular adhesion molecules. Thus, the results of this study demonstrate that diabetes exacerbates periodontal disease.

  14. Endothelial Dysfunction and Preeclampsia: Role of Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Lissette Carolina eSánchez-Aranguren

    2014-10-01

    Full Text Available Preeclampsia (PE is an often fatal pathology characterized by hypertension and proteinuria at the 20th week of gestation that affects 5-10% of the pregnancies. The problem is particularly important in developing countries in where the incidence of hypertensive disorders of pregnancy is higher and maternal mortality rates are twenty times higher than those reported in developed countries. Risk factors for the development of PE includes obesity, insulin resistance and hyperlipidemia that stimulate inflammatory cytokine release and oxidative stress leading to endothelial dysfunction (ED. However, how all these clinical manifestations concur to develop PE is still not very well understood. The related poor trophoblast invasion and uteroplacental artery remodeling described in PE, increases reactive oxygen species (ROS, hypoxia and ED. Here we aim to review current literature from research showing the interplay between oxidative stress, ED and PE to the outcomes of current clinical trials aiming to prevent PE with antioxidant supplementation.

  15. Soluble vascular endothelial growth factor in various blood transfusion components

    DEFF Research Database (Denmark)

    Nielsen, Hans Jørgen; Werther, K; Mynster, T

    1999-01-01

    BACKGROUND: Blood transfusion may reduce survival after curative surgery for solid tumors. This may be related to extracellular content of cancer growth factors present in transfusion components. Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis in solid tumors....... The potential content of VEGF in various blood components for transfusion was evaluated. STUDY DESIGN AND METHODS: Soluble VEGF (sVEGF, isotype 165) was determined by an enzyme-linked immunosorbent assay (EIA) in serum and plasma samples and in lysed cells from healthy volunteers. Subsequently, total content......-reduced PRP. The sVEGF accumulated significantly in WB, SAGM blood, and BCP pools, depending on the storage time. CONCLUSION: The sVEGF (isotype 165) appears to be present in various blood transfusion components, depending on storage time....

  16. Role of platelet CD40 ligand for endothelial cell-monocyte interaction in the presence of flow

    Science.gov (United States)

    Wagner, Andreas H.; Schwarz, Manuel; König, Gerd; Hecker, Markus

    2014-11-01

    CD40 ligand (CD154)-induced ultra-large von Willebrand factor (vWF) multimer-mediated endothelial cell-platelet-monocyte interaction may play an important role in adaptive and maladaptive vascular remodeling processes. Here we analyzed the impact of and conditions favouring the deposition of these multimers on the endothelial cell (EC) surface by way of CD40-CD154 co-stimulation in settings mimicking different forms of blood flow. Upon exposure to low oscillatory shear stress and sCD154, a release of vWF multimers comparable to histamine stimulation was monitored on the EC surface in a string-like fashion. Moreover, ex vivo perfused carotid arteries of wild type mice at low laminar shear stress rates showed a luminal release of vWF as ultra-large vWF multimers (ULVWF) upon stimulation with sCD154 which was absent in blood vessels of CD40 knockout mice. The observed CD40- and flow-dependent vWF release from intact endothelial cells and subsequent vWF multimer formation may facilitate adhesion and subsequent activation of circulating platelets at atherosclerotic predilection sites, which are characterized by disturbed flow patterns. This in turn may amplify endothelial cell-monocyte interaction, thus possibly initiating or promoting early atherosclerotic lesion formation.

  17. Heterologous corneal endothelial cell transplantation--human corneal endothelial cell transplantation in Lewis rats.

    OpenAIRE

    Tchah, H.

    1992-01-01

    A heterologous corneal endothelial transplantation was attempted using human endothelial cells and a Lewis rat penetrating keratoplasty model. Cultured human endothelial cells were seeded to a Lewis rat cornea, which was denuded of its endothelium. When grafted into the syngeneic Lewis rat, the graft remained clear for at least five days, and then became opaque and edematous because of immune rejection reaction. In contrast, corneas denuded of their endothelium became opaque and edematous imm...

  18. Endothelial Nitric Oxide Synthase Uncoupling: A Novel Pathway in OSA Induced Vascular Endothelial Dysfunction

    OpenAIRE

    Varadharaj, Saradhadevi; Porter, Kyle; Pleister, Adam; Wannemacher, Jacob; Sow, Angela; Jarjoura, David; Zweier, Jay L.; Khayat, Rami N.

    2014-01-01

    The mechanism of vascular endothelial dysfunction (VED) and cardiovascular disease in obstructive sleep apnea (OSA) is unknown. We performed a comprehensive evaluation of endothelial nitric oxide synthase (eNOS) function directly in the microcirculatory endothelial tissue of OSA patients who have very low cardiovascular risk status. Nineteen OSA patients underwent gluteal biopsies before, and after effective treatment of OSA. We measured superoxide (O2−·) and nitric oxide (NO) in the microcir...

  19. Polyphenol-enriched diet prevents coronary endothelial dysfunction by activating the Akt/eNOS pathway.

    Science.gov (United States)

    Vilahur, Gemma; Padró, Teresa; Casaní, Laura; Mendieta, Guiomar; López, José A; Streitenberger, Sergio; Badimon, Lina

    2015-03-01

    The Mediterranean diet, rich in polyphenols, has shown to be cardioprotective. However the mechanisms involved remain unknown. We investigated whether supplementation with a pomegranate extract rich in polyphenols renders beneficial effects on coronary function in a clinically relevant experimental model and characterized the underlying mechanisms. Pigs were fed a 10-day normocholesterolemic or hypercholesterolemic diet. Half of the animals were given a supplement of 625 mg/day of a pomegranate extract (Pomanox; 200 mg punicalagins/day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide-synthase inhibitor) were measured using flow Doppler. Akt/endothelial nitric oxide-synthase axis activation, monocyte chemoattractant protein-1 expression, oxidative deoxyribonucleic acid damage in the coronary artery, and lipoprotein resistance to oxidation were evaluated. In dyslipidemic animals, Pomanox supplementation prevented diet-induced impairment of endothelial relaxation, reaching vasodilatory values comparable to normocholesterolemic animals upon stimulation with acetylcholine and/or calcium ionophore. These beneficial effects were associated with vascular Akt/endothelial nitric oxide-synthase activation and lower monocyte chemoattractant protein-1 expression. Pomanox supplementation reduced systemic oxidative stress (higher high-density lipoprotein-antioxidant capacity and higher low-density lipoprotein resistance to oxidation) and coronary deoxyribonucleic acid damage. Normocholesterolemic animals elicited similar drug-related vasodilation regardless of Pomanox supplementation. All animals displayed a similar vasodilatory response to sodium nitroprusside and L-NG-monomethylarginine blunted all vasorelaxation responses except for sodium nitroprusside. Pomanox supplementation hinders hyperlipemia-induced coronary endothelial dysfunction by activating

  20. [Corneal endothelial decompensation of iridocorneal endothelial syndrome treated by penetrating keratoplasty].

    Science.gov (United States)

    Chen, J; Liu, Z; Yu, L

    1996-07-01

    To evaluate the effect of penetrating keratoplasty in treatment of iridocorneal endothelial syndrome. The clinical and pathologic evaluation records were retrospectively analyzed for 8 cases with iridocorneal endothelial syndrome treated by penetrating keratoplasty. Postoperatively, they were followed for 3 months to 6 years. Seven cases had visual improvement, five buttons kept being transparent and three buttons became opaque. The penetrating keratoplasty is an effective measure for treatment of corneal endothelial decompensation of iridocorneal endothelial syndrome. However, the successful operative rate of this syndrome is lower than that in cases with corneal leucoma without vascularization.

  1. Leukocytes Breach Endothelial Barriers by Insertion of Nuclear Lobes and Disassembly of Endothelial Actin Filaments

    Directory of Open Access Journals (Sweden)

    Sagi Barzilai

    2017-01-01

    Full Text Available The endothelial cytoskeleton is a barrier for leukocyte transendothelial migration (TEM. Mononuclear and polymorphonuclear leukocytes generate gaps of similar micron-scale size when squeezing through inflamed endothelial barriers in vitro and in vivo. To elucidate how leukocytes squeeze through these barriers, we co-tracked the endothelial actin filaments and leukocyte nuclei in real time. Nuclear squeezing involved either preexistent or de novo-generated lobes inserted into the leukocyte lamellipodia. Leukocyte nuclei reversibly bent the endothelial actin stress fibers. Surprisingly, formation of both paracellular gaps and transcellular pores by squeezing leukocytes did not require Rho kinase or myosin II-mediated endothelial contractility. Electron-microscopic analysis suggested that nuclear squeezing displaced without condensing the endothelial actin filaments. Blocking endothelial actin turnover abolished leukocyte nuclear squeezing, whereas increasing actin filament density did not. We propose that leukocyte nuclei must disassemble the thin endothelial actin filaments interlaced between endothelial stress fibers in order to complete TEM.

  2. Cataract phacoemulsification and corneal endothelial cell damage

    Directory of Open Access Journals (Sweden)

    Ni Zhu

    2013-07-01

    Full Text Available Phacoemulsification with small incision, reduced number of inflammation cells, and better postoperative recovery has been recognized as the world's most popular option for cataract surgery. Modern cataract surgery is developing gradually from sight rehabilitating to refractive surgery with better vision acuity. Being the most important part of the eye refractive system, maintenance of the cornea's transparency relies heavily upon the healthy endothelial cells. It is well known that there will be endothelial cell loss after phacoemulsification and the damage of the endothelial cells may lead to corneal swellings and opacity, or even the corneal descompensation, which often severely influenced the postoperative vision recovery. This is a review of phacoemulsification and the risk factors of corneal endothelial damage pre-and postoperation.

  3. Apicobasal polarity of brain endothelial cells.

    Science.gov (United States)

    Worzfeld, Thomas; Schwaninger, Markus

    2016-02-01

    Normal brain homeostasis depends on the integrity of the blood-brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood-brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. © The Author(s) 2015.

  4. Bacteria and endothelial cells: a toxic relationship.

    Science.gov (United States)

    Lubkin, Ashira; Torres, Victor J

    2017-02-01

    Pathogenic bacteria use the bloodstream as a highway for getting around the body, and thus have to find ways to enter and exit through the endothelium. Many bacteria approach this problem by producing toxins that can breach the endothelial barrier through diverse creative mechanisms, including directly killing endothelial cells (ECs), weakening the cytoskeleton within ECs, and breaking the junctions between ECs. Toxins can also modulate the immune response by influencing endothelial biology, and can modulate endothelial function by influencing the response of leukocytes. Understanding these interactions, in both the in vitro and in vivo contexts, is of critical importance for designing new therapies for sepsis and other severe bacterial diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Specular Microscopic Features of Corneal Endothelial Vacuolation

    Directory of Open Access Journals (Sweden)

    Mozhgan Rezaei Kanavi

    2011-01-01

    Full Text Available Purpose: To introduce a specular microscopic reference image for endothelial vacuolation in donated corneas. Methods: Two corneas from a donor with diffuse, round to oval dark areas at the endothelial level on slit lamp biomicroscopy and one normal-appearing donor cornea underwent specular microscopy, histopathologic evaluation and transmission electron microscopy. Results: Specular microscopy of the two corneas with abnormal-looking endothelium revealed large numbers of dark, round to oval structures within the endothelium in favor of endothelial vacuolation. Light microscopy disclosed variable sized cyst-like structures within the cytoplasm. Transmission electron microscopy showed electronlucent and relatively large-sized intracytoplasmic vacuoles. These features were not observed in the endothelium of the normal cornea. Conclusion: The specular microscopic features of endothelial vacuolation in donated corneas were confirmed by light microscopy and transmission electron microscopy, therefore the specular image may be proposed as a reference to eye banks.

  6. Endothelial replacement: the limbal pocket approach.

    Science.gov (United States)

    Terry, Mark A

    2003-03-01

    The limbal pocket approach to endothelial replacement shows extraordinary promise in fulfilling the ideal goals of surgical treatment for endothelial dysfunction. From its inception, DLEK surgery has demonstrated that, by preserving the limbus and eliminating surface corneal incisions and sutures, the corneal topography can be stabilized with little change from preoperative measurements of astigmatism and corneal power. The surgical dissection planes of DLEK surgery are inherently stronger than that of PK or any other form of endothelial replacement, allowing early stability of the refraction and making the eye safer from blunt trauma over the long term. Further work with DLEK must be performed in perfecting the optical properties of the stromal interface before this technique can be used widely; however, as technology and techniques improve, DLEK surgery holds the promise of being the ideal method for endothelial replacement in the 21st century.

  7. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

    Science.gov (United States)

    Schuett, Jutta; Schuett, Harald; Oberoi, Raghav; Koch, Ann-Kathrin; Pretzer, Silke; Luchtefeld, Maren; Schieffer, Bernhard; Grote, Karsten

    2017-06-01

    NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells. © FASEB.

  8. The use of adipose mesenchymal stem cells and human umbilical vascular endothelial cells on a fibrin matrix for endothelialized skin substitute.

    Science.gov (United States)

    Sánchez-Muñoz, Isabel; Granados, Rosario; Holguín Holgado, Purificación; García-Vela, José Antonio; Casares, Celia; Casares, Miguel

    2015-01-01

    In recent years, the reconstruction of human skin by tissue engineering represents a clinical challenge and has offered a therapeutic alternative. Avascular engineered skin equivalents have been available for several years and used to treat wounds due to burns, nonhealing ulcers, and surgical excisions. They are constituted by different types of cultured cells included in a three-dimensional structure that permits cellular proliferation to create tissue substitutes. The major drawback of these artificial skin substitutes is their lack of blood supply, since the endurance and cell proliferation of the substitute depend on an adequate oxygen and nutrient supply and on toxin removal. These functions are served by the vascular system. We have produced a new model of endothelialized skin substitute that promotes the formation of capillary-like structures by seeding human umbilical vein endothelial cells (HUVECs) with dermal fibroblasts and human adipose-derived mesenchymal stem cells (hADMSCs) in a fibrin matrix. Dermal fibroblasts and hADMSCs produce extracellular matrix that stimulates cellular growth and proliferation. hADMSCs secrete significant quantities of angiogenic and antiapoptotic factors (vascular endothelial growth factor and hepatocyte growth factor), which induce in vitro differentiation of these cells into endothelial cells promoting angiogenesis and participating in tissue repair and skin regeneration processes. We obtained the artificial skin substitute with similar structure to native skin, including dermis and epidermis. We demonstrated that endothelial cells (CD31 and von Willebrand factor positive) proliferated and organized themselves into capillary-like structures within the fibrin matrix. The epidermis showed a complete epithelization by squamous cells (AE1/AE3 cytokeratin positive) with intracytoplasmic keratohyalin granules, hyperkeratosis, and parakeratosis. We have established a novel artificial skin substitute that facilitates the formation

  9. Derangements in endothelial cell-to-cell junctions involved in the pathogenesis of hypercholesterolemia-induced erectile dysfunction.

    Science.gov (United States)

    Ryu, Ji-Kan; Zhang, Lu Wei; Jin, Hai-Rong; Piao, Shuguang; Choi, Min Ji; Tuvshintur, Buyankhuu; Tumurbaatar, Munkhbayar; Shin, Sun Hwa; Han, Jee-Young; Kim, Woo Jean; Suh, Jun-Kyu

    2009-07-01

    Endothelial cell-to-cell junctions are crucial for vascular formation, networking, and remodeling of blood vessels as well as for inducing and integrating intracellular signals. We investigated the differential expression and distribution of endothelial cell-to-cell junction proteins in the penis of mice with hypercholesterolemia-induced erectile dysfunction. Two-month-old C57BL/6J mice were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. We performed dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) (Seegene, Seoul, Korea) to screen the differential gene expression of 21 endothelial cell-to-cell junctions. At 5 months, erectile function was measured by electrical stimulation of the cavernous nerve, and the penis was harvested and stained with antibody to claudin-5, vascular endothelial (VE)-cadherin, and platelet/endothelial cell adhesion molecule (PECAM)-1 (N = 8 per group). Cavernous specimens from a separate group of animals were used for claudin-5, VE-cadherin, and PECAM-1 reverse transcriptase-PCR and Western blot analysis. Erectile function was significantly lower in hypercholesterolemic mice than in controls. DPO-based multiplex PCR revealed a profound decrease in the gene expression of endothelium-specific cell-to-cell junction proteins, including claudin-5, VE-cadherin, and PECAM-1, in hypercholesterolemic mice compared with that in controls. The expression of claudin-5, VE-cadherin, and PECAM-1 protein evaluated by Western blot or immunohistochemistry was significantly lower in hypercholesterolemic mice than in controls. These endothelial cell-to-cell junction proteins were more sparsely distributed in the endothelium of cavernous sinusoids than in the endothelium of cavernous artery and dorsal blood vessels. Down-regulation of the endothelial cell-to-cell junctions and decreased endothelial content in the corpus cavernosum might play a major

  10. [Endothelial dysfunction in pathogenesis of duodenal ulcer].

    Science.gov (United States)

    Oparin, A G; Oparin, A A

    2002-01-01

    It is shown that in patients with ulcer associated with Helicobacter pylori (HP) there is a close correlation between the severity of the lesion of gastroduodenal protective mucous barrier and that of endothelial dysfunction manifesting in elevated level of endothelin-1, serum levels of TBK-active products, inhibition of blood flow and narrowing of the celiac trunk. The correlation becomes stronger with expanding contamination of gastroduodenal mucosa with HP. Thus, HP may participate in breaking the protective mucous barrier in endothelial dysfunction.

  11. Endothelial microparticles: Sophisticated vesicles modulating vascular function

    Science.gov (United States)

    Curtis, Anne M; Edelberg, Jay; Jonas, Rebecca; Rogers, Wade T; Moore, Jonni S; Syed, Wajihuddin; Mohler, Emile R

    2015-01-01

    Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation. PMID:23892447

  12. Endothelial Dysfunction in Chronic Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Curtis M. Steyers

    2014-06-01

    Full Text Available Chronic inflammatory diseases are associated with accelerated atherosclerosis and increased risk of cardiovascular diseases (CVD. As the pathogenesis of atherosclerosis is increasingly recognized as an inflammatory process, similarities between atherosclerosis and systemic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel diseases, lupus, psoriasis, spondyloarthritis and others have become a topic of interest. Endothelial dysfunction represents a key step in the initiation and maintenance of atherosclerosis and may serve as a marker for future risk of cardiovascular events. Patients with chronic inflammatory diseases manifest endothelial dysfunction, often early in the course of the disease. Therefore, mechanisms linking systemic inflammatory diseases and atherosclerosis may be best understood at the level of the endothelium. Multiple factors, including circulating inflammatory cytokines, TNF-α (tumor necrosis factor-α, reactive oxygen species, oxidized LDL (low density lipoprotein, autoantibodies and traditional risk factors directly and indirectly activate endothelial cells, leading to impaired vascular relaxation, increased leukocyte adhesion, increased endothelial permeability and generation of a pro-thrombotic state. Pharmacologic agents directed against TNF-α-mediated inflammation may decrease the risk of endothelial dysfunction and cardiovascular disease in these patients. Understanding the precise mechanisms driving endothelial dysfunction in patients with systemic inflammatory diseases may help elucidate the pathogenesis of atherosclerosis in the general population.

  13. Interaction between Endothelial Protein C Receptor and Intercellular Adhesion Molecule 1 to Mediate Binding of Plasmodium falciparum-Infected Erythrocytes to Endothelial Cells.

    Science.gov (United States)

    Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell; Brazier, Andrew Jay; Smith, Joseph D

    2016-07-12

    Intercellular adhesion molecule 1 (ICAM-1) and the endothelial protein C receptor (EPCR) are candidate receptors for the deadly complication cerebral malaria. However, it remains unclear if Plasmodium falciparum parasites with dual binding specificity are involved in cytoadhesion or different parasite subpopulations bind in brain microvessels. Here, we investigated this issue by studying different subtypes of ICAM-1-binding parasite lines. We show that two parasite lines expressing domain cassette 13 (DC13) of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family have dual binding specificity for EPCR and ICAM-1 and further mapped ICAM-1 binding to the first DBLβ domain following the PfEMP1 head structure in both proteins. As PfEMP1 head structures have diverged between group A (EPCR binders) and groups B and C (CD36 binders), we also investigated how ICAM-1-binding parasites with different coreceptor binding traits influence P. falciparum-infected erythrocyte binding to endothelial cells. Whereas levels of binding to tumor necrosis factor alpha (TNF-α)-stimulated endothelial cells from the lung and brain by all ICAM-1-binding parasite lines increased, group A (EPCR and ICAM-1) was less dependent than group B (CD36 and ICAM-1) on ICAM-1 upregulation. Furthermore, both group A DC13 parasite lines had higher binding levels to brain endothelial cells (a microvascular niche with limited CD36 expression). This study shows that ICAM-1 is a coreceptor for a subset of EPCR-binding parasites and provides the first evidence of how EPCR and ICAM-1 interact to mediate parasite binding to both resting and TNF-α-activated primary brain and lung endothelial cells. Cerebral malaria is a severe neurological complication of P. falciparum infection associated with infected erythrocyte (IE) binding in cerebral vessels. Yet little is known about the mechanisms by which parasites adhere in the brain or other microvascular sites. Here, we studied parasite lines

  14. Regulation of endothelial barrier function during flow-induced conversion to an arterial phenotype.

    Science.gov (United States)

    Seebach, Jochen; Donnert, Gerald; Kronstein, Romy; Werth, Sebastian; Wojciak-Stothard, Beata; Falzarano, Darryl; Mrowietz, Christof; Hell, Stefan W; Schnittler, Hans-J

    2007-08-01

    Flow-induced conversion of endothelial cells into an elongated arterial phenotype requires a coordinated regulation of cell junctions. Here we investigated the effect of acute and chronic flow on junction regulation. Using an extended experimental setup that allows analyses of endothelial barrier function under flow conditions, we found a flow-induced upregulation of the transendothelial electrical resistance within minutes. This was accompanied by an increase in actin filaments along the junctions and vascular endothelial (VE)-cadherin clustering, which was identified at nanoscale resolution by stimulated emission depletion microscopy. In addition, a transient tyrosine phosphorylation of VE-cadherin and catenins occurred within minutes following the onset of flow. VE-cadherin and actin distribution were maintained under chronic flow over 24 h and associated with the upregulation of VE-cadherin and alpha-catenin expression, thus compensating for the cell elongation-mediated increase in cell border length. Importantly, all observed effects were rac1 dependent as verified by the inhibitory effect of dominant negative N17rac1. These results show that flow-induced conversion of endothelial cells into an arterial phenotype occurs while intercellular junctions remain intact. The data place rac1 in a central multimodal regulatory position that might be important in the development of vascular diseases, such as arteriosclerosis.

  15. Smooth muscle–endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

    Science.gov (United States)

    Lutter, Sophie; Xie, Sherry; Tatin, Florence

    2012-01-01

    Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels—smooth muscle and endothelial cells—and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function. PMID:22665518

  16. Nicorandil prevents sirolimus-induced production of reactive oxygen species, endothelial dysfunction, and thrombus formation

    Directory of Open Access Journals (Sweden)

    Ken Aizawa

    2015-03-01

    Full Text Available Sirolimus (SRL is widely used to prevent restenosis after percutaneous coronary intervention. However, its beneficial effect is hampered by complications of thrombosis. Several studies imply that reactive oxygen species (ROS play a critical role in endothelial dysfunction and thrombus formation. The present study investigated the protective effect of nicorandil (NIC, an anti-angina agent, on SRL-associated thrombosis. In human coronary artery endothelial cells (HCAECs, SRL stimulated ROS production, which was prevented by co-treatment with NIC. The preventive effect of NIC on ROS was abolished by 5-hydroxydecanoate but not by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. NIC also inhibited SRL-induced up-regulation of NADPH oxidase subunit p22phox mRNA. Co-treatment with NIC and SRL significantly up-regulated superoxide dismutase 2. NIC treatment significantly improved SRL-induced decrease in viability of HCAECs. The functional relevance of the preventive effects of NIC on SRL-induced ROS production and impairment of endothelial viability was investigated in a mouse model of thrombosis. Pretreatment with NIC inhibited the SRL-induced acceleration of FeCl3-initiated thrombus formation and ROS production in the testicular arteries of mice. In conclusion, NIC prevented SRL-induced thrombus formation, presumably due to the reduction of ROS and to endothelial protection. The therapeutic efficacy of NIC could represent an additional option in the prevention of SRL-related thrombosis.

  17. A novel compound, NP-184, inhibits the vascular endothelial growth factor induced angiogenesis.

    Science.gov (United States)

    Lin, Kuan-Ting; Lien, Jin-Cherng; Chung, Ching-Hu; Kuo, Sheng-Chu; Huang, Tur-Fu

    2010-03-25

    Angiogenesis is observed in many diseases, such as tumor progression, diabetes and rheumatoid arthritis; it is a process that involves proliferation, migration, differentiation and tube formation of endothelial cells. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by induction of these endothelial functions. Thus, inhibition of these critical angiogenic steps is a practical therapeutic strategy for those diseases. NP-184 is a substituted benzimidazole analogue which exhibits a potent anti-thrombotic activity. In this report, NP-184 inhibited the viability of human umbilical vascular endothelial cells (HUVEC) in a concentration-dependent manner, and caused cell apoptosis as examined by cell-cycle analysis and Annexin V staining with flow cytometry. NP-184 also concentration-dependently inhibited the HUVEC migration, tube formation on Matrigel, and rat aortic ring sprouting stimulated by VEGF. Regarding the intracellular signal transduction, NP-184 concentration-dependently interfered with the activation of AKT, ERK and the nuclear translocation of NF-kappaB. In vivo study showed that NP-184 dose-dependently reduced angiogenesis in Matrigel plug assay. These results indicate that NP-184 is a potential candidate for developing the treatment of angiogenesis related-diseases.

  18. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Science.gov (United States)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

    2016-11-01

    Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  19. Small and Intermediate Calcium-Activated Potassium Channel Openers Improve Rat Endothelial and Erectile Function

    Science.gov (United States)

    Comerma-Steffensen, Simon G.; Carvacho, Ingrid; Hedegaard, Elise R.; Simonsen, Ulf

    2017-01-01

    Modulation of endothelial calcium-activated potassium (KCa) channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of KCa channels with small (KCa2.x) and intermediate (KCa3.1) conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC) strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP), intracavernosal pressure (ICP), and electrocardiographic (ECG) measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of KCa2.3 and large KCa conductance (KCa1.1) channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of KCa2.x and KCa3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of KCa2.3 channels has a potential for treatment of erectile dysfunction. PMID:28993731

  20. Spinal cord stimulation

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007560.htm Spinal cord stimulation To use the sharing features on this page, please enable JavaScript. Spinal cord stimulation is a treatment for pain that uses a ...

  1. Feldspar, Infrared Stimulated Luminescence

    DEFF Research Database (Denmark)

    Jain, Mayank

    2014-01-01

    This entry primarily concerns the characteristics and the origins of infrared-stimulated luminescence in feldspars.......This entry primarily concerns the characteristics and the origins of infrared-stimulated luminescence in feldspars....

  2. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  3. Anti-atherogenic effects of statins: Impact on angiopoietin-2 release from endothelial cells.

    Science.gov (United States)

    Hilbert, Tobias; Poth, Jens; Frede, Stilla; Klaschik, Sven; Hoeft, Andreas; Baumgarten, Georg; Knuefermann, Pascal

    2013-11-15

    Beyond lipid lowering, statins are supposed to exert pleiotropic effects positively influencing the progression of atherosclerotic lesions. The development of such lesions is associated with increased release of angiopoietin-2 (Ang-2), an endothelial cell-specific protein growth factor stored in Weibel-Palade bodies (WPBs). The aim of our study was to examine whether statin pretreatment influences the release of Ang-2 from endothelial cells. Stimulation of HUVECs and HMVECs with PMA, thrombin or histamine resulted in significant release of Ang-2, as evidenced by ELISA. Pretreatment with simvastatin and mevastatin suppressed this release to basal level, while pravastatin had no effect. Simvastatin itself increased nitric oxide (NO, EC number 1.14.13.39) synthesis, measured by Griess reaction. Combining the statin pretreatment with the eNOS inhibitor L-NNA as well as bypassing the HMG-CoA reductase (EC number: 1.1.1.34) by adding mevalonic acid or geranyl pyrophosphate restored the exocytotic effect of PMA. Immunofluorescence microscopy showed that depletion of WPBs upon PMA stimulation ceased after pretreatment with simvastatin. This study demonstrates a potent suppressive effect of statins on the release of Ang-2 from endothelial cells. Regarding its harmful effects in the development of atherosclerotic lesions, our data provide further insight into the mechanisms of the anti-atherogenic potential of statins. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Endothelial progenitor cells express PAF receptor and respond to PAF via Ca(2+)-dependent signaling.

    Science.gov (United States)

    Balestrieri, Maria Luisa; Giovane, Alfonso; Milone, Lara; Servillo, Luigi

    2010-10-01

    Endothelial progenitor cell (EPC) therapy is a promising approach to promote angiogenesis and endothelial repair in patients with cardiovascular diseases (CVD). However, their release of proinflammatory mediators may compromise the therapeutic efficacy. Little is known about the role of Platelet-Activating Factor (PAF) in EPC functional response. Here, we investigated the expression of PAF receptor (PAF-R) in early EPC and the release of PAF under stimulation with factors involved in endothelial dysfunction. Results indicated that early EPC express the PAF-R and respond to PAF signaling via a transient increase of cytoplasmic Ca(2+) concentration. EPC release PAF in a time dependent manner upon stimulation with tumor necrosis factor-alpha (TNF-alpha) or high-glucose concentration with a peak at 30 min and 10 min (pPAF, starting at concentration of 50 ng/ml, exerted a detrimental effect on EPC number with a concomitant increase of p38 activity. Furthermore, both the reduction of early EPC number and the enhanced p38 activity induced by PAF were abolished by CV3988, a PAF receptor antagonist. These novel findings, revealing that early EPC respond to PAF signaling, unveil an inflammatory pathway that may play a crucial role in the outcome of cardiovascular cell therapy with EPC. Copyright @ 2010 Elsevier B.V. All rights reserved.

  5. Endothelial endoglin is involved in inflammation: role in leukocyte adhesion and transmigration.

    Science.gov (United States)

    Rossi, Elisa; Sanz-Rodriguez, Francisco; Eleno, Nelida; Düwell, Annette; Blanco, Francisco J; Langa, Carmen; Botella, Luisa M; Cabañas, Carlos; Lopez-Novoa, José M; Bernabeu, Carmelo

    2013-01-10

    Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5β1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5β1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.

  6. Vascular Growth in Ischemic Limbs: A Review of Mechanisms and Possible Therapeutic Stimulation

    NARCIS (Netherlands)

    Weel, V. van; Tongeren, R.B. van; Hinsbergh, V.W.M. van; Bockel, J.H. van; Quax, P.H.A.

    2008-01-01

    Stimulation of vascular growth to treat limb ischemia is promising, and early results obtained from uncontrolled clinical trials using angiogenic agents, e.g., vascular endothelial growth factor, led to high expectations. However, negative results from recent placebo-controlled trials warrant

  7. Protection of Coronary Endothelial Function during Cardiac Surgery: Potential of Targeting Endothelial Ion Channels in Cardioprotection

    Directory of Open Access Journals (Sweden)

    Qin Yang

    2014-01-01

    Full Text Available Vascular endothelium plays a critical role in the control of blood flow by producing vasoactive factors to regulate vascular tone. Ion channels, in particular, K+ channels and Ca2+-permeable channels in endothelial cells, are essential to the production and function of endothelium-derived vasoactive factors. Impairment of coronary endothelial function occurs in open heart surgery that may result in reduction of coronary blood flow and thus in an inadequate myocardial perfusion. Hyperkalemic exposure and concurrent ischemia-reperfusion during cardioplegic intervention compromise NO and EDHF-mediated function and the impairment involves alterations of K+ channels, that is, KATP and KCa, and Ca2+-permeable TRP channels in endothelial cells. Pharmacological modulation of these channels during ischemia-reperfusion and hyperkalemic exposure show promising results on the preservation of NO and EDHF-mediated endothelial function, which suggests the potential of targeting endothelial K+ and TRP channels for myocardial protection during cardiac surgery.

  8. Endothelial toxicity of unusual nucleotide metabolites.

    Science.gov (United States)

    Pelikant-Malecka, Iwona; Sielicka, Alicja; Kaniewska, Ewa; Smolenski, Ryszard T; Slominska, Ewa M

    2015-08-01

    Endothelium plays a pivotal role in the vascular tone regulation, platelet aggregation, regulation of immune response, inflammation and angiogenesis and its dysfunction is an earliest event in the development of cardiovascular disease. All these processes are affected by endothelial dysfunction. Endothelial toxicity induced by metabolites present in blood is a common scenario in pathology. This involves physiological metabolites such as asymmetric dimethylarginine or homocysteine that are normally excreted by kidneys, but accumulate in pathological conditions, adversely affecting function of endothelium. Our group identified new molecule with potential endothelial toxicity: 4-pirydone-3-carboxamide-1-β-d-ribonucleoside (4PYR). This nucleoside is most likely produced by oxidation of nicotinamide containing precursor by aldehyde oxidase. 4PYR easy crosses cell membrane and become phosphorylated inside the cell giving rise to mono-, di- and triphospates (4PYMP, 4PYDP and 4PYTP). There is considerable evidence that 4PYR is toxic in endothelium and other cell types by disrupting cell energetics evident as ATP depletion. Endothelial dysfunction in the in vitro and in vivo experiments is, however, evident only after prolonged exposure to 4PYR while acute cardiovascular effects are minor. 4PYR endothelial toxicity could be particularly important in patients with chronic renal disease where accumulation of 4PYR and its metabolites is particularly prominent. 4PYR metabolism and toxicity could be blocked by application of nucleoside transport inhibitors and we have proven efficiency of such intervention. We believe that blocking metabolism of endothelial nucleoside toxins such as 4PYR could become important strategy for endothelial targeted therapy. Copyright © 2015. Published by Elsevier Urban & Partner Sp. z o.o.

  9. Interleukin-34 restores blood-brain barrier integrity by upregulating tight junction proteins in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Shijie Jin

    Full Text Available Interleukin-34 (IL-34 is a newly discovered cytokine as an additional ligand for colony stimulating factor-1 receptor (CSF1R, and its functions are expected to overlap with colony stimulating factor-1/macrophage-colony stimulating factor. We have previously shown that the IL-34 is primarily produced by neurons in the central nervous system (CNS and induces proliferation and neuroprotective properties of microglia which express CSF1R. However, the functions of IL-34 in the CNS are still elucidative. Here we show that CNS capillary endothelial cells also express CSF1R. IL-34 protected blood-brain barrier integrity by restored expression levels of tight junction proteins, which were downregulated by pro-inflammatory cytokines. The novel function of IL-34 on the blood-brain barrier may give us a clue for new therapeutic strategies in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis and Alzheimer's disease.

  10. Engineering an endothelialized vascular graft: a rational approach to study design in a non-human primate model.

    Directory of Open Access Journals (Sweden)

    Deirdre E J Anderson

    Full Text Available After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data-correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.

  11. Brain Stimulation in Addiction.

    Science.gov (United States)

    Salling, Michael C; Martinez, Diana

    2016-11-01

    Localized stimulation of the human brain to treat neuropsychiatric disorders has been in place for over 20 years. Although these methods have been used to a greater extent for mood and movement disorders, recent work has explored brain stimulation methods as potential treatments for addiction. The rationale behind stimulation therapy in addiction involves reestablishing normal brain function in target regions in an effort to dampen addictive behaviors. In this review, we present the rationale and studies investigating brain stimulation in addiction, including transcranial magnetic stimulation, transcranial direct current stimulation, and deep brain stimulation. Overall, these studies indicate that brain stimulation has an acute effect on craving for drugs and alcohol, but few studies have investigated the effect of brain stimulation on actual drug and alcohol use or relapse. Stimulation therapies may achieve their effect through direct or indirect modulation of brain regions involved in addiction, either acutely or through plastic changes in neuronal transmission. Although these mechanisms are not well understood, further identification of the underlying neurobiology of addiction and rigorous evaluation of brain stimulation methods has the potential for unlocking an effective, long-term treatment of addiction.

  12. Endothelial monolayer permeability under controlled oxygen tension.

    Science.gov (United States)

    Funamoto, Kenichi; Yoshino, Daisuke; Matsubara, Kento; Zervantonakis, Ioannis K; Funamoto, Kiyoe; Nakayama, Masafumi; Masamune, Jun; Kimura, Yoshitaka; Kamm, Roger D

    2017-06-19

    Endothelial permeability has been extensively investigated in the context of pathologies such as cancer and also in studies of drug delivery from the circulation. Hypoxia is a critical regulator of endothelial cell (EC) behavior and affects the barrier function of endothelial linings, yet its role has been little studied. This paper reveals the effect of hypoxia on the permeability of an EC monolayer by cellular experiments using a microfluidic device and a conventional cell culture dish. Human umbilical vein endothelial cells (HUVECs) were seeded into one microfluidic channel, creating an EC monolayer on each vertical surface of a collagen gel confined to a central chamber. Oxygen tension was regulated to produce normoxic (21% O2) or hypoxic (3% O2) conditions by the supply of gas mixtures of oxygen, carbon dioxide, and nitrogen at predefined ratios into channels fabricated into the device. Permeability of the EC monolayer quantified by analyzing diffusion of fluorescence-labelled dextrans into the collagen gel increases with barrier function loss by 6 hour hypoxic exposure, showing 11-fold and 4-fold increases for 70 kDa and 10 kDa dextrans, respectively, on average. Consistent with this, subsequent immunofluorescent staining and separate western blot analysis of HUVECs on a culture dish demonstrate loose cell-cell adhesion resulting from internalization of VE-cadherin under hypoxia. Thus, hypoxic stress increases endothelial permeability by altering cell-cell junction integrity.

  13. Dietary phosphorus acutely impairs endothelial function.

    Science.gov (United States)

    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.

  14. [Assessment of endothelial function in autoimmune diseases].

    Science.gov (United States)

    Benhamou, Y; Bellien, J; Armengol, G; Gomez, E; Richard, V; Lévesque, H; Joannidès, R

    2014-08-01

    Numerous autoimmune-inflammatory rheumatic diseases have been associated with accelerated atherosclerosis or other types of vasculopathy leading to an increase in cardiovascular disease incidence. In addition to traditional cardiovascular risk factors, endothelial dysfunction is an important early event in the pathogenesis of atherosclerosis, contributing to plaque initiation and progression. Endothelial dysfunction is characterized by a shift of the actions of the endothelium toward reduced vasodilation, a proinflammatory and a proadhesive state, and prothrombic properties. Therefore, assessment of endothelial dysfunction targets this vascular phenotype using several biological markers as indicators of endothelial dysfunction. Measurements of soluble adhesion molecules (ICAM-1, VCAM-1, E-selectin), pro-thrombotic factors (thrombomodulin, von Willebrand factor, plasminogen activator inhibitor-1) and inflammatory cytokines are most often performed. Regarding the functional assessment of the endothelium, the flow-mediated dilatation of conduit arteries is a non-invasive method widely used in pathophysiological and interventional studies. In this review, we will briefly review the most relevant information upon endothelial dysfunction mechanisms and explorations. We will summarize the similarities and differences in the biological and functional assessments of the endothelium in different autoimmune diseases. Copyright © 2013 Société nationale française de médecine interne (SNFMI). Published by Elsevier SAS. All rights reserved.

  15. Endothelial Extracellular Vesicles-Promises and Challenges.

    Science.gov (United States)

    Hromada, Carina; Mühleder, Severin; Grillari, Johannes; Redl, Heinz; Holnthoner, Wolfgang

    2017-01-01

    Extracellular vesicles, including exosomes, microparticles, and apoptotic bodies, are phospholipid bilayer-enclosed vesicles that have once been considered as cell debris lacking biological functions. However, they have recently gained immense interest in the scientific community due to their role in intercellular communication, immunity, tissue regeneration as well as in the onset, and progression of various pathologic conditions. Extracellular vesicles of endothelial origin have been found to play a versatile role in the human body, since they are on the one hand known to contribute to cardiovascular diseases, but on the other hand have also been reported to promote endothelial cell survival. Hence, endothelial extracellular vesicles hold promising therapeutic potential to be used as a new tool to detect as well as treat a great number of diseases. This calls for clinically approved, standardized, and efficient isolation and characterization protocols to harvest and purify endothelial extracellular vesicles. However, such methods and techniques to fulfill stringent requirements for clinical trials have yet to be developed or are not harmonized internationally. In this review, recent advances and challenges in the field of endothelial extracellular vesicle research are discussed and current problems and limitations regarding isolation and characterization are pointed out.

  16. Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

    Science.gov (United States)

    Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

    2017-01-01

    Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

  17. Protective Effect of Allium tuberosum Extract on Vascular Inflammation in Tumor Necrosis Factor-α-induced Human Vascular Endothelial Cells.

    Science.gov (United States)

    Hur, Haeng Jeon; Lee, Ae Sin

    2017-12-01

    Endothelial adhesion molecule expression induced by pro-inflammatory cytokine plays an important role in vascular endothelial cell injury, leading to vascular disease. Allium tuberosum (AT), which is used as a functional food, has a thrombolytic effect. It contains vitamin A, vitamin C, carbohydrate, calcium, iron, and phosphorus. There are many carotenes that turn into vitamin A in the body. Also, it helps blood circulation and stimulates metabolism. The purpose of the this study was to estimate the anti-inflammatory effects of the AT extract. Human vascular endothelial cells were pre-treated with 100 μg/mL AT extract for 30 minutes and subsequently co-treated with TNF-α (10 ng/mL) and AT extract (100 μg/mL) for 1, 4, and 6 hours. After treatment, the cells were lysed and used for quantitative reverse transcription PCR, Western blot analysis, and monocyte adhesion assay. We examined the effect of the AT extract on inflammatory gene expression in TNF-α-induced human umbilical vein endothelial cells (HUVECs). The extract reduced the expression levels of mRNA and protein of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in TNF-α-stimulated HUVECs. It also inhibited the TNF-α-induced phosphorylation of the NF-κB p65 subunit and degradation of IκBα. Furthermore, the AT extract prevented the increased adhesion capacity of monocyte to TNF-α-stimulated vascular endothelial cells by reducing ICAM-1 and VCAM-1 expression. The AT extract has preventive and anti-inflammatory effect against vascular disease and has potential for supporting prevention against the early process of atherosclerosis.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  20. IL-1 beta and TGF beta 2 synergistically induce endothelial to mesenchymal transition in an NF kappa B-dependent manner

    NARCIS (Netherlands)

    Maleszewska, Monika; Moonen, Jan-Renier A. J.; Huijkman, Nicolette; van de Sluis, Bart; Krenning, Guido; Harmsen, Martin C.

    Endothelial to mesenchymal transition (EndMT) contributes to fibrotic diseases. The main inducer of EndMT is TGF beta signaling. TGF beta 2 is the dominant isoform in the physiological embryonic EndMT, but its role in the pathological EndMT in the context of inflammatory co-stimulation is not known.

  1. Improvement of endothelial dysfunction in experimental heart failure by chronic RAAS-blockade : ACE-inhibition or AT(1)-receptor blockade?

    NARCIS (Netherlands)

    Buikema, H; Pinto, YM; van Gilst, WH

    Chronic heart failure (CHF) is associated with endothelial dysfunction. Activation of the renin-angiotensin-aldosterone system (RAAS) is believed to be important in the deterioration of endodiehal dysfunction in CHF through stimulation of oxidative stress. Whereas angiotensin-converting enzyme

  2. Induction of angiogenesis and modulation of vascular endothelial growth factor receptor-2 by simvastatin after traumatic brain injury.

    Science.gov (United States)

    Wu, Hongtao; Jiang, Hao; Lu, Dunyue; Qu, Changsheng; Xiong, Ye; Zhou, Dong; Chopp, Michael; Mahmood, Asim

    2011-05-01

    Our previous studies demonstrated that simvastatin reduced neuronal death, increased neurogenesis, and promoted functional recovery after traumatic brain injury (TBI). To investigate the effect of simvastatin on angiogenesis after TBI and the related signaling pathways. Saline or simvastatin (1 mg/kg) was administered orally to rats starting at day 1 after TBI or sham surgery and then daily for 14 days. Rats were sacrificed at 3 and 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining, enzyme-linked immunosorbent assay, and Western blot analysis. Cultured rat brain microvascular endothelial cells were subjected to oxygen-glucose deprivation followed by immunocytochemical staining with phallotoxins and vascular endothelial growth factor receptor-2 (VEGFR-2). Western blot analysis was carried out to examine the simvastatin-induced activation of the v-akt murine thymoma viral oncogene homolog (Akt) signaling pathway. The expression of VEGFR-2 was detected by enzyme-linked immunosorbent assay. Simvastatin significantly increased the length of vascular perimeter, promoted the proliferation of endothelial cells, and improved the sensorimotor function after TBI. Simvastatin stimulated endothelial cell tube formation after oxygen-glucose deprivation in vitro. VEGFR-2 expression in both brain tissues and cultured rat brain microvascular endothelial cells was enhanced after simvastatin treatment, which may be modulated by activation of Akt. Akt-dependent endothelial nitric oxide synthase phosphorylation was also induced by simvastatin in vivo and in vitro. Simvastatin augments TBI-induced angiogenesis in the lesion boundary zone and hippocampus and improves functional recovery. Simvastatin also promotes angiogenesis in vitro. These beneficial effects on angiogenesis may be related to simvastatin-induced activation of the VEGFR-2/Akt/endothelial nitric oxide synthase signaling pathway.

  3. Complement component C3a plays a critical role in endothelial activation and leukocyte recruitment into the brain.

    Science.gov (United States)

    Wu, Fengjiao; Zou, Qiang; Ding, Xiaodan; Shi, Dongyan; Zhu, Xingxing; Hu, Weiguo; Liu, Lixin; Zhou, Hong

    2016-01-28

    The complement system is becoming increasingly recognized as a key participant in many neurodegenerative diseases of the brain. Complement-deficient animals exhibit reduced neuroinflammation. In the present study, we administered intracerebroventricularly lipopolysaccharide (LPS) to mimic local infection of the brain and investigated the role of key complement component C3 in brain vasculature endothelial activation and leukocyte recruitment. The degree of neutrophil infiltration was determined by esterase staining. Leukocyte-endothelial interactions were measured using intravital microscopy. Cerebral endothelial activation was evaluated using real-time PCR and Western blotting. Neutrophil infiltration into the brain cortex and hippocampus was significantly reduced in C3(-/-) mice and C3aR(-/-) mice but not in C6(-/-) mice. We detected markedly attenuated leukocyte-endothelial interactions in the brain microvasculature of C3(-/-) mice. Accordingly, in response to LPS administration, the brain microvasculature in these mice had decreased expression of P-selectin, E-selectin, intercellular cell adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Depletion of C3 from the circulation also caused reduction in VCAM-1 and E-selectin expression and leukocyte recruitment, suggesting that C3 in the circulation contributed to brain endothelial activation. Furthermore, C3(-/-) mice exhibited decreased leukocyte recruitment into the brain upon tumor necrosis factor-α (TNF-α) stimulation. C3a activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) and induced the upregulation of VCAM-1 and ICAM-1 expression in murine primary cerebral endothelial cells in vitro. Our study provides the first evidence that C3a plays a critical role in cerebral endothelial activation and leukocyte recruitment during inflammation in the brain.

  4. Inflammation-induced endothelial cell-derived extracellular vesicles modulate the cellular status of pericytes.

    Science.gov (United States)

    Yamamoto, Seiji; Niida, Shumpei; Azuma, Erika; Yanagibashi, Tsutomu; Muramatsu, Masashi; Huang, Ting Ting; Sagara, Hiroshi; Higaki, Sayuri; Ikutani, Masashi; Nagai, Yoshinori; Takatsu, Kiyoshi; Miyazaki, Kenji; Hamashima, Takeru; Mori, Hisashi; Matsuda, Naoyuki; Ishii, Yoko; Sasahara, Masakiyo

    2015-02-17

    Emerging lines of evidence have shown that extracellular vesicles (EVs) mediate cell-to-cell communication by exporting encapsulated materials, such as microRNAs (miRNAs), to target cells. Endothelial cell-derived EVs (E-EVs) are upregulated in circulating blood in different pathological conditions; however, the characteristics and the role of these E-EVs are not yet well understood. In vitro studies were conducted to determine the role of inflammation-induced E-EVs in the cell-to-cell communication between vascular endothelial cells and pericytes/vSMCs. Stimulation with inflammatory cytokines and endotoxin immediately induced release of shedding type E-EVs from the vascular endothelial cells, and flow cytometry showed that the induction was dose dependent. MiRNA array analyses revealed that group of miRNAs were specifically increased in the inflammation-induced E-EVs. E-EVs added to the culture media of cerebrovascular pericytes were incorporated into the cells. The E-EV-supplemented cells showed highly induced mRNA and protein expression of VEGF-B, which was assumed to be a downstream target of the miRNA that was increased within the E-EVs after inflammatory stimulation. The results suggest that E-EVs mediate inflammation-induced endothelial cell-pericyte/vSMC communication, and the miRNAs encapsulated within the E-EVs may play a role in regulating target cell function. E-EVs may be new therapeutic targets for the treatment of inflammatory diseases.

  5. Endothelial nitric oxide synthase uncoupling: a novel pathway in OSA induced vascular endothelial dysfunction.

    Science.gov (United States)

    Varadharaj, Saradhadevi; Porter, Kyle; Pleister, Adam; Wannemacher, Jacob; Sow, Angela; Jarjoura, David; Zweier, Jay L; Khayat, Rami N

    2015-02-01

    The mechanism of vascular endothelial dysfunction (VED) and cardiovascular disease in obstructive sleep apnea (OSA) is unknown. We performed a comprehensive evaluation of endothelial nitric oxide synthase (eNOS) function directly in the microcirculatory endothelial tissue of OSA patients who have very low cardiovascular risk status. Nineteen OSA patients underwent gluteal biopsies before, and after effective treatment of OSA. We measured superoxide (O2(•-)) and nitric oxide (NO) in the microcirculatory endothelium using confocal microscopy. We evaluated the effect of the NOS inhibitor l-Nitroarginine-Methyl-Ester (l-NAME) and the NOS cofactor tetrahydrobiopterin (BH4) on endothelial O2(•-) and NO in patient endothelial tissue before and after treatment. We found that eNOS is dysfunctional in OSA patients pre-treatment, and is a source of endothelial O2(•-) overproduction. eNOS dysfunction was reversible with the addition of BH4. These findings provide a new mechanism of endothelial dysfunction in OSA patients and a potentially targetable pathway for treatment of cardiovascular risk in OSA. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Pre-Descemet's endothelial keratoplasty (PDEK).

    Science.gov (United States)

    Agarwal, Amar; Dua, Harminder S; Narang, Priya; Kumar, Dhivya A; Agarwal, Ashvin; Jacob, Soosan; Agarwal, Athiya; Gupta, Ankur

    2014-09-01

    A new surgical technique for endothelial keratoplasty is described, in which the composite of pre-Descemet's layer (Dua's layer) with Descemet's membrane and endothelium is transplanted subsequent to the removal of the recipient's Descemet's membrane. The technique was performed in five eyes of five patients, with successful attachment of the graft and good postoperative visual recovery in all cases. Postoperative optical coherence tomography showed good graft attachment without interface abnormalities and a mean graft thickness was 28±5.6 μm. This study demonstrates the practicality of the technique, termed pre-Descemet's endothelial keratoplasty (PDEK), which can be a viable option in endothelial keratoplasty with some potential advantages. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  7. Levosimendan exerts anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro.

    Science.gov (United States)

    Krychtiuk, Konstantin A; Watzke, Lukas; Kaun, Christoph; Buchberger, Elisabeth; Hofer-Warbinek, Renate; Demyanets, Svitlana; Pisoni, Julia; Kastl, Stefan P; Rauscher, Sabine; Gröger, Marion; Aliabadi, Arezu; Zuckermann, Andreas; Maurer, Gerald; de Martin, Rainer; Huber, Kurt; Wojta, Johann; Speidl, Walter S

    2015-02-01

    Levosimendan is a positive inotropic drug for the treatment of acute decompensated heart failure (HF). Clinical trials showed that levosimendan was particularly effective in HF due to myocardial infarction. Myocardial necrosis induces a strong inflammatory response, involving chemoattractants guiding polymorphonuclear neutrophils (PMN) into the infarcted myocardial tissue. Our aim was to examine whether levosimendan exhibits anti-inflammatory effects on human adult cardiac myocytes (HACM) and human heart microvascular endothelial cells (HHMEC). Cardiac myocytes and endothelial cells were stimulated with interleukin-1β (IL)-1β (200 U/ml) and treated with levosimendan (0.1-10 µM) for 2-48 hours. IL-1β strongly induced expression of IL-6 and IL-8 in HACM and E-selectin and intercellular adhesion molecule-1 (ICAM-1) in HHMEC and human umbilical vein endothelial cells (HUVEC). Treatment with levosimendan strongly attenuated IL-1β-induced expression of IL-6 and IL-8 in HACM as well as E-selectin and ICAM-1 in ECs. Levosimendan treatment further reduced adhesion of PMN to activated endothelial cells under both static and flow conditions by approximately 50 %. Incubation with 5-hydroxydecanoic acid, a selective blocker of mitochondrial ATP-dependent potassium channels, partly abolished the above seen anti-inflammatory effects. Additionally, levosimendan strongly diminished IL-1β-induced reactive oxygen species and nuclear factor-κB (NF-κB) activity through inhibition of S536 phosphorylation. In conclusion, levosimendan exhibits anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro. These findings could explain, at least in part, the beneficial effects of levosimendan after myocardial infarction.

  8. Testosterone Deficiency Causes Endothelial Dysfunction via Elevation of Asymmetric Dimethylarginine and Oxidative Stress in Castrated Rats.

    Science.gov (United States)

    Kataoka, Tomoya; Hotta, Yuji; Maeda, Yasuhiro; Kimura, Kazunori

    2017-12-01

    Testosterone is believed to mediate the penile erectile response by producing adequate nitric oxide; therefore, testosterone deficiency results in erectile dysfunction through decreased nitric oxide bioavailability. However, the mechanisms underlying endothelial dysfunction in testosterone deficiency remain unclear. To investigate the mechanism of endothelial dysfunction in a rat model of testosterone deficiency. Rats were distributed into 3 groups: castrated (Cast), castrated and supplemented with testosterone (Cast + T), and sham (Sham). In the Cast + T group, castrated rats were treated daily with subcutaneous testosterone (3 mg/kg daily) for 4 weeks; Sham and Cast rats received only the vehicle. Erectile function using intracavernosal pressure and mean arterial pressure measurements after electrical stimulation of the cavernous nerve, endothelial function using isometric tension, asymmetric dimethylarginine (ADMA) levels using ultra-performance liquid chromatography and tandem mass spectrometry, and inflammatory biomarker expression were performed 4 weeks after the operation. In the Cast group, the ratio of intracavernosal pressure to mean arterial pressure significantly decreased, acetylcholine-induced relaxation was lower, and serum ADMA, oxidative stress, and inflammation biomarker levels were significantly increased (P Testosterone injection significantly improved each of these parameters (P testosterone deficiency on erectile function and the effect of testosterone replacement therapy. This study provides evidence of the influence of testosterone deficiency on endothelial function by investigating ADMA and oxidative stress. A major limitation of this study is the lack of a direct link of increased ADMA by oxidative stress to inflammation. Testosterone deficiency increased not only ADMA levels but also oxidative stress and inflammation in castrated rats, which can cause damage to the corpus cavernosum, resulting in erectile dysfunction. Kataoka T, Hotta Y

  9. Small and Intermediate Calcium-Activated Potassium Channel Openers Improve Rat Endothelial and Erectile Function

    Directory of Open Access Journals (Sweden)

    Simon G. Comerma-Steffensen

    2017-09-01

    Full Text Available Modulation of endothelial calcium-activated potassium (KCa channels has been proposed as an approach to restore endothelial function. The present study investigated whether novel openers of KCa channels with small (KCa2.x and intermediate (KCa3.1 conductance, NS309 and NS4591, improve endothelium-dependent relaxation and erectile function. Rat corpus cavernosum (CC strips were mounted for isometric tension recording and processed for immunoblotting. Mean arterial pressure (MAP, intracavernosal pressure (ICP, and electrocardiographic (ECG measurements were conducted in anesthetized rats. Immunoblotting revealed the presence of KCa2.3 and large KCa conductance (KCa1.1 channels in the corpus cavernosum. NS309 and NS4591 increased current in CC endothelial cells in whole cell patch clamp experiments. Relaxation induced by NS309 (<1 μM was inhibited by endothelial cell removal and high extracellular potassium. An inhibitor of nitric oxide (NO synthase, and blockers of KCa2.x and KCa1.1 channels, apamin and iberiotoxin also inhibited NS309 relaxation. Incubation with NS309 (0.5 μM markedly enhanced acetylcholine relaxation. Basal erectile function (ICP/MAP increased during administration of NS309. Increases in ICP/MAP after cavernous nerve stimulation with NS309 were unchanged, whereas NS4591 significantly improved erectile function. Administration of NS309 and NS4591 caused small changes in the electrocardiogram, but neither arrhythmic events nor prolongation of the QTc interval were observed. The present study suggests that openers of KCa2.x and KCa3.1 channels improve endothelial and erectile function. The effects of NS309 and NS4591 on heart rate and ECG are small, but will require additional safety studies before evaluating whether activation of KCa2.3 channels has a potential for treatment of erectile dysfunction.

  10. Adenovirus vectors can induce activation of endothelial cells: CD40 ...

    African Journals Online (AJOL)

    Adenovirus vectors can induce activation of endothelial cells: CD40-CD40L interactions partly participate in the endothelial cells activation induced by adenovirus vectors in an NF-kappaB-dependent manner.

  11. Identification of epigenetically silenced genes in tumor endothelial cells

    NARCIS (Netherlands)

    Hellebrekers, Debby M. E. I.; Melotte, Veerle; Vire, Emmanuelle; Langenkamp, Elise; Molema, Grietje; Fuks, Francois; Herman, James G.; Van Criekinge, Wim; Griffioen, Arjan W.; van Engeland, Manon

    2007-01-01

    Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated

  12. Adipokine expression and endothelial function in subclinical hypothyroidism rats

    Directory of Open Access Journals (Sweden)

    Ningning Gong

    2018-02-01

    Full Text Available The purpose of our study was to observe adipokine expression and endothelial function in subclinical hypothyroidism (sHT rats and to determine whether levothyroxine (LT4 treatment affects these changes. Sixty-five male Wistar rats were randomly divided into five groups: the control group; sHT A, B and C groups and the sHT + T4 group. The sHT rats were induced by methimazole (MMI and the sHT + T4 rats were administered LT4 treatment after 8 weeks of MMI administration. Thyroid function and lipid levels were measured using radioimmunoassays and enzymatic colorimetric methods, respectively. Serum adiponectin (APN, chemerin, TNF-α, endothelin (ET-1 and nitric oxide (NO levels were measured using ELISA kits and a nitric-reductive assay. The expression of APN, chemerin and TNF-α in visceral adipose tissue (VAT was measured in experimental rats using RT-PCR and Western blotting. Hematoxylin–eosin (HE staining was used to observe changes in adipose tissue. The sHT rats had significantly higher levels of thyroid-stimulating hormone (TSH, TNF-α, chemerin, ET-1, total cholesterol (TC and low-density lipoprotein cholesterol (LDL-C and lower levels of APN and NO than those in control and sHT + T4 rats. Based on Pearson correlation analysis, the levels of chemerin, TNF-α, ET-1, LDL-C, TC and triglyceride (TG were positively correlated with TSH, but APN and NO levels were negatively correlated with TSH. These findings demonstrated that high TSH levels contribute to the changes of adipokines and endothelial dysfunction in sHT, but LT4 treatment ameliorates those changes.

  13. Intravaginal stimulation randomized trial.

    Science.gov (United States)

    Smith, J J

    1996-01-01

    The effectiveness of intravaginal electrical stimulation was compared to standard therapy in the treatment of genuine stress urinary incontinence and detrusor instability. A total of 57 women with urinary incontinence was evaluated with video urodynamics and voiding diaries before and after treatment. Of the women 18 with stress urinary incontinence were randomized to electrical stimulation or Kegel exercise and 38 with detrusor instability were randomized to anticholinergic therapy or electrical stimulation. Of patients using electrical stimulation in the stress urinary incontinence group 66% improved and 72% of the patients with detrusor instability treated with electrical stimulation improved. These rates were not statistically significant when compared to traditional therapy. Electrical stimulation is safe and at least as effective as properly performed Kegel and anticholinergic therapy in the treatment of stress urinary incontinence and detrusor instability.

  14. Resistance exercise increases endothelial progenitor cells and angiogenic factors.

    Science.gov (United States)

    Ross, Mark D; Wekesa, Antony L; Phelan, John P; Harrison, Michael

    2014-01-01

    Bone marrow-derived endothelial progenitor cells (EPC) are involved in vascular growth and repair. They increase in the circulation after a single bout of aerobic exercise, potentially related to muscle ischemia. Muscular endurance resistance exercise (MERE) bouts also have the potential to induce muscle ischemia if appropriately structured. The objective of this study is to determine the influence of a single bout of MERE on circulating EPC and related angiogenic factors. Thirteen trained men age 22.4 ± 0.5 yr (mean ± SEM) performed a bout of MERE consisting of three sets of six exercises at participants' 15-repetition maximum without resting between repetitions or exercises. The MERE bout duration was 12.1 ± 0.6 min. Blood lactate and HR were 11.9 ± 0.9 mmol·L and 142 ± 5 bpm, respectively, at the end of MERE. Blood was sampled preexercise and at 10 min, 2 h, and 24 h postexercise. Circulating EPC and serum concentrations of vascular endothelial growth factors (VEGF-A, VEGF-C, and VEGF-D), granulocyte colony stimulating factor, soluble Tie-2, soluble fms-like tyrosine kinase-1, and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, and MMP-9) were higher (P < 0.05) in the postexercise period. Circulating EPC levels were unchanged at 10 min postexercise but higher at 2 h postexercise (P < 0.05). The concentration of most angiogenic factors and metalloproteinases were higher at 10 min postexercise (VEGF-A, +38%; VEGF-C, +40%; VEGF-D, +9%; soluble Tie-2, +15%; soluble fms-like tyrosine kinase-1, +24%; MMP-1, +62%; MMP-2, +3%; MMP-3, +54%; and MMP-9, +45%; all P < 0.05). Soluble E-selectin was lower (P < 0.05) at 2 and 24 h postexercise, with endothelial microparticles and thrombomodulin unchanged. Short intense bouts of MERE can trigger increases in circulating EPC and related angiogenic factors, potentially contributing to vascular adaptation and vasculoprotection.

  15. High glucose derived endothelial microparticles increase active caspase-3 and reduce microRNA-Let-7a expression in endothelial cells.

    Science.gov (United States)

    Bammert, Tyler D; Hijmans, Jamie G; Reiakvam, Whitney R; Levy, Ma'ayan V; Brewster, Lillian M; Goldthwaite, Zoe A; Greiner, Jared J; Stockelman, Kelly A; DeSouza, Christopher A

    2017-11-18

    The experimental aim of this study was to determine the effects of high glucose-induced endothelial microparticles (EMPs) on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 10 5  cells/condition. Cells were incubated with media containing 25 mM d-glucose (concentration representing a diabetic glycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144 + expression) and concentration was determined by flow cytometry. HUVECs (3 × 10 6  cells/condition) were treated with EMPs generated from either the normal or high glucose conditions for 24 h. Intracellular concentration of active caspase-3 was determined by enzyme immunoassay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. High glucose-derived EMPs significantly increased both basal (1.5 ± 0.1 vs 1.0 ± 0.1 ng/mL) and staurosporine-stimulated (2.2 ± 0.2 vs 1.4 ± 0.1 ng/mL) active caspase-3 compared with normal glucose EMPs. Additionally, the expression of miR-Let-7a was markedly reduced (∼140%) by high glucose EMPs (0.43 ± 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell active caspase-3. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let-7a expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Endothelial dysfunction – A predictor of atherosclerosis | Chhabra ...

    African Journals Online (AJOL)

    Endothelial dysfunction is a systemic disorder and a critical element in the pathogenesis of atherosclerotic diseases and its complications. Growing evidences suggest that the individual burden of currently known cardiovascular risk factors is not the only determinant of endothelial function; rather endothelial integrity ...

  17. (-)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huang-Joe [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China); Division of Cardiology, Department of Medicine, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Lo, Wan-Yu [Department of Medical Research, China Medical University Hospital, No. 2, Yuh-Der Road, Taichung 40447, Taiwan (China); Graduate Integration of Chinese and Western Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lu, Te-Ling [School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan (China); Huang, Haimei, E-mail: hmhuang@life.nthu.edu.tw [Institute of Biotechnology, National Tsing Hua University, No. 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan (China)

    2010-01-01

    Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (-)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6 h up to a concentration of 25 {mu}mol/L. At a concentration of 25 {mu}mol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60 min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-{kappa}B nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

  18. Spreading endothelial cell dysfunction in response to necrotic trophoblasts. Soluble factors released from endothelial cells that have phagocytosed necrotic shed trophoblasts reduce the proliferation of additional endothelial cells.

    Science.gov (United States)

    Chen, Q; Ding, J X; Liu, B; Stone, P; Feng, Y J; Chamley, L

    2010-11-01

    The pathogenesis of preeclampsia is not clear but the disease is characterised by systemic endothelial cell dysfunction that is considered to be triggered by a placental factor. Necrotic trophoblastic debris that is deported in the maternal blood is one possible placental trigger for preeclampsia. Syncytial knots were first associated with preeclampsia over 100 years ago. However, syncytial knots are very large and most are trapped in the pulmonary capillaries making it difficult to envisage how they could lead to widespread systemic endothelial cell dysfunction. This study was undertaken to examine whether conditioned medium from endothelial cells that have phagocytosed necrotic trophoblastic debris could adversely affect the proliferation or survival of fresh endothelial cells. Trophoblastic cellular debris, harvested from placental explants was added to endothelial cell monolayers directly or after induction of necrosis by freeze-thawing. Conditioned medium from the endothelial cell cultures was exposed to fresh endothelial cells and their proliferation measured by Alamar Blue, and CyQUANTNF cell proliferation assays. Endothelial cell death was examined by a fluorogenic caspase-3 activity assay and LDH release. Conditioned medium from endothelial cells that had phagocytosed necrotic but not apoptotic trophoblastic debris significantly inhibited the proliferation of fresh endothelial cells but did not induce their death. The conditioned medium also reduced cell-surface endoglin expression by fresh endothelial cells. These results confirm that phagocytosis of necrotic trophoblastic debris by endothelial cells results in the secretion of soluble factors that might explain how necrotic trophoblastic debris trapped in the pulmonary capillaries could induce systemic endothelial cell dysfunction. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Effect and possible mechanism of monocyte-derived VEGF on monocyte-endothelial cellular adhesion after electrical burns.

    Science.gov (United States)

    Ruan, Qiongfang; Zhao, Chaoli; Ye, Ziqing; Ruan, Jingjing; Xie, Qionghui; Xie, Weiguo

    2015-06-01

    One of the major obstacles in the treatment of severe electrical burns is properly handling the resulting uncontrolled inflammation. Such inflammation often causes secondary injury and necrosis, thus complicating patient outcomes. Vascular endothelial grow factor (VEGF) has emerged as an important mediator for the recruitment of monocytes to the site inflammation. This study was designed to explore the effects and possible mechanism of VEGF on monocyte-endothelial cellular adhesion. To do so, we used a cultured human monocytic cell line (THP-1) that was stimulated with serum derived from rats that had received electrical burns. Serum was obtained from rats that had received electrical burns. Both the VEGF and soluble flt-1 (sflt-1) concentrations of the serum were determined by double-antibody sandwich ELISA. The concentrations of VEGF, sflt-1, and TNF-α obtained from the cell-free cultured supernatant of THP-1 cells that had been exposed to the serum were then determined by double-antibody sandwich ELISA. Serum-stimulated THP-1 cells were added to wells with a monolayer of endothelial cells to detect the level of monocyte-endothelial cells adhesion. Finally, the state of phosphorylation of AKT was determined by Western blotting. Both in vivo and in vitro studies showed that compared to controls, the levels of VEGF were significantly increased after electrical burns. This increased was accompanied by a reduction of sflt-1 levels. Furthermore, the serum of rats that had received electrical burns was able to both activate monocytes to secrete TNF-α and enhance monocyte-endothelial cell adhesion. Treatment with the serum also resulted in an up-regulation of the phosphorylation of AKT, but had no effect on the total levels of AKT. Phosphatidylinositide 3-kinases (PI3K) inhibition decreased the number of THP-1 cells that were adhered to endothelial cells. Finally, sequestering VEGF with sflt-1 was able to reduce the effect on monocyte-endothelial cells adhesion by

  20. ORIGINAL ARTICLE Relationship between endothelial nitric oxide ...

    African Journals Online (AJOL)

    salah

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  1. Protective role of endothelial nitric oxide synthase

    NARCIS (Netherlands)

    Albrecht, Ester W J A; Stegeman, Coen A; Heeringa, Peter; Henning, Robert; van Goor, Harry

    Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively

  2. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  3. Endothelial derived hyperpolarization in renal interlobar arteries

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Sørensen, Charlotte M.

    2015-01-01

    In small arteries, vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are connect by myoendothelial junctions (MEJ), usually extending from the EC. Ca2+ activated K+ channels (IKCa and SKCa) located in the MEJ are suggested to play a role in NO-independent endothelium derived...

  4. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  5. Endothelial Changes in Individuals with Prehypertension.

    Science.gov (United States)

    Valente, Flavia Mariana; Vespasiano, Patricia; Barbosa, Juliana Aparecida; Cesarino, Claudia Bernardi; de Andrade, Days Oliveira; Barufi Fernandes, Leticia Aparecida; Cosenso-Martin, Luciana Neves; Balduino-Mendes, Alessandra Beatriz; Gaglianone, Camila; Vilela-Martin, José Fernando

    2016-01-01

    Prehypertension is considered a precursor of systemic arterial hypertension and a predictor of morbidity-mortality due to cardiovascular diseases, which are the main causes of death in Brazil and the world. Thus, early diagnosis and the adoption of therapeutic measures in cases of prehypertension can reduce cardiovascular risk. The aim of the present study was to perform a selective review of the literature to identify and discuss early endothelial changes in individuals with pre-hypertension. The findings indicate an increase in ET-1-mediated vasoconstrictor tone in prehypertension, with endothelial-dependent vasodilatation impairment. Moreover, significantly high levels of angiotensin, arginine and vasopressin were found in this group of patients. A reduction in endothelial fibrinolytic capacity was another important change found in patients with prehypertention and was associated with an increased risk for atherothrombotic events. The present findings demonstrate endothelial changes in individuals with prehypertension that contribute to the development of arterial hypertension as well as a high risk for cardiovascular events, underscoring the importance of the early adoption of optimized therapeutic measures for this population.

  6. Protective effects of vascular endothelial growth factor in cultured brain endothelial cells against hypoglycemia.

    Science.gov (United States)

    Zhao, Fei; Deng, Jiangshan; Yu, Xiaoyan; Li, Dawei; Shi, Hong; Zhao, Yuwu

    2015-08-01

    Hypoglycemia is a common and serious problem among patients with type 1 diabetes receiving treatment with insulin. Clinical studies have demonstrated that hypoglycemic edema is involved in the initiation of hypoglycemic brain damage. However, the mechanisms of this edema are poorly understood. Vascular endothelial growth factor (VEGF), a potent regulator of blood vessel function, has been observed an important candidate hormone induced by hypoglycemia to protect neurons by restoring plasma glucose. Whether VEGF has a protective effect against hypoglycemia-induced damage in brain endothelial cells is still unknown. To investigate the effects of hypoglycemia on cerebral microvascular endothelial cells and assess the protective effect of exogenous VEGF on endothelial cells during hypoglycemia, confluent monolayers of the brain endothelial cell line bEnd.3 were treated with normal (5.5 mM glucose), hypoglycemic (0, 0.5, 1 mM glucose) medium or hypoglycemic medium in the presence of VEGF. The results clearly showed that hypoglycemia significantly downregulated the expression of claudin-5 in bEnd.3 cells, without affecting ZO-1 and occludin expression and distribution. Besides, transendothelial permeability significantly increased under hypoglycemic conditions compared to that under control conditions. Moreover, the hypoglycemic medium in presence of VEGF decreased endothelial permeability via the inhibition of claudin-5 degradation and improved hypoglycemia-induced cell toxicity. Furthermore, Glucose transporter-1 (Glut-1) and apoptosis regulator Bcl-2 expression were significantly upregulated. Taken together, hypoglycemia can significantly increase paraendocellular permeability by downregulating claudin-5 expression. We further showed that VEGF protected brain endothelial cells against hypoglycemia by enhancing glucose passage, reducing endothelial cell death, and ameliorating paraendocellular permeability.

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

    Science.gov (United States)

    Suschek, C; Kolb, H; Kolb-Bachofen, V

    1997-12-01

    1. Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants. 2. In each of the different endothelial cells Mg-Dobesilate incubation (0.25-1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor N(G)-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects. 3. iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT-PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT-PCR.

  8. Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities

    OpenAIRE

    Aman, Jurjan; Weijers, Ester M.; Geerten P van Nieuw Amerongen; Malik, Asrar B.; van Hinsbergh, Victor W.M.

    2016-01-01

    Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the micr...

  9. Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

    Directory of Open Access Journals (Sweden)

    Li Cui

    2014-06-01

    Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

  10. Genetic Variation in the Platelet Endothelial Aggregation Receptor 1 Gene Results in Endothelial Dysfunction.

    Directory of Open Access Journals (Sweden)

    Adam S Fisch

    Full Text Available Platelet Endothelial Aggregation Receptor 1 (PEAR1 is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02. Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG and phenotypes (e.g. endothelial cell migration, angiogenesis that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04. Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease.

  11. Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ming-Chung [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan (China); Chen, Chia-Ling [Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Yang, Tsan-Tzu; Choi, Pui-Ching [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Hsing, Chung-Hsi [Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan (China); Department of Anesthesiology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lin, Chiou-Feng, E-mail: cflin@mail.ncku.edu.tw [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China)

    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

  12. Akt-dependent phosphorylation of endothelial nitric-oxide synthase mediates penile erection

    OpenAIRE

    Hurt, K. Joseph; Musicki, Biljana; Palese, Michael A.; Crone, Julie K.; Becker, Robyn E.; Moriarity, John L.; Snyder, Solomon H.; Burnett, Arthur L.

    2002-01-01

    In the penis, nitric oxide (NO) can be formed by both neuronal NO synthase and endothelial NOS (eNOS). eNOS is activated by viscous drag/shear stress in blood vessels to produce NO continuously, a process mediated by the phosphatidylinositol 3-kinase (PI3kinase)/Akt pathway. Here we show that PI3-kinase/Akt physiologically mediates erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vasorelaxant drug papaverine cause rapid increases in phos...

  13. Corneal donor tissue preparation for endothelial keratoplasty.

    Science.gov (United States)

    Woodward, Maria A; Titus, Michael; Mavin, Kyle; Shtein, Roni M

    2012-06-12

    Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection. Initially, cornea donor posterior lamellar dissection for DSEK was performed manually resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing. Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in

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

    Science.gov (United States)

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

    2013-04-01

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

  15. Selective endothelial overexpression of arginase II induces endothelial dysfunction and hypertension and enhances atherosclerosis in mice.

    Directory of Open Access Journals (Sweden)

    Boris L Vaisman

    Full Text Available Cardiovascular disorders associated with endothelial dysfunction, such as atherosclerosis, have decreased nitric oxide (NO bioavailability. Arginase in the vasculature can compete with eNOS for L-arginine and has been implicated in atherosclerosis. The aim of this study was to evaluate the effect of endothelial-specific elevation of arginase II expression on endothelial function and the development of atherosclerosis.Transgenic mice on a C57BL/6 background with endothelial-specific overexpression of human arginase II (hArgII gene under the control of the Tie2 promoter were produced. The hArgII mice had elevated tissue arginase activity except in liver and in resident peritoneal macrophages, confirming endothelial specificity of the transgene. Using small-vessel myography, aorta from these mice exhibited endothelial dysfunction when compared to their non-transgenic littermate controls. The blood pressure of the hArgII mice was 17% higher than their littermate controls and, when crossed with apoE -/- mice, hArgII mice had increased aortic atherosclerotic lesions.We conclude that overexpression of arginase II in the endothelium is detrimental to the cardiovascular system.

  16. Deep lamellar endothelial keratoplasty (DLEK): pursuing the ideal goals of endothelial replacement.

    Science.gov (United States)

    Terry, M A

    2003-11-01

    Endothelial dysfunction is a leading cause of corneal vision loss and treatment requires surgical replacement with donor endothelium. Standard penetrating keratoplasty (PK) suffers from the inherent problems of surface corneal incisions and sutures and poor wound healing of vertical stromal wounds. This often results in high irregular astigmatism, unpredictable corneal power, and the risk of long-term visual loss from suture-induced vascularization, ulceration, rejection, and late wound rupture. This paper delineates five ideal goals of endothelial replacement, which include: (1) a smooth surface topography without significant change in astigmatism from preoperative to postoperative; (2) a highly predictable and stable corneal power; (3) a healthy donor endothelium that resolves all oedema; (4) a tectonically stable globe, safe from injury and infection; and (5) an optically pure cornea. Deep lamellar endothelial keratoplasty (DLEK) is a surgical method of endothelial replacement that is performed through a limbal scleral incision that leaves the surface of the recipient cornea untouched. The early results of this innovative surgery are discussed and compared to the results of PK in terms of fulfillment of the five ideal goals of endothelial replacement. With further refinement of interface creation, DLEK surgery may be the ideal method for endothelial replacement.

  17. Rapid induction of neutrophil-endothelial adhesion by endothelial complement fixation.

    Science.gov (United States)

    Marks, R M; Todd, R F; Ward, P A

    1989-05-25

    The adhesion of neutrophils to vascular endothelium is an early event in their recruitment into acute inflammatory lesions. In evaluating potential neutrophil-endothelial adhesive mechanisms in acute inflammation, important considerations are that adhesion in vivo may occur very rapidly following injury and that the specificity of the reaction resides in altered endothelium. That is, neutrophils adhere only to altered endothelium adjacent to an inflammatory focus, rather than at random as would be expected if activation of neutrophils were the initiator of adhesion. We have explored a possible bridging role for complement in causing early neutrophil-endothelial cell adhesion. The complement system is involved in inflammatory processes, is capable of rapid amplification, and endothelial complement fixation at sites of inflammation could generate an endothelium-restricted signal for neutrophil adhesion. We have now developed a model in which this can be investigated without complicating factors such as immunoglobulin deposition, by constructing a novel molecule, a hybrid of the endothelial binding lectin Ulex europaeus I and of the complement activator cobra venom factor. This molecule has the capacity to cause fixation of complement on human umbilical vein endothelial cells. We show that complement fixation is a potent and rapid stimulus for neutrophil adhesion. Neutrophil adhesion requires only endothelial deposition of C3, and is mediated through the type 3 complement receptor.

  18. Endothelial progenitor cell biology in ankylosing spondylitis.

    Science.gov (United States)

    Verma, Inderjeet; Syngle, Ashit; Krishan, Pawan

    2015-03-01

    Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). EPCs were depleted in AS patients as compared to healthy controls (CD34(+) /CD133(+) : 0.027 ± 0.010% vs. 0.044 ± 0.011%, P < 0.001). EPC depletions were significantly associated with disease duration (r = -0.52, P = 0.01), BASDAI (r = -0.45, P = 0.04) and C-reactive protein (r = -0.5, P = 0.01). This is the first study to demonstrate endothelial progenitor cell depletion in AS patients. EPC depletions inversely correlate with disease duration, disease activity and inflammation, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  19. Endothelial dysfunction in rheumatic autoimmune diseases.

    Science.gov (United States)

    Murdaca, Giuseppe; Colombo, Barbara Maria; Cagnati, Paola; Gulli, Rossella; Spanò, Francesca; Puppo, Francesco

    2012-10-01

    Rheumatic autoimmune diseases have been associated with accelerated atherosclerosis and various types of vasculopathies. Atherosclerosis is an inflammatory condition which starts as a "response to injury" favoring endothelial dysfunction which is associated with increased expression of adhesion molecules, pro-inflammatory cytokines, pro-thrombotic factors, oxidative stress upregulation and abnormal vascular tone modulation. Endothelial dysfunction in rheumatic autoimmune diseases involves innate immune responses, including macrophages and dendritic cells expression of scavenger and toll-like receptors for modified or native LDL as well as neutrophil and complement activation, and dysregulation of adaptive immune responses, including proliferation of autoreactive T-helper-1 lymphocytes and defective function of dendritic and regulatory T cells. Specific differences for endothelial function among different disorders include: a) increased amounts of pro-atherogenic hormones, decreased amounts of anti-atherogenic hormones and increased insulin resistance in rheumatoid arthritis; b) autoantibodies production in systemic lupus erythematosus and antiphospholipid syndrome; c) smooth muscle cells proliferation, destruction of internal elastic lamina, fibrosis and coagulation and fibrinolytic system dysfunction in systemic sclerosis. Several self-antigens (i.e. high density lipoproteins, heat shock proteins, β2-glycoprotein1) and self-molecules modified by oxidative events (i.e. low density lipoproteins and oxidized hemoglobin) have been identified as targets of autoimmune responses. Endothelial dysfunction leads to accelerated atherosclerosis in rheumatoid arthritis, systemic lupus erythematosus and spondyloarthropaties whereas obliterative vasculopathy is associated with systemic sclerosis. In this paper, we will briefly review the most relevant information upon endothelial dysfunction and inflammatory mechanisms in atherosclerosis and we will summarize the similarities

  20. Corneal endothelial cytotoxicity of the Calotropis procera (ushaar) plant.

    Science.gov (United States)

    Al-Mezaine, Hani S; Al-Amry, Mohammed A; Al-Assiri, Abdullah; Fadel, Talal S; Tabbara, Khalid F; Al-Rajhi, Ali A

    2008-05-01

    To report 6 eyes of 5 patients with transient corneal edema after exposure to the milky latex of Calotropis procera (ushaar). Interventional case series. Intracorneal penetration of ushaar latex can lead to permanent endothelial cell loss with morphologic alteration. Corneal edema resolved completely in approximately 2 weeks in all cases, despite reduced endothelial cell count and abnormal morphology. Corneal endothelial toxicity of ushaar latex is caused by its ability to penetrate the corneal stroma and induce permanent loss of endothelial cells. Corneal edema resolves if sufficient endothelial cell viability is still present after resolution of ushaar keratitis.

  1. Rapamycin inhibits ox-LDL-induced inflammation in human endothelial cells in vitro by inhibiting the mTORC2/PKC/c-Fos pathway.

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    Sun, Juan-Juan; Yin, Xiao-Wei; Liu, Hui-Hui; Du, Wen-Xiu; Shi, Lu-Yao; Huang, Ya-Bo; Wang, Fen; Liu, Chun-Feng; Cao, Yong-Jun; Zhang, Yan-Lin

    2017-10-26

    Rapamycin and its derivative possess anti-atherosclerosis activity, but its effects on adhesion molecule expression and macrophage adhesion to endothelial cells during atherosclerosis remain unclear. In this study we explored the effects of rapamycin on ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells in vitro and the underlying mechanisms. Ox-LDL (6-48 μg/mL) dose-dependently increased the protein levels of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in human umbilical vein endothelial cells (HUVECs), whereas pretreatment with rapamycin (1-10 μmol/L) dose-dependently inhibited ox-LDL-induced increase in the adhesion molecule expression and macrophage adhesion to endothelial cells. Knockdown of mTOR or rictor, rather than raptor, mimicked the effects of rapamycin. Ox-LDL (100 μg/mL) time-dependently increased PKC phosphorylation in HUVECs, which was abolished by rapamycin or rictor siRNA. Pretreatment with PKC inhibitor staurospo¬rine significantly reduced ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells, whereas pretreatment with PKC activator PMA/TPA attenuated the inhibitory effect of rapamycin on adhesion molecule expression. Ox-LDL (100 μg/mL) time-dependently increased c-Fos levels in HUVECs, and pretreatment with rapamycin or rictor siRNA significantly decreased expression of c-Fos. Knockdown of c-Fos antagonized ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells. Our results demonstrate that rapamycin reduces ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells by inhibiting mTORC2, but not mTORC1, and mTORC2 acts through the PKC/c-Fos signaling pathway.

  2. Effects of the green tea polyphenol epigallocatechin-3-gallate on high-fat diet-induced insulin resistance and endothelial dysfunction.

    Science.gov (United States)

    Jang, Hyun-Ju; Ridgeway, Simone D; Kim, Jeong-A

    2013-12-01

    Insulin resistance, a hallmark of metabolic disorders, is a risk factor for diabetes and cardiovascular disease. Impairment of insulin responsiveness in vascular endothelium contributes to insulin resistance. The reciprocal relationship between insulin resistance and endothelial dysfunction augments the pathophysiology of metabolism and cardiovascular functions. The most abundant green tea polyphenol, epigallocatechin-3-gallate (EGCG), has been shown to have vasodilator action in vessels by activation of endothelial nitric oxide synthase (eNOS). However, it is not known whether EGCG has a beneficial effect in high-fat diet (HFD)-induced endothelial dysfunction. Male C57BL/6J mice were fed either a normal chow diet (NCD) or HFD with or without EGCG supplement (50 mg·kg(-1)·day(-1)) for 10 wk. Mice fed a HFD with EGCG supplement gained less body weight and showed improved insulin sensitivity. In vehicle-treated HFD mice, endothelial function was impaired in response to insulin but not to acetylcholine, whereas the EGCG-treated HFD group showed improved insulin-stimulated vasodilation. Interestingly, EGCG intake reduced macrophage infiltration into aortic tissues in HFD mice. Treatment with EGCG restored the insulin-stimulated phosphorylation of eNOS, insulin receptor substrate-1 (IRS-1), and protein kinase B (Akt), which was inhibited by palmitate (200 μM, 5 h) in primary bovine aortic endothelial cells. From these results, we conclude that supplementation of EGCG improves glucose tolerance, insulin sensitivity, and endothelial function. The results suggest that EGCG may have beneficial health effects in glucose metabolism and endothelial function through modulating HFD-induced inflammatory response.

  3. Heat stress-induced disruption of endothelial barrier function is via PAR1 signaling and suppressed by Xuebijing injection.

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    Qiulin Xu

    Full Text Available Increased vascular permeability leading to acute lung injury (ALI and acute respiratory distress syndrome (ARDS is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1, the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs, investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1, specific inhibitor(RWJ56110, and Xuebijing injection (XBJ, a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a

  4. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling

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    Chia-Wei Huang

    2015-01-01

    Full Text Available Neonatal hypoxic-ischemic (HI brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs from adipose-derived stem cells (ASCs and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1 and vascular endothelial growth factor receptor 2 (VEGFR2 was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.

  5. Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF-α mediated NF-κB pathway.

    Science.gov (United States)

    Gao, Wei; Liu, Haibo; Yuan, Jie; Wu, Chaoneng; Huang, Dong; Ma, Yuanji; Zhu, Jianbing; Ma, Leilei; Guo, Junjie; Shi, Hongtao; Zou, Yunzeng; Ge, Junbo

    2016-12-01

    Whether dendritic cell (DC) derived exosomes play a role in the progression of endothelial inflammation and atherosclerosis remains unclear. Using a transwell system and exosome release inhibitor GW4869, we demonstrated that mature DCs contributed to endothelial inflammation and exosomes were involved in the process. To further confirm this finding, we isolated exosomes from bone marrow dendritic cell (BMDC) culture medium (named DC-exos) and stimulated human umbilical vein endothelial cell (HUVEC) with these DC-exos. We observed that mature DC-exos increased HUVEC inflammation through NF-κB pathway in a manner similar to that of lipopolysaccharide. After a protein array analysis of exosomes, we identified and confirmed tumour necrosis factor (TNF)-α on exosome membrane being the trigger of NF-κB pathway in HUVECs. We then performed an in vivo study and found that the aorta endothelial of mice could uptake intravenously injected exosomes and was activated by these exosomes. After a period of 12 weeks of mature DC-exos injection into ApoE-/- mice, the atherosclerotic lesions significantly increased. Our study demonstrates that mature DCs derived exosomes increase endothelial inflammation and atherosclerosis via membrane TNF-α mediated NF-κB pathway. This finding extends our knowledge on how DCs affect inflammation and provides a potential method to prevent endothelial inflammation and atherosclerosis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  6. Kinin B1 receptor regulates interactions between neutrophils and endothelial cells by modulating the levels of Mac-1, LFA-1 and intercellular adhesion molecule-1.

    Science.gov (United States)

    Figueroa, Carlos D; Matus, Carola E; Pavicic, Francisca; Sarmiento, Jose; Hidalgo, Maria A; Burgos, Rafael A; Gonzalez, Carlos B; Bhoola, Kanti D; Ehrenfeld, Pamela

    2015-04-01

    Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  7. Proteomic identification of VEGF-dependent protein enrichment to membrane caveolar-raft microdomains in endothelial progenitor cells.

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    Chillà, Anastasia; Magherini, Francesca; Margheri, Francesca; Laurenzana, Anna; Gamberi, Tania; Bini, Luca; Bianchi, Laura; Danza, Giovanna; Mazzanti, Benedetta; Serratì, Simona; Modesti, Alessandra; Del Rosso, Mario; Fibbi, Gabriella

    2013-07-01

    Endothelial cell caveolar-rafts are considered functional platforms that recruit several pro-angiogenic molecules to realize an efficient angiogenic program. Here we studied the differential caveolar-raft protein composition of endothelial colony-forming cells following stimulation with VEGF, which localizes in caveolae on interaction with its type-2 receptor. Endothelial colony-forming cells are a cell population identified in human umbilical blood that show all the properties of an endothelial progenitor cell and a high proliferative rate. Two-dimensional gel electrophoresis analysis was coupled with mass spectrometry to identify candidate proteins. The twenty-eight differentially expressed protein spots were grouped according to their function using Gene Ontology classification. In particular, functional categories relative to cell death inhibition and hydrogen peroxide metabolic processes resulted enriched. In these categories, Peroxiredoxin-2 and 6, that control hydrogen peroxide metabolic processes, are the main enriched molecules together with the anti-apoptotic 78 kDa glucose regulated protein. Some of the proteins we identified had never before identified as caveolar-raft components. Other identified proteins include calpain small subunit-1, known to mediates angiogenic response to VEGF, gelsolin, which regulates stress fiber assembly, and annexin A3, an angiogenic mediator that induces VEGF production. We validated the functional activity of the above proteins, showing that the siRNA silencing of these resulted in the inhibition of capillary morphogenesis. Overall, our data show that VEGF stimulation triggers the caveolar-raft recruitment of proteins that warrant a physiological amount of reactive oxygen species to maintain a proper angiogenic function of endothelial colony-forming cells and preserve the integrity of the actin cytoskeleton.

  8. Streptococcal-vimentin cross-reactive antibodies induce microvascular cardiac endothelial proinflammatory phenotype in rheumatic heart disease.

    Science.gov (United States)

    Delunardo, F; Scalzi, V; Capozzi, A; Camerini, S; Misasi, R; Pierdominici, M; Pendolino, M; Crescenzi, M; Sorice, M; Valesini, G; Ortona, E; Alessandri, C

    2013-09-01

    Rheumatic heart disease (RHD) is characterized by the presence of anti-streptococcal group A antibodies and anti-endothelial cell antibodies (AECA). Molecular mimicry between streptococcal antigens and self proteins is a hallmark of the pathogenesis of rheumatic fever. We aimed to identify, in RHD patients, autoantibodies specific to endothelial autoantigens cross-reactive with streptococcal proteins and to evaluate their role in inducing endothelial damage. We used an immunoproteomic approach with endothelial cell-surface membrane proteins in order to identify autoantigens recognized by AECA of 140 RHD patients. Cross-reactivity of purified antibodies with streptococcal proteins was analysed. Homologous peptides recognized by serum cross-reactive antibodies were found through comparing the amino acid sequence of streptococcal antigens with human antigens. To investigate interleukin (IL)-1R-associated kinase (IRAK1) and nuclear factor-κB (NF-κB) activation, we performed a Western blot analysis of whole extracts proteins from unstimulated or stimulated human microvascular cardiac endothelial cells (HMVEC-C). Adhesion molecule expression and release of proinflammatory cytokines and growth factors were studied by multiplex bead based immunoassay kits. We observed anti-vimentin antibodies in sera from 49% RHD AECA-positive patients. Cross-reactivity of purified anti-vimentin antibodies with heat shock protein (HSP)70 and streptopain streptococcal proteins was shown. Comparing the amino acid sequence of streptococcal HSP70 and streptopain with human vimentin, we found two homologous peptides recognized by serum cross-reactive antibodies. These antibodies were able to stimulate HMVEC-C inducing IRAK and NF-κB activation, adhesion molecule expression and release of proinflammatory cytokines and growth factors. In conclusion, streptococcal-vimentin cross-reactive antibodies were able to activate microvascular cardiac endothelium by amplifying the inflammatory response

  9. Chronic shear induces caveolae formation and alters ERK and Akt responses in endothelial cells

    Science.gov (United States)

    Boyd, Nolan L.; Park, Heonyong; Yi, Hong; Boo, Yong Chool; Sorescu, George P.; Sykes, Michelle; Jo, Hanjoong

    2003-01-01

    Caveolae are plasmalemmal domains enriched with cholesterol, caveolins, and signaling molecules. Endothelial cells in vivo are continuously exposed to shear conditions, and their caveolae density and location may be different from that of static cultured cells. Here, we show that chronic shear exposure regulates formation and localization of caveolae and caveolin-1 in bovine aortic endothelial cells (BAEC). Chronic exposure (1 or 3 days) of BAEC to laminar shear increased the total number of caveolae by 45-48% above static control. This increase was due to a rise in the luminal caveolae density without changing abluminal caveolae numbers or increasing caveolin-1 mRNA and protein levels. Whereas some caveolin-1 was found in the plasma membrane in static-cultured cells, it was predominantly localized in the Golgi. In contrast, chronic shear-exposed cells showed intense caveolin-1 staining in the luminal plasma membrane with minimum Golgi association. The preferential luminal localization of caveolae may play an important role in endothelial mechanosensing. Indeed, we found that chronic shear exposure (preconditioning) altered activation patterns of two well-known shear-sensitive signaling molecules (ERK and Akt) in response to a step increase in shear stress. ERK activation was blunted in shear preconditioned cells, whereas the Akt response was accelerated. These results suggest that chronic shear stimulates caveolae formation by translocating caveolin-1 from the Golgi to the luminal plasma membrane and alters cell signaling responses.

  10. Prednisolone inhibits cytokine-induced adhesive and cytotoxic interactions between endothelial cells and neutrophils in vitro.

    Science.gov (United States)

    Heimbürger, M; Lärfars, G; Bratt, J

    2000-03-01

    We assessed whether prednisolone influenced the ability of human polymorphonuclear neutrophils (PMN) to adhere to and cause lysis of human umbilical vein endothelial cells (HUVEC) in vitro (as measured by the release of 51Cr). Pretreatment of the endothelium with IL-1beta or tumour necrosis factor-alpha (TNF-alpha) caused prominent endothelial E-selectin expression and endothelial hyperadhesiveness for neutrophils, as well as PMN-mediated cytotoxicity. All these processes were dose-dependently reduced when prednisolone was added to the assay system. This protective effect remained when HUVEC alone were pretreated with the drug prior to washing and cytokine activation. Likewise, when HUVEC cytotoxicity was induced by the nitric oxide (NO) donor S-nitroso-acetyl-penicillamine (SNAP), prednisolone reduced cell injury significantly. In contrast, prednisolone did not interfere with signalling systems between TNF-alpha-stimulated HUVEC and quiescent PMN such as IL-8 generation and release of cytosolic Ca2 + in the PMN. Thus, in this in vitro model of vasculitis, prednisolone dose-dependently reduced cytokine-induced E-selectin expression and HUVEC hyperadhesiveness for neutrophils, as well as reducing neutrophil-dependent cytotoxicity against HUVEC via NO-dependent steps.

  11. Integrin-substrate interactions underlying shear-induced inhibition of the inflammatory response of endothelial cells.

    Science.gov (United States)

    Luu, N Thin; Glen, Katie E; Egginton, Stuart; Rainger, G Ed; Nash, Gerard B

    2013-02-01

    Conditioning of endothelial cells by shear stress suppresses their response to inflammatory cytokines. We questioned whether signalling through different integrin-matrix interactions, previously associated with the pathogenic effects of disturbed flow, supported the anti-inflammatory action of steady shear. Primary human endothelial cells were cultured on different substrates and exposed to shear stress (2.0Pa) for varying periods before stimulation with tumour necrosis factor-α (TNF). Shear-conditioning inhibited cytokine-induced recruitment of flowing neutrophils. However, the effect was similar for culture on collagen, laminin or fibronectin, even when seeding was reduced to 2 hours, and shear to 3 hours before TNF treatment (to minimise deposition of endothelial matrix). Nevertheless, in short- or longer-term cultures, reduction in expression of β(1)-integrin (but not β(3)-integrin) using siRNA essentially ablated the effect of shear-conditioning on neutrophil recruitment. Studies of focal adhesion kinase (FAK) phosphorylation, siRNA against FAK and a FAK-inhibitor (PF573228) indicated that FAK activity was an essential component downstream of β(1)-integrin. In addition, MAP-kinase p38 was phosphorylated downstream of FAK and also required for functional modification. Mechanotransduction through β(1)-integrins, FAK and p38 is required for anti-inflammatory effects of steady shear stress. Separation of the pathways which underlie pathological versus protective responses of different patterns of flow is required to enable therapeutic modification or mimicry, respectively.

  12. Monocytes initiate a cycle of leukocyte recruitment when cocultured with endothelial cells.

    Science.gov (United States)

    Tsouknos, Andreas; Nash, Gerard B; Rainger, G Ed

    2003-09-01

    During the development of atherosclerotic plaque, monocytes and T-lymphocytes are recruited to the arterial intima by endothelial cells (EC) lining the vessel. This process is associated with chronic arterial inflammation and requires the activation-dependent expression of adhesion receptors and chemokines on EC. Here we show that monocytes can activate cocultured EC so that they support the adhesion, activation and transmigration of a secondary bolus of flowing peripheral blood monocytes or lymphocytes. The number of adherent leukocytes and their behaviour was comparable to that seen on EC activated with tumour necrosis factor-alpha (TNF-alpha). Depending upon the duration of endothelial cell/monocyte coculture different patterns of adhesion receptors were utilised by leukocytes. After 4 h coculture, antibodies against E-selectin, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) reduced mononuclear leukocyte adhesion. After 24 h coculture, antibodies against E-selectin and VCAM-1 but not P-selectin were effective. Immunofluorescence analysis confirmed that monocyte coculture induced endothelial expression of E-selectin and VCAM-1, while P-selectin was at the limit of detection. We conclude that EC stimulated by monocytes can support the adhesion of flowing mononuclear leukocytes. We hypothesise that this mode of EC activation and leukocyte recruitment could initiate a self-perpetuating cycle of inflammation that could be relevant to atherogenesis and other chronic inflammatory disease states.

  13. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    Science.gov (United States)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  14. Hydrogen sulfide increases nitric oxide production from endothelial cells by an Akt-dependent mechanism

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    Arturo J Cardounel

    2011-12-01

    Full Text Available Hydrogen sulfide (H2S and nitric oxide (NO are both gasotransmitters that can elicit synergistic vasodilatory responses in the in the cardiovascular system, but the mechanisms behind this synergy are unclear. In the current study we investigated the molecular mechanisms through which H2S regulates endothelial NO production. Initial studies were performed to establish the temporal and dose-dependent effects of H2S on NO generation using EPR spin trapping techniques. H2S stimulated a two-fold increase in NO production from endothelial nitric oxide synthase (eNOS, which was maximal 30 min after exposure to 25-150 µM H2S. Following 30 min H2S exposure, eNOS phosphorylation at Ser 1177 was significantly increased compared to control, consistent with eNOS activation. Pharmacological inhibition of Akt, the kinase responsible for Ser 1177 phosphorylation, attenuated the stimulatory effect of H2S on NO production. Taken together, these data demonstrate that H2S up-regulates NO production from eNOS through an Akt-dependent mechanism. These results implicate H2S in the regulation of NO in endothelial cells, and suggest that deficiencies in H2S signaling can directly impact processes regulated by NO.

  15. Cerebellar stimulation in ataxia.

    Science.gov (United States)

    Groiss, Stefan Jun; Ugawa, Yoshikazu

    2012-06-01

    The cerebellum plays an important role in movement execution and motor control by modulation of the primary motor cortex (M1) through cerebello-thalamo-cortical connections. Transcranial magnetic stimulation (TMS) allows direct investigations of neural networks by stimulating neural structures in humans noninvasively. The motor evoked potential to single-pulse TMS of M1 is used to measure the motor cortical excitability. A conditioning stimulus over the cerebellum preceding a test stimulus of the contralateral M1 enables us to study the cerebellar regulatory functions on M1. In this brief review, we describe this cerebellar stimulation method and its usefulness as a diagnostic tool in clinical neurophysiology.

  16. Infection of human endothelial cells by Japanese encephalitis virus: increased expression and release of soluble HLA-E.

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    Shwetank

    Full Text Available Japanese encephalitis virus (JEV is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP. In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-α as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-α and IFN-β as well as the dsRNA analog, poly (I:C. Both IFN-β and TNF-α further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

  17. Chronic consumption of flavanol-rich cocoa improves endothelial function and decreases vascular cell adhesion molecule in hypercholesterolemic postmenopausal women.

    Science.gov (United States)

    Wang-Polagruto, Janice F; Villablanca, Amparo C; Polagruto, John A; Lee, Luke; Holt, Roberta R; Schrader, Heather R; Ensunsa, Jodi L; Steinberg, Francene M; Schmitz, Harold H; Keen, Carl L

    2006-01-01

    Endothelial dysfunction characterizes many disease states including subclinical atherosclerosis. The consumption of flavanol-rich cocoa and cocoa-based products has been shown to improve endothelial function in both compromised and otherwise normal, healthy individuals when administered either acutely or over a period of several days, or weeks. Women experience increased risk for cardiovascular disease after menopause, which can be associated with endothelial dysfunction. Whether a flavanol-rich cocoa-based product can improve endothelial function in hypercholesterolemic postmenopausal women is not known. The purpose of the present study was to determine whether chronic dietary administration of flavanol-rich cocoa improves endothelial function and markers of cardiovascular health in hypercholesterolemic postmenopausal women. Thirty-two postmenopausal hypercholesterolemic women were randomly assigned to consume a high-flavanol cocoa beverage (high cocoa flavanols (CF)--446 mg of total flavanols), or a low-flavanol cocoa beverage (low CF--43 mg of total flavanols) for 6 weeks in a double-blind study (n=16 per group). Endothelial function was determined by brachial artery-reactive hyperemia. Plasma was analyzed for lipids (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol), hormones (follicle-stimulating hormone), total nitrate/nitrite, activation of cellular adhesion markers (vascular cell adhesion molecule 1, intercellular adhesion molecule 1, E-Selectin, P-Selectin), and platelet function and reactivity. Changes in these plasma markers were then correlated to brachial reactivity. Brachial artery hyperemic blood flow increased significantly by 76% (Pflavanol-rich cocoa consumption in hypercholesterolemic postmenopausal women. In addition, our results suggest that reductions in plasma soluble vascular cell adhesion molecule-1 after chronic consumption of a flavanol-rich cocoa may be mechanistically linked to improved

  18. Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage.

    Science.gov (United States)

    Smit, Kirsten F; Kerindongo, Raphaela P; Böing, Anita; Nieuwland, Rienk; Hollmann, Markus W; Preckel, Benedikt; Weber, Nina C

    2015-09-10

    Helium induces preconditioning in human endothelium protecting against postischemic endothelial dysfunction. Circulating endothelial microparticles are markers of endothelial dysfunction derived in response to injury. Another noble gas, xenon, protected human umbilical vein endothelial cells (HUVEC) against inflammatory stress in vitro. We hypothesised that helium protects the endothelium in vitro against inflammatory and oxidative stress. HUVEC were isolated from fresh umbilical cords and grown upon confluence. Cells were subjected to starving medium for 12h before the experiment and treated for either 3 × 5 min or 1 × 30 min with helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2) in a specialised gas chamber. Subsequently, cells were stimulated with TNF-α (40 ng/ml for 24h or 10 ng/ml for 2h) or H2O2 (500 μM for 2h) or left untreated. Adhesion molecule expression was analysed using real-time quantitative polymerase chain reaction. Caspase-3 expression and viability of the cells was measured by flowcytometry. Microparticles were investigated by nanoparticle tracking analysis. Helium had no effect on adhesion molecule expression after TNF-α stimulation but in combination with oxidative stress decreased cell viability (68.9 ± 1.3% and 58 ± 1.9%) compared to control. Helium further increased TNF-α induced release of caspase-3 containing particles compared to TNF-α alone (6.4 × 10(6) ± 1.1 × 10(6) and 2.9 × 10(6) ± 0.7 × 10(6), respectively). Prolonged exposure of helium increased microparticle formation (2.4 × 10(9) ± 0.5 × 10(9)) compared to control (1.7 × 10(9) ± 0.2 × 10(9)). Summarized, helium increases inflammatory and oxidative stress-induced endothelial damage and is thus not biologically inert. A possible noxious effects on the cellular level causing alterations in microparticle formation both in number and content should be acknowledged. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. 20-Hydroxyeicosatetraenoic acid impairs endothelial insulin signaling by inducing phosphorylation of the insulin receptor substrate-1 at Ser616.

    Directory of Open Access Journals (Sweden)

    Xuguang Li

    Full Text Available 20-Hydroxyeicosatetraenoic acid (20-HETE induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser616; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr632 and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser1177 site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser616 is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.

  20. Release of eicosanoids from white blood cells, platelets, smooth muscle cells, and endothelial cells in response to endotoxin and A23187.

    Science.gov (United States)

    Bottoms, G D; Johnson, M; Ward, D; Fessler, J; Lamar, C; Turek, J

    1986-01-01

    Endotoxin produces numerous pathophysiologic changes in animals, including vascular endothelial cell damage and hematologic changes. Direct effects of endotoxin on arachidonic acid metabolism and the release of eicosanoids from endothelial cells and neutrophils have been reported. A rapid release of these autocoids occurs when cells are incubated with endotoxin, and this appears to be one of the earliest endotoxin-induced changes. Some of these eicosanoids may result in beneficial effects, and others may result in detrimental effects. This study was to determine the release of eicosanoids from white blood cells, platelets, smooth muscle cells, and endothelial cells in response to varying amounts of endotoxin and the calcium ionophore A23187. The results indicate that endotoxin has a major direct effect on vascular endothelial cells and smooth muscle cells as indicated by its ability to increase the synthesis of predominately i6-keto-PGF1 alpha by these cells. These effects were seen within a dose range of endotoxin that is lethal in horses. Very high concentrations of endotoxin (100 micrograms/ml) were required to stimulate a small increase in the production of i6-keto-PGF1 alpha and iLTC4 by freshly isolated neutrophils. Stimulation of cells with A23187 revealed that, of the eicosanoids measured, the one produced predominately by endothelial cells and smooth muscle cells was 6-keto-PGF1 alpha, by platelets was TxB2, and by neutrophils was LTC4 (LTB4 was not measured). A mixture of all white blood cells including platelets when incubated with A23187 produced large amounts of TxB2, LTB4, and LTC4 with smaller amounts of 6-keto-PGF1 alpha. The results indicate that endotoxin directly affects cells and stimulates them to produce thromboxane and prostacyclin, but very high concentrations of endotoxin were required to stimulate neutrophils to produce rather small increases in iLTC4.

  1. β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier.

    Science.gov (United States)

    Gorina, Roser; Lyck, Ruth; Vestweber, Dietmar; Engelhardt, Britta

    2014-01-01

    In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

  2. Intracavernous delivery of a designed angiopoietin-1 variant rescues erectile function by enhancing endothelial regeneration in the streptozotocin-induced diabetic mouse.

    Science.gov (United States)

    Jin, Hai-Rong; Kim, Woo Jean; Song, Jae Sook; Piao, Shuguang; Choi, Min Ji; Tumurbaatar, Munkhbayar; Shin, Sun Hwa; Yin, Guo Nan; Koh, Gou Young; Ryu, Ji-Kan; Suh, Jun-Kyu

    2011-03-01

    Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals. Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days -3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test. Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47(phox) and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1-treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin. These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as

  3. Oscillatory shear alters endothelial hydraulic conductivity and nitric oxide levels.

    Science.gov (United States)

    Hillsley, Mechteld V; Tarbell, John M

    2002-05-24

    This study addresses the role of nitric oxide (NO) and downstream signaling pathways in mediating the influences of oscillatory shear stress on the hydraulic conductivity (L(p)) of bovine aortic endothelial cell (BAEC) monolayers. Exposure of BAEC monolayers to 20 dyne/cm2 steady shear stress for 3 h induced a 3.3-fold increase in L(p). When an oscillatory shear amplitude of 10 dyne/cm2 was superimposed on a steady shear of 10 dyne/cm2 to produce a non-reversing oscillatory shear pattern (10+/-10 dyne/cm2), L(p) increased by 3.0-fold within 90 min. When the amplitude was increased to 15 dyne/cm2, resulting in a reversing oscillatory shear pattern (10+/-15 dyne/cm2), the increase in L(p) over 3 h was completely suppressed. Twenty and 10+/-10 dyne/cm2 induced 2.9- and 2.6-fold increases in NO production above non-sheared controls, respectively, whereas 10+/-15 dyne/cm2 stimulated a 14-fold increase in NO production. The inhibition of L(p) with reversing oscillatory shear may be associated with alterations in cyclic guanosine monophosphate (cGMP) production downstream of NO which is up-regulated by reversing oscillatory shear, but is unaffected by steady shear.

  4. Stimulants and narcotic drugs.

    Science.gov (United States)

    King, P; Coleman, J H

    1987-04-01

    This article focuses on the use of stimulant and narcotic drugs, as well as lookalike preparations that are subject to abuse. There is a brief discussion of the pharmacology of these agents, demographic use patterns, and treatment modalities.

  5. Geothermal Well Stimulation

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, D. A.; Morris, C. W.; Sinclair, A. R.; Hanold, R. J.; Vetter, O. J.

    1981-03-01

    The stimulation of geothermal wells presents some new and challenging problems. Formation temperatures in the 300-600 F range can be expected. The behavior of stimulation fluids, frac proppants, and equipment at these temperatures in a hostile brine environment must be carefully evaluated before performance expectations can be determined. In order to avoid possible damage to the producing horizon of the formation, high temperature chemical compatibility between the in situ materials and the stimulation materials must be verified. Perhaps most significant of all, in geothermal wells the required techniques must be capable of bringing about the production of very large amounts of fluid. This necessity for high flow rates represents a significant departure from conventional petroleum well stimulation and demands the creation of very high near-wellbore permeability and/or fractures with very high flow conductivity.

  6. Arterial endothelial function measurement method and apparatus

    Science.gov (United States)

    Maltz, Jonathan S; Budinger, Thomas F

    2014-03-04

    A "relaxoscope" (100) detects the degree of arterial endothelial function. Impairment of arterial endothelial function is an early event in atherosclerosis and correlates with the major risk factors for cardiovascular disease. An artery (115), such as the brachial artery (BA) is measured for diameter before and after several minutes of either vasoconstriction or vasorelaxation. The change in arterial diameter is a measure of flow-mediated vasomodification (FMVM). The relaxoscope induces an artificial pulse (128) at a superficial radial artery (115) via a linear actuator (120). An ultrasonic Doppler stethoscope (130) detects this pulse 10-20 cm proximal to the point of pulse induction (125). The delay between pulse application and detection provides the pulse transit time (PTT). By measuring PTT before (160) and after arterial diameter change (170), FMVM may be measured based on the changes in PTT caused by changes in vessel caliber, smooth muscle tone and wall thickness.

  7. Circulating Endothelial Microparticles in Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    A. F. Tramontano

    2010-01-01

    Full Text Available Background. Endothelial Microparticles (EMPs are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM is not known. We studied the cellular adhesion molecule (CAM profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization. Methods and Results. EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/L specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process. Conclusion. Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

  8. Targeted endothelial nanomedicine for common acute pathological conditions.

    Science.gov (United States)

    Shuvaev, Vladimir V; Brenner, Jacob S; Muzykantov, Vladimir R

    2015-12-10

    Endothelium, a thin monolayer of specialized cells lining the lumen of blood vessels is the key regulatory interface between blood and tissues. Endothelial abnormalities are implicated in many diseases, including common acute conditions with high morbidity and mortality lacking therapy, in part because drugs and drug carriers have no natural endothelial affinity. Precise endothelial drug delivery may improve management of these conditions. Using ligands of molecules exposed to the bloodstream on the endothelial surface enables design of diverse targeted endothelial nanomedicine agents. Target molecules and binding epitopes must be accessible to drug carriers, carriers must be free of harmful effects, and targeting should provide desirable sub-cellular addressing of the drug cargo. The roster of current candidate target molecules for endothelial nanomedicine includes peptidases and other enzymes, cell adhesion molecules and integrins, localized in different domains of the endothelial plasmalemma and differentially distributed throughout the vasculature. Endowing carriers with an affinity to specific endothelial epitopes enables an unprecedented level of precision of control of drug delivery: binding to selected endothelial cell phenotypes, cellular addressing and duration of therapeutic effects. Features of nanocarrier design such as choice of epitope and ligand control delivery and effect of targeted endothelial nanomedicine agents. Pathological factors modulate endothelial targeting and uptake of nanocarriers. Selection of optimal binding sites and design features of nanocarriers are key controllable factors that can be iteratively engineered based on their performance from in vitro to pre-clinical in vivo experimental models. Targeted endothelial nanomedicine agents provide antioxidant, anti-inflammatory and other therapeutic effects unattainable by non-targeted counterparts in animal models of common acute severe human disease conditions. The results of animal

  9. Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-12-15

    Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

  10. Metformin improves endothelial function in aortic tissue and microvascular endothelial cells subjected to diabetic hyperglycaemic conditions.

    Science.gov (United States)

    Ghosh, Suparna; Lakshmanan, Arun P; Hwang, Mu Ji; Kubba, Haidar; Mushannen, Ahmed; Triggle, Chris R; Ding, Hong

    2015-12-01

    The cellular mechanisms whereby metformin, the first line drug for type 2 diabetes (T2DM), mediates its antidiabetic effects remain elusive, particularly as to whether metformin has a direct protective action on the vasculature. This study was designed to determine if a brief 3-h exposure to metformin protects endothelial function against the effects of hyperglycaemia. We investigated the protective effects of metformin on endothelial-dependent vasodilatation (EDV) in thoracic aortae from T2DM db/db mice and on high glucose (HG, 40 mM) induced changes in endothelial nitric oxide synthase (eNOS) signaling in mouse microvascular endothelial cells (MMECs) in culture. Exposure of aortae from db+/? non-diabetic control mice to high glucose (HG, 40 mM) containing Krebs for 3-h significantly (Pmetformin; metformin also improved ACh-induced EDV in aortae from diabetic db/db mice. Immunoblot analysis of MMECs cultured in HG versus NG revealed a significant reduction of the ratio of phosphorylated (p-eNOS)/eNOS and p-Akt/Akt, but not the expression of total eNOS or Akt. The 3-h exposure of MMECs to metformin significantly (Pmetformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase.

    Science.gov (United States)

    Boo, Yong Chool; Sorescu, George P; Bauer, Philip M; Fulton, David; Kemp, Bruce E; Harrison, David G; Sessa, William C; Jo, Hanjoong

    2003-10-01

    Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology.

  12. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    Science.gov (United States)

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  13. Corneal Donor Tissue Preparation for Endothelial Keratoplasty

    OpenAIRE

    Woodward, Maria A.; Titus, Michael; Mavin, Kyle; Shtein, Roni M.

    2012-01-01

    Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK...

  14. Endothelial microparticles (EMP in physiology and pathology

    Directory of Open Access Journals (Sweden)

    Ewa Sierko

    2015-08-01

    Full Text Available Endothelial microparticles (EMP are released from endothelial cells (ECs in the process of activation and/or apoptosis. They harbor adhesive molecules, enzymes, receptors and cytoplasmic structures and express a wide range of various constitutive antigens, typical for ECs, at their surface. Under physiological conditions the concentration of EMP in the blood is clinically insignificant. However, it was reported that under pathological conditions EMP concentration in the blood might slightly increase and contribute to blood coagulation, angiogenesis and inflammation. It has been shown that EMP directly and indirectly contribute to the activation of blood coagulation. Endothelial microparticles directly participate in blood coagulation through their surface tissue factor (TF – a major initiator of blood coagulation. Furthermore, EMP exhibit procoagulant potential via expression of negatively charged phospholipids at their surface, which may promote assembly of coagulation enzymes (TF/VII, tenases and prothrombinase complexes, leading to thrombus formation. In addition, they provide a binding surface for coagulation factors: IXa, VIII, Va and IIa. Moreover, it is possible that EMP transfer TF from TF-bearing EMP to activated platelets and monocytes by binding them through adhesion molecules. Also, EMP express von Willebrand factor, which may facilitate platelet aggregation. Apart from their procoagulant properties, it was demonstrated that EMP may express adhesive molecules and metalloproteinases (MMP-2, MMP-9 at their surface and release growth factors, which may contribute to angiogenesis. Additionally, surface presence of C3 and C4 – components of the classical pathway – suggests pro-inflammatory properties of these structures. This article contains a summary of available data on the biology and pathophysiology of endothelial microparticles and their potential role in blood coagulation, angiogenesis and inflammation.

  15. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Endothelial Dysfunction as a Factor of Renal Impairment Development in Patients with Hypothyroidism

    Directory of Open Access Journals (Sweden)

    O.N. Didushko

    2016-06-01

    Full Text Available Introduction. The endothelial dysfunction was found to be detected even within normal range of thyroid-stimulating hormone and it worsens while the growth of thyroid-stimulating hormone levels. The objective of the investigation was to study the condition of the endothelium and its influence on renal function in patients with primary hypothyroidism. Materials and methods. 188 patients with manifested hypothyroidism were examined. They were divided into following groups: group I involved 45 non-obese patients with hypothyroidism on the background of autoimmune thyroiditis; group II included 46 obese patients with autoimmune thyroiditis and hypothyroidism; group III consisted of 47 non-obese patients with post-operative hypothyroidism and group IV consisted of obese patients with postoperative hypothyroidism, n = 50. Results. Investigating endothelial dysfunction in all groups of patients suffering from hypothyroidism, a significant difference in its indices has been revealed, moreover, endothelial dysfunction has appeared to be more pronounced in patients from group II and group IV. Increase in the diameter of the brachial artery after compression in group II and group IV has turned out to be twice less and respectively, endothelium-dependent vasodilation (EDVD has become 46.4 % and 47.7 % less than the one in healthy people. An inverse correlation of medium strength has been established between thyroid-stimulating hormone and EDVD levels as well as between EDVD and total cholesterol and low-density lipoproteins in a joint group that involved patients with manifested hypothyroidism. The same inverse correlation of medium strength has been revealed in group I and group II; and weak, but positive inverse correlation between vasculoendothelial growth factor and glomerular filtration rate has been established in group III and group IV. Conclusions. Revealed correlations indicate mutually aggravating influence of thyroid hypofunction, obesity

  17. A simple method for isolating and culturing the rat brain microvascular endothelial cells.

    Science.gov (United States)

    Liu, Yang; Xue, Qiang; Tang, Qing; Hou, Min; Qi, Hongyi; Chen, Gang; Chen, Weihai; Zhang, Jifen; Chen, Yi; Xu, Xiaoyu

    2013-11-01

    Brain microvascular endothelial cells (BMECs), a main component of the blood-brain barrier, play a critical role in the pathogenesis of many brain diseases. The primary culture of BMECs has been used in various models for studying cerebrovascular diseases in vitro. However, there are still several problems existing in the isolation and cultivation of primary rat BMECs, such as low yield, contamination with other cell types, and requirement of a large number of animals and expensive growth factor. In this study, we describe a simple, economical (without any growth factor) and repeatable method to obtain endothelial cells with high purity (>99%) and yield (about 2.2×10(7) per rat) from cerebral cortexes of neonatal rat, mainly from gray matter. In vitro examinations determined that the isolated cells expressed typical phenotypic markers of differentiated brain endothelium such as multiple drug resistant protein, von Willebrand factor, platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), and intercellular adhesion molecule (ICAM). These cells also possessed morphological and ultra-structural characteristics that were observed by phase contrast microscope and electric microscope. Then GFAP and α-SMA were used, respectively, to identify astrocyte and pericyte which were potential to contaminate primary culturing of BMECs. And specific reaction of endothelial cells to external stimulation was tested by culture with TNF-α for 24h. All these results of our experiments supply that our protocol provides an effective and reliable method to obtain high purity and yield of rat BMECs and offers a useful tool for studying cellular physiology, cerebrovascular diseases, brain tumors, blood-brain barrier and neurovascular units, etc. © 2013. Published by Elsevier Inc. All rights reserved.

  18. Release of endothelial cell associated VEGFR2 during TGF-β modulated angiogenesis in vitro.

    Science.gov (United States)

    Jarad, M; Kuczynski, E A; Morrison, J; Viloria-Petit, A M; Coomber, B L

    2017-01-23

    Sprouting angiogenesis requires vascular endothelial proliferation, migration and morphogenesis. The process is regulated by soluble factors, principally vascular endothelial growth factor (VEGF), and via bidirectional signaling through the Jagged/Notch system, leading to assignment of tip cell and stalk cell identity. The cytokine transforming growth factor beta (TGF-β) can either stimulate or inhibit angiogenesis via its differential surface receptor signaling. Here we evaluate changes in expression of angiogenic signaling receptors when bovine aortic endothelial cells were exposed to TGF-β1 under low serum conditions. TGF-β1 induced a dose dependent inhibition of tip cell assignment and subsequent angiogenesis on Matrigel, maximal at 5.0 ng/ml. This occurred via ALK5-dependent pathways and was accompanied by significant upregulation of the TGF-β co-receptor endoglin, and SMAD2 phosphorylation, but no alteration in Smad1/5 activation. TGF-β1 also induced ALK5-dependent downregulation of Notch1 but not of its ligand delta-like ligand 4. Cell associated VEGFR2 (but not VEGFR1) was significantly downregulated and accompanied by reciprocal upregulation of VEGFR2 in conditioned medium. Quantitative polymerase chain reaction analysis revealed that this soluble VEGFR2 was not generated by a selective shift in mRNA isoform transcription. This VEGFR2 in conditioned medium was full-length protein and was associated with increased soluble HSP-90, consistent with a possible shedding of microvesicles/exosomes. Taken together, our results suggest that endothelial cells exposed to TGF-β1 lose both tip and stalk cell identity, possibly mediated by loss of VEGFR2 signaling. The role of these events in physiological and pathological angiogenesis requires further investigation.

  19. Aging-related changes in oxidative stress response of human endothelial cells.

    Science.gov (United States)

    Conti, Valeria; Corbi, Graziamaria; Simeon, Vittorio; Russomanno, Giusy; Manzo, Valentina; Ferrara, Nicola; Filippelli, Amelia

    2015-08-01

    Oxidative stress is strongly associated with aging and age-related diseases and plays a crucial role in endothelial dysfunction development. To better understand the molecular mechanisms of aging and stress response in humans, we examined changes to young and older human endothelial cells over time (72, 96 and 120 h), before and after H2O2-induced stress. We measured the expression of the deacetylase Sirtuin 1 (Sirt1) and its transcriptional target Forkhead box O3a (Foxo3a); TBARS, a well-known marker of overall oxidative stress, and catalase activity as index of antioxidation. Moreover, we quantified levels of cellular senescence by senescence-associated β galactosidase (SA-βgal) assay. Under oxidative stress induction older cells showed a progressive decrease of Sirt1 and Foxo3a expression, persistently high TBARS levels with high, but ineffective Cat activity to counteract such levels. In addition cellular senescence drastically increased in older cells compared with Young cells both in presence and in the absence of oxidative stress. By following the cell behavior during the time course, we can hypothesize that while in young cells an oxidative stress induction stimulated an adequate response through activation of molecular factor crucial to counteract oxidative stress, the older cells are not able to adequately adapt themselves to external stress stimuli. During their life, endothelial cells impair the ability to defend themselves from oxidative stress stimuli. This dysfunction involves the pathway of Sirt1 a critical regulator of oxidative stress response and cellular lifespan, underlining its crucial role in endothelial homeostasis control during aging and age-associated diseases.

  20. The small GTPase RhoB regulates TNFα signaling in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Jeffrey Kroon

    Full Text Available The inflammatory response of endothelial cells triggered by cytokines such as TNFα and IL1β plays a pivotal role in innate immunity. Upon pro-inflammatory cytokine stimulation, endothelial cells produce chemokines and cytokines that attract and activate leukocytes, and express high levels of leukocyte adhesion molecules. This process is mediated by intracellular signaling cascades triggered by activation of e.g. the TNFα receptor (TNFR that lead to the activation of the NFκB transcription factor and of MAP kinases, which in turn activate inflammatory gene transcription. We found that the small GTPase RhoB was strongly and rapidly upregulated in primary human endothelial cells by TNFα, IL1β and LPS. We subsequently investigated the role of RhoB in the regulation of TNFR signaling in endothelial cells by silencing RhoB expression with siRNA. We provide evidence that the TNFα-induced activation of p38 MAP kinase is strongly dependent on RhoB, but not on RhoA, while JNK activation is regulated by both RhoB and RhoA. Consistent with the important role of p38 MAP kinase in inflammation, we demonstrate that loss of RhoB impairs TNFα-induced ICAM-1 expression and reduces cell production of IL6 and IL8. In addition, we show that RhoB silencing alters the intracellular traffic of TNFα after endocytosis. Since RhoB is a known regulator of the intracellular traffic of membrane receptors, our data suggest that RhoB controls TNFα signaling through the regulation of the TNFR traffic.

  1. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2016-11-15

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  2. Proanthocyanidins, from Ribes nigrum leaves, reduce endothelial adhesion molecules ICAM-1 and VCAM-1

    Science.gov (United States)

    Garbacki, N; Kinet, M; Nusgens, B; Desmecht, D; Damas, J

    2005-01-01

    Background The effects of proanthocyanidins (PACs), isolated from blackcurrant (Ribes nigrum L.) leaves, on neutrophil accumulation during inflammatory processes were investigated in vivo and in vitro. Methods In vivo studies were performed using carrageenin-induced pleurisy in rats pre-treated with PACs. Exudate volume and PMNs accumulation were measured. Leukocyte cell adhesion molecules (LFA-1, Mac-1 and VLA-4) mobilization in circulating granulocytes were analysed by flow cytometry and endothelial cell adhesion molecules (ICAM-1 and VCAM-1) were detected by immunohistochemistry on lung sections. In vitro studies were conducted on endothelial LT2 cells, stimulated with TNF-α, to evaluate ICAM-1, IL-8 and VEGF mRNA expression upon PACs treatment. Data sets were examined by one-way analysis of variance (ANOVA) followed by a Scheffe post-hoc test. Results Pretreatment of the animals with PACs (10, 30 and 60 mg/kg) inhibited dose-dependently carrageenin-induced pleurisy in rats by reducing pleural exudate formation and PMNs infliltration. Leukocyte cell adhesion molecules mobilization was not down-regulated on granulocytes by PACs. Immunohistochemistry on lung sections showed a decreased production of endothelial cell adhesion molecules. In vitro experiments demonstrated that PACs were able to significantly inhibit ICAM-1 but not IL-8 and VEGF165 mRNA expression. Moreover, VEGF121 mRNA expression was dose-dependently enhanced. Conclusion This study provides evidence to support the anti-inflammatory activity of proanthocyanidins is related to an inhibition of leukocyte infiltration which can be explained at least in part by a down-regulation of endothelial adhesion molecules, ICAM-1 and VCAM-1 and that these compounds are capable of modulating TNF-α-induced VEGF transcription. PMID:16091140

  3. Proanthocyanidins, from Ribes nigrum leaves, reduce endothelial adhesion molecules ICAM-1 and VCAM-1

    Directory of Open Access Journals (Sweden)

    Desmecht D

    2005-08-01

    Full Text Available Abstract Background The effects of proanthocyanidins (PACs, isolated from blackcurrant (Ribes nigrum L. leaves, on neutrophil accumulation during inflammatory processes were investigated in vivo and in vitro. Methods In vivo studies were performed using carrageenin-induced pleurisy in rats pre-treated with PACs. Exudate volume and PMNs accumulation were measured. Leukocyte cell adhesion molecules (LFA-1, Mac-1 and VLA-4 mobilization in circulating granulocytes were analysed by flow cytometry and endothelial cell adhesion molecules (ICAM-1 and VCAM-1 were detected by immunohistochemistry on lung sections. In vitro studies were conducted on endothelial LT2 cells, stimulated with TNF-α, to evaluate ICAM-1, IL-8 and VEGF mRNA expression upon PACs treatment. Data sets were examined by one-way analysis of variance (ANOVA followed by a Scheffe post-hoc test. Results Pretreatment of the animals with PACs (10, 30 and 60 mg/kg inhibited dose-dependently carrageenin-induced pleurisy in rats by reducing pleural exudate formation and PMNs infliltration. Leukocyte cell adhesion molecules mobilization was not down-regulated on granulocytes by PACs. Immunohistochemistry on lung sections showed a decreased production of endothelial cell adhesion molecules. In vitro experiments demonstrated that PACs were able to significantly inhibit ICAM-1 but not IL-8 and VEGF165 mRNA expression. Moreover, VEGF121 mRNA expression was dose-dependently enhanced. Conclusion This study provides evidence to support the anti-inflammatory activity of proanthocyanidins is related to an inhibition of leukocyte infiltration which can be explained at least in part by a down-regulation of endothelial adhesion molecules, ICAM-1 and VCAM-1 and that these compounds are capable of modulating TNF-α-induced VEGF transcription.

  4. Descemet membrane endothelial keratoplasty and refractive surgery.

    Science.gov (United States)

    Tong, C Maya; Baydoun, Lamis; Melles, Gerrit R J

    2017-07-01

    Descemet membrane endothelial keratoplasty (DMEK) has become a first-line treatment in corneal endothelial diseases because of its exceptional clinical outcomes and low complication rates. Because of its improved refractive predictability, DMEK is now also considered for managing cases with endothelial decompensation following previous refractive procedures, or in combination with those. This article reviews the clinical outcomes in these cases and discusses the possibility of refractive interventions following DMEK. DMEK has been successfully performed in eyes after laser in-situ keratomileusis, eyes after anterior chamber intraocular lens (IOL) implantation and aphakic eyes. Often, DMEK is combined with cataract surgery (triple-DMEK). Initial reports on reducing the refractive cylinder by toric IOL implantation are available. Although there are some reports on phacoemulsification and IOL implantation after phakic DMEK, reports on laser refractive procedures following DMEK are lacking. In contrast to earlier keratoplasty techniques, DMEK induces on average only mild refractive shifts owing to the 'natural' restoration of the cornea. As such, DMEK may be ideal in managing corneal decompensation in refractive patients. However, further studies are required to assess the safety and efficacy of DMEK after refractive treatment and of refractive procedures following DMEK.

  5. Doinseunggitang Ameliorates Endothelial Dysfunction in Diabetic Atherosclerosis

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    Jung Joo Yoon

    2013-01-01

    Full Text Available Atherosclerosis, a chronic and progressive disease characterized by vascular inflammation, is a leading cause of death in diabetes patients. Doinseunggitang (DYSGT, traditional prescription, has been used for promoting blood circulation to remove blood stasis. The aim of this study was to investigate the beneficial effects of DYSGT on endothelial dysfunction in diabetic atherosclerosis animal model. Apolipoprotein E knockout (ApoE KO mice fed on a Western diet were treated with DYSGT (200 mg/kg/day. DYSGT significantly lowered blood glucose level and glucose tolerance as well as systolic blood pressure. Metabolic parameter showed that DYSGT markedly decreased triglyceride and LDL-cholesterol levels. In the thoracic aorta, the impairment of vasorelaxation response to acetylcholine and atherosclerotic lesion was attenuated by DYSGT. Furthermore, DYSGT restored the reduction of endothelial nitric oxide synthase (eNOS expression, leading to the inhibition of intracellular adhesion molecule-1 (ICAM-1 and endothelin-1 (ET-1 expression. In conclusion, DYSGT improved the development of diabetic atherosclerosis via attenuation of the endothelial dysfunction, possibly by inhibiting ET-1, cell adhesion molecules, and lesion formation. Therefore, these results suggest that Korean traditional prescription Doinseunggitang may be useful in the treatment and prevention of diabetic vascular complications.

  6. Protective effects of Donepezil against endothelial permeability.

    Science.gov (United States)

    Tang, Xuelu; Di, Xiuhua; Liu, Yilin

    2017-09-15

    The endothelium lines the interior surface of blood vessels, and under pathophysiologic conditions, its integrity can be compromised due to a disturbance in the expression of tight junctions. Donepezil is a licensed drug used in the palliative treatment of Alzheimer's disease (AD). Increasing evidence has reported that donepezil has an anti-inflammatory activity. However, little information is available regarding the role of donepezil in vascular diseases. In this study, we found that pretreatment with donepezil significantly ameliorated endothelial permeability induced by tumor necrosis factor (TNF-α) by restoring the expression of the tight junction proteins vascular endothelial cadherin (VE-cadherin) and zonula occludens-1 (ZO-1) in human umbilical vein endothelial cells (HUVECs). Mechanistically, our results indicate that donepezil regulates the expression and activity of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1), but not matrix metalloproteinase-2 (MMP-2) or tissue inhibitor of metalloproteinases 2 (TIMP-2). Importantly, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ serine-threonine kinase (AKT)/ nuclear factor kappa B (NF-κB) pathway was found to be involved in this process. These results suggest that donepezil may potentially play an important therapeutic role in vascular diseases. Copyright © 2017. Published by Elsevier B.V.

  7. The Effect of Shiga Toxin on Weibel-Palade Bodies in Primary Human Endothelial Cells

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    Joyce Geelen

    2014-07-01

    Full Text Available Background/Aims: Diarrhea-associated hemolytic uremic syndrome is associated with the presence of Shiga toxin (Stx1, Stx2 and several variants in the circulation. The aim of this study is to examine the possible triggering effect of Stx1 on the exocytosis of Weibel-Palade bodies (WPbs. Methods: Cultured human umbilical venous endothelial cells (HUVECs and glomerular microvascular endothelial cells (GMVECs were stimulated by thrombin and Stx1 in both static and flowing conditions. The amount of secreted von Willebrand factor (VWF in the supernatant as well as the remaining intracellular fraction was determined. Results: In HUVECs and in 2 out of 4 GMVECs, the stimulation of Stx1 in flow at 1 dyne/cm2 resulted in a decrease of intracellular VWF. This is contrary to the results of Stx1 applied in static conditions. At a higher flow rate of 5 dyne/cm2, no effect in GMVECs was observed. Conclusion: Stx1 can contribute, via an effect on WPbs, to the exocytosis of WPbs in flow conditions in HUVECs and probably in GMVECs. This results in the release of VWF, suggesting an initiating role of the coagulation system in the pathogenesis.

  8. Dehydroepiandrosterone sulfate augments blood-brain barrier and tight junction protein expression in brain endothelial cells.

    Science.gov (United States)

    Papadopoulos, Dimitrios; Scheiner-Bobis, Georgios

    2017-08-01

    Tight junctions (TJ) between brain endothelial cells are essential for formation and maintenance of the blood-brain barrier (BBB). Although loss of BBB integrity is associated with several neuropathological disorders, treatments that augment or stabilise the BBB are scarce. Here we show that physiological concentrations of dehydroepiandrosterone sulfate (DHEAS) stimulate the expression of the TJ proteins zonula occludens-1 (ZO-1) and claudin-3 in the brain-derived endothelial cell line bEnd.3 and promote TJ formation between neighbouring cells, demonstrated by augmented transendothelial resistance across cell monolayers. Silencing androgen receptor expression by siRNA does not prevent DHEAS-induced stimulation of ZO-1 expression, indicating that conversion of DHEAS into testosterone is not required for its actions. Suppression of Gnα11 expression by siRNA prevents DHEAS actions, pointing towards a G-protein-coupled receptor as being a mediator of the DHEAS effects. These results are consistent with the idea that DHEAS, acting as a hormone in its own right, supports the integrity of the BBB. The current findings might help in developing new strategies for the prevention or treatment of neurological disorders associated with BBB defects. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Prolonged culture of endothelial cells and deposition of basement membrane modify the recruitment of neutrophils.

    Science.gov (United States)

    Butler, Lynn M; Rainger, G Ed; Rahman, Mahbub; Nash, Gerard B

    2005-10-15

    We tested whether endothelial cell conditioning during prolonged culture and deposition of basement membrane (BM) could modify neutrophil recruitment induced by the inflammatory cytokine, tumour necrosis factor-alpha (TNF). Confluent endothelial cells (EC) from human umbilical veins were cultured for 1 to 20 days and then stimulated with 1, 10 or 100 U/ml of TNF for 4 h. When isolated neutrophils were settled on EC stimulated with the lower doses of TNF, the levels of adhesion and the proportion of adherent cells that transmigrated increased markedly with time of culture. At 100 U/ml TNF, time of culture had little effect on recruitment, but the transmigrated neutrophils moved more slowly under the monolayer in longer-term cultures. The inhibitory effects of function-blocking antibodies against E-selectin and beta2-integrin, and studies in which neutrophils were perfused over short- or long-term cultures, suggested that increased adhesion and migration arose from increased efficiency of neutrophil activation by the EC. Prolonged culture was also associated with deposition of a distinct BM. When fresh EC were seeded on day 20 BM, transmigrated neutrophils moved more slowly under the EC than under control monolayers. Thus, EC change their pro-inflammatory phenotype during prolonged culture, and the deposited basement membrane influences neutrophil migration.

  10. Aspirin Inhibits Platelet-Derived Sphingosine-1-Phosphate Induced Endothelial Cell Migration.

    Science.gov (United States)

    Polzin, Amin; Knoop, Betül; Böhm, Andreas; Dannenberg, Lisa; Zurek, Mark; Zeus, Tobias; Kelm, Malte; Levkau, Bodo; Rauch, Bernhard H

    2018-01-01

    Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies. © 2017 S. Karger AG, Basel.

  11. Low Anticoagulant Heparin Blocks Thrombin-Induced Endothelial Permeability in a PAR-Dependent Manner

    Science.gov (United States)

    Gonzales, Joyce N.; Kim, Kyung-mi; Zemskova, Marina A.; Rafikov, Ruslan; Heeke, Brenten; Varn, Matthew N.; Black, Stephen; Kennedy, Thomas P.; Verin, Alexander D.; Zemskov, Evgeny A.

    2014-01-01

    Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhances lung inflammation, activates endothelial cells and disrupts lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (EC). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin, were also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH, can interfere with thrombin-activated signaling. PMID:24469066

  12. Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells

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    W-J. Wang

    2017-10-01

    Full Text Available Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA using human umbilical vein endothelial cells (HUVEC. HUVECs (5×106 cells/mL were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS and incubated with RA-supplemented hemodialysis fluid (HDF. Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.

  13. Effect of a rosmarinic acid supplemented hemodialysis fluid on inflammation of human vascular endothelial cells.

    Science.gov (United States)

    Wang, W-J; Cheng, M-H; Lin, J-H; Weng, C-S

    2017-10-19

    Chronic systemic inflammation and repetitive damage of vascular endothelia by incompatible dialysis system are probable causes of cardiovascular disease in patients on dialysis. The present study aimed to assess in vitro biocompatibility and anti-inflammatory effect of hemodialysis fluid supplemented with rosmarinic acid (RA) using human umbilical vein endothelial cells (HUVEC). HUVECs (5×106 cells/mL) were pre-exposed to 1 μg/mL of lipopolysaccharides (LPS) and incubated with RA-supplemented hemodialysis fluid (HDF). Cytotoxicity was assessed qualitatively by morphologic assessment and quantitatively by MTT assay. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR and production of NO was quantified. Phosphorylation of AKT and nuclear localization of nuclear factor kappa B (NF-κB) were examined using western blotting. Exposure of HUVECs to RA-supplemented HDF had no influence on morphology and viability. Inhibition of proinflammatory mediator production in HUVECs by RA supplementation to HDF was significant in a dose-dependent manner. Exposure to RA-supplemented HDF resulted in a decrease in nitric oxide synthase expression and reduction of NO production in LPS-stimulated HUVECs. RA supplementation of HDF suppressed Akt activation in LPS-stimulated HUVECs. In addition, the level of cellular IκB was increased in parallel to a reduced nuclear translocation of NF-κB in LPS-induced endothelial cells. Our results suggest that RA-supplemented HDF is biocompatible and significantly suppressed inflammation induced in endothelial cells. In this respect, the use of HDF supplemented with RA could alleviate inflammation and improve long-term treatment of patients with renal failure on dialysis. Further clinical studies are required to confirm the effects.

  14. Laminar shear stress up-regulates peroxiredoxins (PRX) in endothelial cells: PRX 1 as a mechanosensitive antioxidant.

    Science.gov (United States)

    Mowbray, Amy L; Kang, Dong-Hoon; Rhee, Sue Goo; Kang, Sang Won; Jo, Hanjoong

    2008-01-18

    Shear stress plays a significant role in endothelial cell biology and atherosclerosis development. Previous work by our group has shown that fluid flow stimulates important functional changes in cells through protein expression regulation. Peroxiredoxins (PRX) are a family of antioxidant enzymes but have yet to be investigated in response to shear stress. Studies have shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, whereas laminar shear stress (LS) blocks this response. We hypothesized that PRX are responsible for the anti-oxidative effect of LS. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to LS (15 dyn/cm(2)), OS (+/-5 dyn/cm(2), 1 Hz), or static conditions for 24 h. Using Western blot and immunofluorescence staining, all six isoforms of PRX were identified in BAEC. When compared with OS and static, exposure to chronic LS up-regulated PRX 1 levels intracellularly. LS also increased expression of PRX 5 relative to static controls, but not OS. PRX exhibited broad subcellular localization, with distribution in the cytoplasm, Golgi, mitochondria, and intermediate filaments. In addition, PRX 1 knock down, using specific small interference RNA, attenuated LS-dependent reactive oxygen species reduction in BAEC. However, PRX 5 depletion did not. Together, these results suggest that PRX 1 is a novel mechanosensitive antioxidant, playing an important role in shear-dependent regulation of endothelial biology and atherosclerosis.

  15. Preeclampsia serum upregulates CD40/CD40L expression and induces apoptosis in human umbilical cord endothelial cells.

    Science.gov (United States)

    Wu, Chun-feng; Huang, Fu-dan; Sui, Ren-fang; Sun, Jing-xia

    2012-04-18

    The endothelial cell dysfunction observed in preeclampsia (PE) may be induced by CD40/CD40L signaling. This study investigated the role of CD40/CD40L in the pathogenesis of PE by comparing the effect of maternal serum obtained from healthy pregnant women and PE patients on HUVEC cell growth, apoptosis and CD40/CD40L expression. Maternal serum was obtained from 20 patients with PE (PE group) as well as 20 healthy pregnant women (control group). The human umbilical endothelial cell line, CRL1730, was cultured in the presence of maternal serum for 24, 48, and 72 h after which cell growth and apoptosis were assessed by MTT and flow cytometry analysis, respectively. CD40/CD40L expression was determined using flow cytometry and RT-PCR analyses. As compared to CRL1730 cells treated with control sera, those treated with PE sera had altered morphology, decreased cell growth, increased apoptosis and greater CD40/CD40L protein and mRNA expression. Stimulation of CD40/CD40L protein and mRNA expression by PE sera was greatest at 24 h. PE sera may induce endothelial cell damage possibly through increased CD40/CD40L expression in early-onset PE. Further studies are necessary to determine the factor(s) in PE sera responsible for the observed changes in endothelial cell viability.

  16. Preeclampsia serum upregulates CD40/CD40L expression and induces apoptosis in human umbilical cord endothelial cells

    Directory of Open Access Journals (Sweden)

    Wu Chun-feng

    2012-04-01

    Full Text Available Abstract Background The endothelial cell dysfunction observed in preeclampsia (PE may be induced by CD40/CD40L signaling. This study investigated the role of CD40/CD40L in the pathogenesis of PE by comparing the effect of maternal serum obtained from healthy pregnant women and PE patients on HUVEC cell growth, apoptosis and CD40/CD40L expression. Methods Maternal serum was obtained from 20 patients with PE (PE group as well as 20 healthy pregnant women (control group. The human umbilical endothelial cell line, CRL1730, was cultured in the presence of maternal serum for 24, 48, and 72 h after which cell growth and apoptosis were assessed by MTT and flow cytometry analysis, respectively. CD40/CD40L expression was determined using flow cytometry and RT-PCR analyses. Results As compared to CRL1730 cells treated with control sera, those treated with PE sera had altered morphology, decreased cell growth, increased apoptosis and greater CD40/CD40L protein and mRNA expression. Stimulation of CD40/CD40L protein and mRNA expression by PE sera was greatest at 24 h. Conclusions PE sera may induce endothelial cell damage possibly through increased CD40/CD40L expression in early-onset PE. Further studies are necessary to determine the factor(s in PE sera responsible for the observed changes in endothelial cell viability.

  17. New Insight Into Metformin Action: Regulation of ChREBP and FOXO1 Activities in Endothelial Cells.

    Science.gov (United States)

    Li, Xiaoyu; Kover, Karen L; Heruth, Daniel P; Watkins, Dara J; Moore, Wayne V; Jackson, Kathyrin; Zang, Mengwei; Clements, Mark A; Yan, Yun

    2015-08-01

    Metformin has been considered a potential adjunctive therapy in treating poorly controlled type 1 diabetes with obesity and insulin resistance, owing to its potent effects on improving insulin sensitivity. However, the underlying mechanism of metformin's vascular protective effects remains obscure. Thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox state induced by high-glucose concentration, decreases thioredoxin reductase activity and mediates apoptosis induced by oxidative stress. Here we report that high glucose-induced endothelial dysfunction is associated with induction of TXNIP expression in primary human aortic endothelial cells exposed to high-glucose conditions, whereas the metformin treatment suppresses high-glucose-induced TXNIP expression at mRNA and protein levels. We further show that metformin decreases the high-glucose-stimulated nuclear entry rate of two transcription factors, carbohydrate response element-binding protein (ChREBP) and forkhead box O1 (FOXO1), as well as their recruitment on the TXNIP promoter. An AMP-activated protein kinase inhibitor partially compromised these metformin effects. Our data suggest that endothelial dysfunction resulting from high-glucose concentrations is associated with TXNIP expression. Metformin down-regulates high-glucose-induced TXNIP transcription by inactivating ChREBP and FOXO1 in endothelial cells, partially through AMP-activated protein kinase activation.

  18. Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

    Science.gov (United States)

    Stewart-Hutchinson, Phillip J; Szasz, Taylor P; Jaeger, Emily R; Onken, Michael D; Cooper, John A; Morley, Sharon Celeste

    2017-09-01

    Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL -/- ) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL -/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration. © Society for Leukocyte Biology.

  19. Therapeutic Angiogenesis via Solar Cell-Facilitated Electrical Stimulation.

    Science.gov (United States)

    Jeong, Gun-Jae; Oh, Jin Young; Kim, Yeon-Ju; Bhang, Suk Ho; Jang, Hyeon-Ki; Han, Jin; Yoon, Jeong-Kee; Kwon, Sang-Mo; Lee, Tae Il; Kim, Byung-Soo

    2017-11-08

    Cell therapy has been suggested as a treatment modality for ischemic diseases, but the poor survival and engraftment of implanted cells limit its therapeutic efficacy. To overcome such limitation, we used electrical stimulation (ES) derived from a wearable solar cell for inducing angiogenesis in ischemic tissue. ES enhanced the secretion of angiogenic growth factors and the migration of mesenchymal stem cells (MSCs), myoblasts, endothelial progenitor cells, and endothelial cells in vitro. In a mouse ischemic hindlimb model, ES generated by a solar cell and applied to the ischemic region promoted migration of MSCs toward the ischemic site and upregulated expression of angiogenic paracrine factors (vascular endothelial, basic fibroblast, and hepatocyte growth factors; and stromal cell-derived factor-1α). Importantly, solar cell-generated ES promoted the formation of capillaries and arterioles at the ischemic region, attenuated muscle necrosis and fibrosis, and eventually prevented loss of the ischemic limb. Solar cell ES therapy showed higher angiogenic efficacy than conventional MSC therapy. This study shows the feasibility of using solar cell ES as a novel treatment for therapeutic angiogenesis.

  20. Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells.

    Science.gov (United States)

    Coso, Sanja; Zeng, Yiping; Sooraj, Dhanya; Williams, Elizabeth D

    2011-10-15

    Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

  1. Tumor Microvasculature: Endothelial Leakiness and Endothelial Pore Size Distribution in a Breast Cancer Model

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    E.E. Uzgiris

    2008-01-01

    Full Text Available Tumor endothelial leakiness is quantified in a rat mammary adenocarcinoma model using dynamic contrast enhancement MRI and contrast agents of widely varying sizes. The contrast agents were constructed to be of globular configuration and have their uptake rate into tumor interstitium be driven by the same diffusion process and limited only by the availability of endothelial pores of passable size. It was observed that the endothelial pore distribution has a steep power law dependence on size, r−β, with an exponent of −4.1. The model of large pore dominance in tumor leakiness as reported in some earlier investigation with fluorescent probes and optical chamber methods is rejected for this tumor model and a number of other tumor types including chemically induced tumors. This steep power law dependence on size is also consistent with observations on human breast cancer.

  2. Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells.

    Science.gov (United States)

    Bernardini, Chiara; Zannoni, Augusta; Turba, Maria Elena; Fantinati, Paolo; Tamanini, Carlo; Bacci, Maria Laura; Forni, Monica

    2005-01-01

    Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any

  3. Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes.

    Science.gov (United States)

    Lansink, M; van Bennekum, A M; Blaner, W S; Kooistra, T

    1997-07-15

    Retinoic acid stimulates the expression of tissue-type plasminogen activator (t-PA) in vascular endothelial cells in vitro and enhances t-PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t-PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that all-trans-retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All-trans-retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9-cis-Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9-cis-retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise all-trans-retinoic acid to 9-cis-retinoic acid and vice versa. In both endothelial cells and hepatocytes all-trans-retinoic acid metabolism was inhibitable by the cytochrome P-450 inhibitors liarozole (10 microM) and ketoconazole (10 microM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10-fold lower all-trans-retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t-PA. In conclusion, our results clearly demonstrate that all-trans-retinoic acid and 9-cis retinoic acid are actively but differently metabolized and isomerised by human

  4. Molecular sieving characteristics of the cultured endothelial monolayer.

    Science.gov (United States)

    Siflinger-Birnboim, A; Del Vecchio, P J; Cooper, J A; Blumenstock, F A; Shepard, J M; Malik, A B

    1987-07-01

    We examined the selectivity of the bovine pulmonary artery endothelial monolayer in vitro to molecules of different sizes. The cultured bovine pulmonary endothelial monolayer was grown on a gelatinized filter and the transendothelial transport was studied by determining the permeability of molecules ranging from 182 to 340,000 daltons under diffusion conditions. The permeabilities across the cultured bovine endothelium were modeled according to cylindrical pore theory. The data were best fit by a two-pore model with radii 65 A and 304 A and a ratio of small to large pores of 160:1. The results indicate that the cultured endothelial monolayer is a selective barrier to molecules of different sizes and that the molecular selectivity is consistent with a diffusional pathway through endothelial pore equivalents. The cultured endothelial monolayer is a useful system for studying the permeability characteristics of the endothelial barrier.

  5. Receptor-Mediated Transport of Insulin across Endothelial Cells

    Science.gov (United States)

    King, George L.; Johnson, Sandra M.

    1985-03-01

    Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.

  6. Descemet stripping and endothelial keratoplasty in endothelial dysfunctions: Three-month results in 75 eyes

    Directory of Open Access Journals (Sweden)

    Basak Samar

    2008-01-01

    Full Text Available Purpose: To analyze the results of Descemet stripping and endothelial keratoplasty (DSEK in the first consecutive 75 cases. Materials and Methods: Prospective, non-randomized, non-comparative interventional case series. Seventy-five eyes of 75 patients with endothelial dysfunctions of different etiology, scheduled for DSEK, were included in this study. Healthy donor cornea with a cell count of> 2000 cells/sq mm was considered for transplantation in each case. Indications, operative problems and postoperative complications were noted. Best corrected visual acuity (BCVA, refractive and keratometric astigmatism, central corneal thickness (CCT and endothelial cell density (ECD were analyzed for each patient after a minimum follow-up of three months. Results: Main indication was pseudophakic corneal edema and bullous keratopathy in 53 (70.7% eyes. Seventeen (22.7% cases had moderate to severe Fuchs′ dystrophy with various grades of cataract; and DSEK was combined with manual small-incision cataract surgery (MSICS with posterior chamber intraocular lens (PCIOL in those cases. After three months, BCVA was 20/60 or better in 62 (82.7% cases. Mean refractive and keratometric astigmatism were 1.10 ± 0.55 diopter cylinder (DCyl and 1.24 ± 0.92 DCyl. The CCT and ECD were 670.8 ± 0.32 µm and 1485.6 ± 168.6/sq mm respectively. The mean endothelial cell loss after three months was 26.8 ± 4.24% (range: 13.3-38.4%. Dislocation of donor lenticule occurred in six (8.0% eyes. Graft failure occurred in one case. Conclusions: Descemet stripping and endothelial keratoplasty is a safe and effective procedure in patients with endothelial dysfunctions with encouraging surgical and visual outcomes. It can be safely combined with MSICS with PCIOL in patients with moderate to severe Fuchs′ dystrophy with cataract.

  7. Soluble endothelial cell molecules and circulating endothelial cells in patients with venous thromboembolism.

    Science.gov (United States)

    Torres, Cláudia; Matos, Rui; Morais, Sara; Campos, Manuel; Lima, Margarida

    2017-12-01

    : To evaluate the plasma levels of soluble endothelial cell molecules in patients with venous thromboembolism (VTE) out of the acute phase as compared with healthy individuals. We also investigated the possible associations of the soluble endothelial cell molecules among them, as well as with other clinical and laboratory data, including the numbers of circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and CEC expressing activation-related [cluster of differentiation (CD)54 and CD62E] and procoagulant (CD142) markers. In total, 15 patients with VTE and 20 normal individuals were studied. The CEC and CEP were quantified and characterized by flow cytometry. The soluble molecules studied included P-selectin, E-selectin, intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1 and tissue factor (ELISA), and von Willebrand factor antigen (immunoturbidimetry). VTE patients had significantly higher levels of vascular cell adhesion molecule 1 and von Willebrand factor antigen and lower levels of soluble E-selectin than controls. They also showed significantly higher numbers of CEC, as of activated/procoagulant CEC and lower numbers of CEP, compared with controls. We did not find any correlation between the levels of soluble molecules and the numbers of endothelial cell in circulation, but there was with several clinical and laboratory data in VTE patients. Our results would suggest that in VTE patients, the endothelium remains activated and in some hypercoagulable state. The levels of soluble endothelial cell molecules did not seem to be directly related to the numbers of CEC and CEP neither reflected the number of activated CEC, which may be because of the different function that surface and soluble molecules may have.

  8. Effects of trovafloxacin on the IL-1-dependent activation of E-selectin in human endothelial cells in vitro.

    Science.gov (United States)

    Zakeri, S M; Meyer, H; Meinhardt, G; Reinisch, W; Schrattbauer, K; Knoefler, M; Block, L H

    2000-06-01

    E-selectin is an endothelial-specific surface protein, which is transiently expressed in response to inflammatory cytokines and plays an important role in the recruitment of leukocytes to the site of infection. The effect of two fluoroquinolones, ciprofloxacin (cipro) and trovafloxacin (trova), on the interleukin-1 (IL-1)-dependent activation of E-Selectin was studied on human umbilical vein endothelial cells (HUVEC) in vitro. Trova, at 80 microg/ml, affected the transient expression of E-selectin mRNA after pro-inflammatory stimulation with IL-1 leading to a sustained expression over 24 h. Surface expression of E-selectin remained upregulated after 24 h in a higher percentage of cells when they were activated in the presence of trova, as determined by flow cytometry analysis. Moreover, the concentration of shedded soluble E-selectin (sE-selectin) in the cell supernatant increased by 3.5 fold compared to those stimulated in the presence of cipro or without fluoroquinolones. Analogously, the antiproliferative effect of trova on endothelial cells was found to be more pronounced compared to cipro leading to an accumulation of cells arrested in G1-phase. These data provide evidence that accumulation of high concentration of trova in vivo in inflamed tissue might alter inflammatory responses.

  9. Alpha-tocopherol and BAY 11-7082 reduce vascular cell adhesion molecule in human aortic endothelial cells.

    Science.gov (United States)

    Catalán, Ursula; Fernández-Castillejo, Sara; Pons, Laia; Heras, Mercedes; Aragonés, Gemma; Anglès, Neus; Morelló, Jose-Ramon; Solà, Rosa

    2012-01-01

    In endothelial dysfunction, vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression (collectively termed cell adhesion molecules; CAMs) increase at sites of atherosclerosis and are stimulated by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α). We evaluated the effect of alpha-tocopherol (AT; 10-150 µM) and BAY 11-7082 (BAY; 0.1 or 1 µM) on CAMs mRNA expression as well as their protein in soluble release form (sCAMs) in human aortic endothelial cells (HAECs) activated by TNF-α (1 or 10 ng/ml). Also, we determined the extent of lymphocyte adhesion to activated HAECs. BAY reduced VCAM-1, E-selectin and ICAM-1 mRNA expression by 30, 30 and 10%, respectively. Furthermore, protein reduction of sVCAM-1 by 70%, sE-selectin by 51% and sICAM-1 by 25% compared to HAECs stimulated by TNF-α was observed (p adhesion to human Jurkat T lymphocytes was higher compared to nonactivated HAECs (p adhesion (p cell adhesion, while AT selectively inhibits VCAM-1; both induce endothelial dysfunction improvement. Copyright © 2012 S. Karger AG, Basel.

  10. MicroRNA-34a regulation of endothelial senescence

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Takashi; Yagi, Shusuke [Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, West Henrietta, NY 14586 (United States); Yamakuchi, Munekazu, E-mail: munekazu_yamakuchi@urmc.rochester.edu [Department of Medicine, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, West Henrietta, NY 14586 (United States)

    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.

  11. Strategies to reverse endothelial dysfunction in diabetic nephropathy

    OpenAIRE

    Badal, Shawn S.; Danesh, Farhad R.

    2012-01-01

    Endothelial dysfunction underlies the basic pathophysiology of microvascular complications of diabetes. Endothelial dysfunction is associated with impaired nitric oxide (NO) availability. Since NO production is tightly regulated by endothelial nitric oxide synthase (eNOS), several therapeutic strategies have been investigated and proposed to improve eNOS bioavailability in the vasculature. The findings of Cheng et al. suggest that increased availability of eNOS may be an effective strategy in...

  12. Endothelial progenitor cells, cardiovascular risk factors and lifestyle modifications.

    Science.gov (United States)

    Di Stefano, Rossella; Felice, Francesca; Feriani, Roberto; Balbarini, Alberto

    2013-04-01

    Endothelial progenitor cells (EPCs) contribute substantially to preservation of a structurally and functionally intact endothelium. EPCs home in to the sites of endothelial injury and ischemia, where they proliferate, differentiate and integrate into the endothelial layer or exert a paracrine function by producing vascular growth factors. This review will focus on successful lifestyle interventions that aim to maintain vascular health through beneficial actions on cell populations with vasculogenic potential. The results of the studies proving the role of healthy lifestyle are particularly emphasized.

  13. Endothelial Cell Implantation and Survival within Experimental Gliomas

    Science.gov (United States)

    Lal, Bachchu; Indurti, Ravi R.; Couraud, Pierre-Olivier; Goldstein, Gary W.; Laterra, John

    1994-10-01

    The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the β-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by β-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

  14. Stimulatory effect of ouabain on VCAM-1 and iNOS expression in murine endothelial cells: involvement of NF-kappa B.

    Science.gov (United States)

    Bereta, J; Cohen, M C; Bereta, M

    1995-12-11

    Endothelial cells play a pivotal role in the development of atherosclerosis. An 'activated' phenotype of these cells is manifested by signal transduction-dependent expression of genes encoding cytokines, pro- and anticoagulant factors, and cell adhesion molecules. In the current study we examined the effect of ouabain, an inhibitor of Na+/K(+)-ATPase, on the process of endothelial cell activation. We demonstrated that ouabain was able to stimulate VCAM-1 expression and potentiate the effect of IFN-gamma on this process. Moreover, ouabain provided a complementary signal for either TNF or IFN-gamma in inducing iNOS expression. Our data also show, for the first time, that inhibition of Na+/K(+)-ATPase led to activation of the transcription factor, NF-kappa B, which may provide an explanation for the effects of ouabain on endothelial cells.

  15. The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.

    Science.gov (United States)

    García-Morales, Verónica; Luaces-Regueira, María; Campos-Toimil, Manuel

    2017-12-01

    3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca2+ concentration ([Ca2+]c) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca2+]c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca2+, was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca2+]c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Syncytin is involved in breast cancer-endothelial cell fusions

    DEFF Research Database (Denmark)

    Bjerregaard, B; Holck, Susanne; Christensen, Ib Jarle

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  17. Femtosecond-laser-assisted Descemet's stripping endothelial keratoplasty.

    Science.gov (United States)

    Cheng, Yanny Y Y; Pels, Elisabeth; Nuijts, Rudy M M A

    2007-01-01

    To our knowledge, we describe the first patient with pseudophakic bullous keratoplasty treated with femtosecond-laser-assisted endothelial keratoplasty. A 5.5 mm corneoscleral tunnel incision was made; after Descemet's membrane was stripped, an 8.0 mm posterior lamellar corneal disk prepared with a femtosecond laser was inserted into the anterior chamber against the recipient cornea without the use of corneal sutures. Four months postoperatively, the posterior corneal disk was clear and the induced astigmatism was 2.1 diopters, demonstrating a functional corneal endothelial layer. The femtosecond laser offers a new surgical approach for minimally invasive endothelial keratoplasty in corneal endothelial disorders.

  18. Vascular Endothelial Growth Factor Levels in Patients with Hypothyroidism

    Directory of Open Access Journals (Sweden)

    O.M. Didushko

    2016-01-01

    Full Text Available The objective of the investigation was to determine the levels of vascular endothelial growth factor in patients with primary hypothyroidism. Having examined 133 patients with manifested hypothyroidism, we discovered that levels of vascular endothelial growth factor significantly increased in blood plasma of both obese and non-obese patients, but the highest indices have been found in patients with hypothyroidism resulting from autoimmune thyroiditis and with obesity. The obtained correlations indicate mutually aggravating effect of thyroid hypofunction, obesity and hypercholesterolemia on the development of endothelial dysfunction in patients with primary hypothyroidism, in particular on the increase of vascular endothelial growth factor levels.

  19. Vagus Nerve Stimulation

    Science.gov (United States)

    Howland, Robert H.

    2014-01-01

    The vagus nerve is a major component of the autonomic nervous system, has an important role in the regulation of metabolic homeostasis, and plays a key role in the neuroendocrine-immune axis to maintain homeostasis through its afferent and efferent pathways. Vagus nerve stimulation (VNS) refers to any technique that stimulates the vagus nerve, including manual or electrical stimulation. Left cervical VNS is an approved therapy for refractory epilepsy and for treatment resistant depression. Right cervical VNS is effective for treating heart failure in preclinical studies and a phase II clinical trial. The effectiveness of various forms of non-invasive transcutaneous VNS for epilepsy, depression, primary headaches, and other conditions has not been investigated beyond small pilot studies. The relationship between depression, inflammation, metabolic syndrome, and heart disease might be mediated by the vagus nerve. VNS deserves further study for its potentially favorable effects on cardiovascular, cerebrovascular, metabolic, and other physiological biomarkers associated with depression morbidity and mortality. PMID:24834378

  20. Thrombin Has Biphasic Effects on the Nitric Oxide-cGMP Pathway in Endothelial Cells and Contributes to Experimental Pulmonary Hypertension

    Science.gov (United States)

    Nickel, Katrin F.; Laux, Volker; Heumann, Rolf; von Degenfeld, Georges

    2013-01-01

    Background A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. Principal Findings Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1). Conclusion These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension. PMID:23785394

  1. Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Katrin F Nickel

    Full Text Available BACKGROUND: A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. PRINCIPAL FINDINGS: Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs. Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS and soluble guanylate cyclase (sGC subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot. In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs. Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1. CONCLUSION: These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.

  2. The Probiotic Mixture VSL#3 Accelerates Gastric Ulcer Healing by Stimulating Vascular Endothelial Growth Factor

    Science.gov (United States)

    Dharmani, Poonam; De Simone, Claudio; Chadee, Kris

    2013-01-01

    Studies assessing the effect and mechanism of probiotics on diseases of the upper gastrointestinal tract (GI) including gastric ulcers are limited despite extensive work and promising results of this therapeutic option for other GI diseases. In this study, we investigated the mechanisms by which the probiotic mixture VSL#3 (a mixture of eight probiotic bacteria including Lactobacilli, Bifidobacteria and Streptococcus species) heals acetic acid induced gastric ulcer in rats. VSL#3 was administered orally at low (6×109 bacteria) or high (1.2×1010 bacteria) dosages from day 3 after ulcer induction for 14 consecutive days. VSL#3 treatments significantly enhanced gastric ulcer healing in a dose-dependent manner. To assess the mechanism(s) whereby VSL#3 exerted its protective effects, we quantified the gene expression of several pro-inflammatory cytokines, protein and expression of stomach mucin-Muc5ac, regulatory cytokine-IL-10, COX-2 and various growth factors. Of all the components examined, only expression and protein production of VEGF was increased 332-fold on day 7 in the ulcerated tissues of animals treated with VSL#3. Predictably, animals treated with VEGF neutralizing antibody significantly delayed gastric ulcer healing in VSL#3 treated animals. This is the first report to demonstrate high efficacy of the probiotic mixture VSL#3 in enhancing gastric ulcer healing. Probiotic efficacy was effective at higher concentrations of VSL#3 by specifically increasing the expression and production of angiogenesis promoting growth factors, primarily VEGF. PMID:23484048

  3. Lysyl Oxidase Plays a Critical Role in Endothelial Cell Stimulation to Drive Tumor Angiogenesis

    DEFF Research Database (Denmark)

    Baker, Ann-Marie; Bird, Demelza; Welti, Jonathan C

    2013-01-01

    Identification of key molecules that drive angiogenesis is critical for the development of new modalities for the prevention of solid tumor progression. Using multiple models of colorectal cancer, we show that activity of the extracellular matrix-modifying enzyme lysyl oxidase (LOX) is essential...

  4. Activation of Secretagogue Independent Gastric Acid Secretion via Endothelial Nitric Oxide Synthase Stimulation in Rats

    Directory of Open Access Journals (Sweden)

    Alice Miriam Kitay

    2017-12-01

    Full Text Available Background/Aims: L-arginine is an important mediator of cell division, wound healing, and immune function. It can be transformed by the nitric oxide synthase (NOS to nitric oxide (NO, an important cell signaling molecule. Recent studies from our laboratory demonstrate specific effects of L-arginine (10mM exposure on gastric acid secretion in rat parietal cells. Methods: Studies were performed with isolated gastric glands and the pH sensitive dye BCECF-AM +/- L-arginine to examine its effects on acid secretion. The direct NO-donor diethylamine NONOate sodium salt hydrate, was also used while monitoring intracellular pH. The specific inhibitor of the intracellular NO signal cascade ODQ was also used. Results: We found that gastric proton extrusion was activated with application of L-arginine (10mM, in a separate series when L-arginine (10mM + L-NAME (30µM were added there was no acid secretion. Addition of the NO-donor diethylamine NONOate sodium salt hydrate (10µM also induced acid secretion. When the selective sGC-inhibitor ODQ was added with NONOate we did not observe acid secretion. Conclusion: We conclude that L-arginine is a novel secretagogue, which can mediate gastric acid secretion. Furthermore, the intake of L-arginine causes direct activation of the H+, K+ ATPase and increased proton extrusion from parietal cells resulting in the increased risk for acid-related diseases. The NO/sGC/cGMP pathway has never been described as a possible intracellular mechanism for H+, K+ ATPase activation before and presents a completely new scientific finding. Moreover, our studies demonstrate a novel role for L-NAME to effectively eliminate NOS induced acid secretion and thereby reducing the risk for L-arginine inducible ulcer disease.

  5. Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

    NARCIS (Netherlands)

    Post, Ivo C. J. H.; de Boon, Wadim M. I.; Heger, Michal; van Wijk, Albert C. W. A.; Kroon, Jeffrey; van Buul, Jaap D.; van Gulik, Thomas M.

    2013-01-01

    Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed

  6. The endothelial surface layer: a new target of research in kidney failure and peritoneal dialysis

    NARCIS (Netherlands)

    Vlahu, C.A.

    2016-01-01

    The endothelial glycocalyx is an important regulator of vascular homeostasis, and damage to this complex structure results in increased vascular vulnerability. Together with associated plasma molecules it forms the endothelial surface layer. Because of its vasculoprotective effects, the endothelial

  7. Adult human dental pulp stem cells promote blood-brain barrier permeability through vascular endothelial growth factor-a expression.

    Science.gov (United States)

    Winderlich, Joshua N; Kremer, Karlea L; Koblar, Simon A

    2016-06-01

    Stem cell therapy is a promising new treatment option for stroke. Intravascular administration of stem cells is a valid approach as stem cells have been shown to transmigrate the blood-brain barrier. The mechanism that causes this effect has not yet been elucidated. We hypothesized that stem cells would mediate localized discontinuities in the blood-brain barrier, which would allow passage into the brain parenchyma. Here, we demonstrate that adult human dental pulp stem cells express a soluble factor that increases permeability across an in vitro model of the blood-brain barrier. This effect was shown to be the result of vascular endothelial growth factor-a. The effect could be amplified by exposing dental pulp stem cell to stromal-derived factor 1, which stimulates vascular endothelial growth factor-a expression. These findings support the use of dental pulp stem cell in therapy for stroke. © The Author(s) 2015.

  8. Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled

    Energy Technology Data Exchange (ETDEWEB)

    De Nucci, G.; Gryglewski, R.J.; Warner, T.D.; Vane, J.R. (William Harvey Research Institute, London (England))

    1988-04-01

    Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F{sub 1{alpha}}. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C. The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. The authors suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.

  9. Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

    2014-01-01

    electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

  10. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver

    NARCIS (Netherlands)

    Filali, Ebtisam El; Hiralall, Johan K.; van Veen, Henk A.; Stolz, Donna B.; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and

  11. Endothelial replacement without surface corneal incisions or sutures: topography of the deep lamellar endothelial keratoplasty procedure.

    Science.gov (United States)

    Terry, M A; Ousley, P J

    2001-01-01

    To evaluate the immediate postoperative corneal topography after the deep lamellar endothelial keratoplasty procedure. Eight eye bank eyes underwent deep lamellar endothelial keratoplasty through a 9.0-mm limbal incision replacing the central 7.0 mm of posterior stroma and endothelium through the lamellar pocket wound. Orbscan topography was performed before and after surgery, and simulated keratometry readings and central corneal diopter power were recorded. The change in astigmatism and corneal power from preoperative to postoperative readings was then determined. The net change in corneal astigmatism averaged 0.4+/-0.5 diopters (range, -0.1 to 1.1 diopters). The net change in corneal power averaged -0.2+/-0.4 diopters of flattening (range, -0.9 to +0.2 diopters). Neither the astigmatism nor the corneal power levels after this surgery were significantly different from the preoperative topography (p = 0.22 and 0.27, respectively). The deep lamellar endothelial keratoplasty procedure, with its absence of corneal surface incisions or sutures, has no significant effect on immediate postoperative corneal topography. The potential advantages of this procedure over penetrating keratoplasty in the treatment of endothelial dysfunction are considerable.

  12. [Deep brain stimulation].

    Science.gov (United States)

    Fraix, V; Pollak, P; Chabardes, S; Ardouin, C; Koudsie, A; Benazzouz, A; Krack, P; Batir, A; Le Bas, J-F; Benabid, A-L

    2004-05-01

    The present renewal of the surgical treatment of Parkinson's disease, almost abandoned for twenty Years, arises from two main reasons. The first is the better understanding of the functional organization of the basal ganglia. It was demonstrated in animal models of Parkinson's disease that the loss of dopaminergic neurons within the substantia nigra, at the origin of the striatal dopaminergic defect, induces an overactivity of the excitatory glutamatergic subthalamo-internal pallidum pathway. The decrease in this hyperactivity might lead to an improvement in the pakinsonian symptoms. The second reason is the improvement in stereotactic neurosurgery in relation with the progress in neuroimaging techniques and with intraoperative electrophysiological microrecordings and stimulations, which help determine the location of the deep brain targets. In the 1970s chronic deep brain stimulation in humans was applied to the sensory nucleus of the thalamus for the treatment of intractable pain. In 1987, Benabid and colleagues suggested high frequency stimulation of the ventral intermediate nucleus of the thalamus in order to treat drug-resistant tremors and to avoid the adverse effects of thalamotomies. How deep brain stimulation works is not well known but it has been hypothetized that it could change the neuronal activities and thus avoid disease-related abnormal neuronal discharges. Potential candidates for deep brain stimulation are selected according to exclusion and inclusion criteria. Surgery can be applied to patients in good general and mental health, neither depressive nor demented and who are severely disabled despite all available drug therapies but still responsive to levodopa. The first session of surgery consists in the location of the target by ventriculography and/or brain MRI. The electrodes are implanted during the second session. The last session consists in the implantation of the neurostimulator. The ventral intermediate nucleus of the thalamus was the

  13. Radioprotection of mouse CNS endothelial cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lyubimova, N.; Coultas, P.; Martin, R. [Peter McCallum Cancer Institute, Melbourne, VIC (Australia)

    1996-12-31

    Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy {gamma}-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

  14. SOCS1 prevents graft arteriosclerosis by preserving endothelial cell function.

    Science.gov (United States)

    Qin, Lingfeng; Huang, Qunhua; Zhang, Haifeng; Liu, Renjing; Tellides, George; Min, Wang; Yu, Luyang

    The aim of this study was to determine the role of suppressor of cytokine signaling 1 (SOCS1) in graft arteriosclerosis (GA). GA, the major cause of late cardiac allograft failure, is initiated by immune-mediated endothelial activation resulting in vascular inflammation and consequent neointima formation. SOCS1, a negative regulator of cytokine signaling, is highly expressed in endothelial cells (ECs) and may prevent endothelial inflammatory responses and phenotypic activation. Clinical specimens of coronary arteries with GA, with atherosclerosis, or without disease were collected for histological analysis. SOCS1 knockout or vascular endothelial SOCS1 (VESOCS1) transgenic mice were used in an aorta transplant model of GA. Mouse aortic ECs were isolated for in vitro assays. Dramatic but specific reduction of endothelial SOCS1 was observed in human GA and atherosclerosis specimens, which suggested the importance of SOCS1 in maintaining normal endothelial function. SOCS1 deletion in mice resulted in basal EC dysfunction. After transplantation, SOCS1-deficient aortic grafts augmented leukocyte recruitment and neointima formation, whereas endothelial overexpression of SOCS1 diminished arterial rejection. Induction of endothelial adhesion molecules in early stages of GA was suppressed by the VESOCS1 transgene, and this effect was confirmed in cultured aortic ECs. Moreover, VESOCS1 maintained better vascular function during GA progression. Mechanistically, endothelial SOCS1, by modulating both basal and cytokine-induced expression of the adhesion molecules platelet/endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, restrained leukocyte adhesion and transendothelial migration during inflammatory cell infiltration. SOCS1 prevents GA progression by preserving endothelial function and attenuating cytokine-induced adhesion molecule expression in vascular endothelium. Copyright © 2014 American College of Cardiology

  15. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

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

  16. Obstructive sleep apnea and endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Wang Q

    2013-10-01

    Full Text Available Qing Wang,1,* Qi Wu,2,* Jing Feng,3,4 Xin Sun5 1The Second Respiratory Department of the First People's Hospital of Kunming, Yunnan, People's Republic of China; 2Tianjin Haihe Hospital, Tianjin, People's Republic of China; 3Respiratory Department of Tianjin Medical University General Hospital, Tianjin, People's Republic of China; 4Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, NC, USA; 5Respiratory Department of Tianjin Haihe Hospital, Tianjin, People's Republic of China *These authors contributed equally to this work Background: Obstructive sleep apnea (OSA occurs in 4% of middle-aged men and 2% of middle-aged women in the general population, and the prevalence is even higher in specific patient groups. OSA is an independent risk factor for a variety of cardiovascular diseases. Endothelial injury could be the pivotal determinant in the development of cardiovascular pathology in OSA. Endothelial damage ultimately represents a dynamic balance between the magnitude of injury and the capacity for repair. Bone marrow–derived endothelial progenitor cells (EPCs within adult peripheral blood present a possible means of vascular maintenance that could home to sites of injury and restore endothelial integrity and normal function. Methods: We summarized pathogenetic mechanisms of OSA and searched for available studies on numbers and functions of EPCs in patients with OSA to explore the potential links between the numbers and functions of EPCs and OSA. In particular, we tried to elucidate the molecular mechanisms of the effects of OSA on EPCs. Conclusion: Intermittent hypoxia cycles and sleep fragmentation are major pathophysiologic characters of OSA. Intermittent hypoxia acts as a trigger of oxidative stress, systemic inflammation, and sympathetic activation. Sleep fragmentation is associated with a burst of sympathetic activation and systemic inflammation. In most studies, a reduction in circulating EPCs has

  17. Coronin 1B serine 2 phosphorylation by p38α is critical for vascular endothelial growth factor-induced migration of human umbilical vein endothelial cells.

    Science.gov (United States)

    Kim, Geun-Young; Park, Jin-Hee; Kim, Hanna; Lim, Hyun-Joung; Park, Hyun-Young

    2016-12-01

    Coronin 1B is an actin-binding protein that regulates various cellular processes including cell motility. However, the role of coronin 1B in vascular cell migration remains controversial. Here, we examined the function of coronin 1B in vascular endothelial growth factor (VEGF)-induced migration of human umbilical vein endothelial cells (HUVECs) and investigated the mechanism by which coronin 1B regulates this cellular process. We found that depletion of coronin 1B increased the VEGF-induced migration of HUVECs. VEGF phosphorylated coronin 1B at Ser2 and stimulated its translocation to the leading edge of stimulated cells. Lentivirus-mediated overexpression of wild-type coronin 1B or a phosphodeficient coronin 1B S2A mutant decreased VEGF-induced transwell migration of HUVECs. Treatment with the p38 inhibitor SB203580 or depletion of p38α by small interfering RNA transfection decreased VEGF-induced coronin 1B phosphorylation. In vitro binding and kinase assays revealed that active p38α directly binds to and phosphorylates coronin 1B at Ser2. In addition, VEGF induced active p38α binding to coronin 1B in HUVECs. VEGF disrupted the interaction between coronin 1B and the actin-related protein (Arp)2/3 complex and p38α depletion prevented this VEGF-induced dissociation. These findings suggest that coronin 1B plays an inhibitory role in VEGF-induced migration of HUVECs and that VEGF-activated p38α phosphorylates coronin 1B at Ser2 and activates the Arp2/3 complex by liberating it from coronin 1B. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Sanguis draconis, a Dragon’s Blood Resin, Attenuates High Glucose-Induced Oxidative Stress and Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Yi Chang

    2014-01-01

    Full Text Available Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by limiting the proliferative potential of these cells. Here we aimed to investigate the effect of an ethanolic extract of Sanguis draconis (SD, a kind of dragon’s blood resin that is obtained from Daemonorops draco (Palmae, on human umbilical vein endothelial cells (HUVEC under high-glucose (HG stimulation and its underlying mechanism. Concentration-dependent (0–50 μg/mL assessment of cell viability showed that SD does not affect cell viability with a similar trend up to 48 h. Remarkably, SD (10–50 μg/mL significantly attenuated the high-glucose (25 and 50 mM induced cell toxicity in a concentration-dependent manner. SD inhibited high glucose-induced nitrite (NO and lipid peroxidation (MDA production and reactive oxygen species (ROS formation in HUVEC. Western blot analysis revealed that SD treatments abolished HG-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2, nuclear transcription factor, κB (NF-κB, VCAM-1, and E-selectin, and it also blocked the breakdown of PARP-116 kDa protein in a dose-dependent manner. Furthermore, we found that SD increased the expression of Bcl-2 and decreased Bax protein expression in HG-stimulated HUVEC. Thus, these results of this study demonstrate for the first time that SD inhibits glucose induced oxidative stress and vascular inflammation in HUVEC by inhibiting the ERK/NF-κB/PARP-1/Bax signaling cascade followed by suppressing the activation of VCAM-1 and E-selectin. These data suggest that SD may have a therapeutic potential in vascular inflammation due to the decreased levels of oxidative stress, apoptosis, and PARP-1 activation.

  19. Sanguis draconis, a dragon's blood resin, attenuates high glucose-induced oxidative stress and endothelial dysfunction in human umbilical vein endothelial cells.

    Science.gov (United States)

    Chang, Yi; Chang, Ting-Chen; Lee, Jie-Jen; Chang, Nen-Chung; Huang, Yung-Kai; Choy, Cheuk-Sing; Jayakumar, Thanasekaran

    2014-01-01

    Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by limiting the proliferative potential of these cells. Here we aimed to investigate the effect of an ethanolic extract of Sanguis draconis (SD), a kind of dragon's blood resin that is obtained from Daemonorops draco (Palmae), on human umbilical vein endothelial cells (HUVEC) under high-glucose (HG) stimulation and its underlying mechanism. Concentration-dependent (0-50 μg/mL) assessment of cell viability showed that SD does not affect cell viability with a similar trend up to 48 h. Remarkably, SD (10-50 μg/mL) significantly attenuated the high-glucose (25 and 50 mM) induced cell toxicity in a concentration-dependent manner. SD inhibited high glucose-induced nitrite (NO) and lipid peroxidation (MDA) production and reactive oxygen species (ROS) formation in HUVEC. Western blot analysis revealed that SD treatments abolished HG-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), nuclear transcription factor, κB (NF-κB), VCAM-1, and E-selectin, and it also blocked the breakdown of PARP-116 kDa protein in a dose-dependent manner. Furthermore, we found that SD increased the expression of Bcl-2 and decreased Bax protein expression in HG-stimulated HUVEC. Thus, these results of this study demonstrate for the first time that SD inhibits glucose induced oxidative stress and vascular inflammation in HUVEC by inhibiting the ERK/NF-κB/PARP-1/Bax signaling cascade followed by suppressing the activation of VCAM-1 and E-selectin. These data suggest that SD may have a therapeutic potential in vascular inflammation due to the decreased levels of oxidative stress, apoptosis, and PARP-1 activation.

  20. A20 functions as mediator in TNFα-induced injury of human umbilical vein endothelial cells through TAK1-dependent MAPK/eNOS pathway.

    Science.gov (United States)

    Li, Lei; Huang, Bingqing; Song, Shiyang; Sohun, Hareshwaree; Rao, Zhiheng; Tao, Luyuan; Jin, Qike; Zeng, Jingjing; Wu, Rongzhou; Ji, Kangting; Lin, Jiafeng; Wu, Lianpin; Chu, Maoping

    2017-09-12

    A20, a negative regulator of nuclear factor κB signaling, has been shown to attenuate atherosclerotic events. Transforming growth factor beta-activated kinase 1 (TAK1) plays a critical role in TNFα-induced atherosclerosis via endothelial nitric oxide (NO) synthase (eNOS) uncoupling and NO reduction. In the study, we investigated the hypothesis that A20 protected endothelial cell injury induced by TNFα through modulating eNOS activity and TAK1 signalling. Human umbilical vein endothelial cells (HUVECs) were stimulated by TNFα. The impact of A20 on cell apoptosis, eNOS expression and NO production and related TAK1 pathway were detected. Both eNOS and NO production were remarkably reduced. TAK1, p38 MAPK phosphorylation and HUVECs apoptosis were enhanced after TNFα stimulation for 2 hrs. Inhibition of A20 significantly activated TAK1, p38 MAPK phosphorylation, and cell apoptosis, but blocked eNOS expression and NO production. Furthermore, p38 MAPK expression was suppressed by A20 over-expression, but re-enhanced by inhibiting A20 or activation of TAK1. Furtherly, TNFα-induced suppression of eNOS and NO production were largely prevented by silencing p38 MAPK. Collectively, our results suggested that A20-mediated TAK1 inactivation suppresses p38 MAPK and regulated MAPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.

  1. Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro

    NARCIS (Netherlands)

    Arts, J.; Herr, I.; Lansink, M.; Angel, P.; Kooistra, T.

    1997-01-01

    Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a

  2. Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells.

    Science.gov (United States)

    Go, Y M; Park, H; Maland, M C; Darley-Usmar, V M; Stoyanov, B; Wetzker, R; Jo, H

    1998-11-01

    Shear stress differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) by mechanisms involving Galphai2 and Gbeta/gamma proteins, respectively, in bovine aortic endothelial cells (BAEC). The early events in this signaling mechanism by which G proteins regulate ERK and JNK in response to shear stress have not been defined. Here we show that BAEC endogenously express a G protein-dependent form of phosphatidylinositol 3-kinase, PI3Kgamma, and its activity is stimulated by shear stress. PI3Kgamma activity was measured in vitro using BAEC that were transiently transfected with an epitope-tagged PI3Kgamma (vsv-PI3Kgamma). Exposure of BAEC to shear stress rapidly and transiently stimulated the activity of vsv-PI3Kgamma (maximum by 15 s, with a return to basal after 1-min exposure to 5 dyn/cm2 shear stress). Activity of vsv-PI3Kgamma was stimulated by shear stress intensities as low as 0.5 dyn/cm2. Treatment of BAEC with an inhibitor of PI3K, wortmannin, inhibited shear-dependent activation of JNK but had no effect on that of ERK. Furthermore, expression of a kinase-inactive mutant (PI3KgammaK799R) in BAEC inhibited the shear-dependent activation of JNK but not ERK. Taken together, these results suggest that PI3Kgamma selectively regulates the shear-sensitive JNK pathway. This differential and novel signaling pathway may be responsible for coordinating various mechanosensitive events in endothelial cells.

  3. Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic and quiescent adult tissues.

    Science.gov (United States)

    Lambeng, Nathalie; Wallez, Yann; Rampon, Christine; Cand, Francine; Christé, Georges; Gulino-Debrac, Danielle; Vilgrain, Isabelle; Huber, Philippe

    2005-02-18

    Vascular endothelial-cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis. Additionally, Src kinase was constitutively associated with VE-cadherin in both quiescent and angiogenic tissues and increased phosphorylation of VE-cadherin-associated Src was detected in uterus and ovary after hormonal treatment. Src-VE-cadherin association was detected in cultured endothelial cells, independent of VE-cadherin phosphorylation state and Src activation level. In this model, Src inhibition impaired VEGF-induced VE-cadherin phosphorylation, indicating that VE-cadherin phosphorylation was dependent on Src activation. We conclude that VE-cadherin is a substrate for tyrosine kinases in vivo and that its phosphorylation, together with that of associated Src, is increased by angiogenic stimulation. Physical association between Flk1, Src, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of VEGF-stimulated angiogenic processes.

  4. From glioblastoma to endothelial cells through extracellular vesicles: messages for angiogenesis.

    Science.gov (United States)

    Giusti, Ilaria; Delle Monache, Simona; Di Francesco, Marianna; Sanità, Patrizia; D'Ascenzo, Sandra; Gravina, Giovanni Luca; Festuccia, Claudio; Dolo, Vincenza

    2016-09-01

    Glioblastoma has one of the highest mortality rates among cancers, and it is the most common and malignant form of brain cancer. Among the typical features of glioblastoma tumors, there is an aberrant vascularization: all gliomas are among the most vascularized/angiogenic tumors. In recent years, it has become clear that glioblastoma cells can secrete extracellular vesicles which are spherical and membrane-enclosed particles released, in vitro or in vivo, by both normal and tumor cells; they are involved in the regulation of both physiological and pathological processes; among the latter, cancer is the most widely studied. Extracellular vesicles from tumor cells convey messages to other tumor cells, but also to normal stromal cells in order to create a microenvironment that supports cancer growth and progression and are implicated in drug resistance, escape from immunosurveillance and from apoptosis, as well as in metastasis formation; they are also involved in angiogenesis stimulation, inducing endothelial cells proliferation, and other pro-angiogenic activities. To this aim, the present paper assesses in detail the extracellular vesicles phenomenon in the human glioblastoma cell line U251 and evaluates extracellular vesicles ability to promote the processes required to achieve the formation of new blood vessels in human brain microvascular endothelial cells, highlighting that they stimulate proliferation, motility, and tube formation in a dose-response manner. Moreover, a molecular characterization shows that extracellular vesicles are fully equipped for angiogenesis stimulation in terms of proteolytic enzymes (gelatinases and plasminogen activators), pro-angiogenic growth factors (VEGF and TGFβ), and the promoting-angiogenic CXCR4 chemokine receptor.

  5. Exposure to febrile temperature modifies endothelial cell response to tumor necrosis factor-alpha.

    Science.gov (United States)

    Hasday, J D; Bannerman, D; Sakarya, S; Cross, A S; Singh, I S; Howard, D; Drysdale, B E; Goldblum, S E

    2001-01-01

    Fever is an important regulator of inflammation that modifies expression and bioactivity of cytokines, including tumor necrosis factor (TNF)-alpha. Pulmonary vascular endothelium is an important target of TNF-alpha during the systemic inflammatory response. In this study, we analyzed the effect of a febrile range temperature (39.5 degrees C) on TNF-alpha-stimulated changes in endothelial barrier function, capacity for neutrophil binding and transendothelial migration (TEM), and cytokine secretion in human pulmonary artery endothelial cells (EC). Permeability for [(14)C]BSA tracer was increased by treatment with TNF-alpha, and this effect was augmented by incubating EC at 39.5 degrees C. Treating EC with 2. 5 U/ml TNF-alpha stimulated an increase in subsequent neutrophil adherence and TEM. Incubating EC at 39.5 degrees C caused a 30% increase in TEM but did not modify the enhancement of neutrophil adherence or TEM by TNF-alpha treatment. Analysis of cytokine expression in EC cultures exposed to TNF-alpha at either 37 degrees or 39.5 degrees C revealed three patterns of temperature and TNF-alpha responsiveness. Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-8 were not detectable in untreated EC but were increased after TNF-alpha exposure, and this increase was enhanced at 39.5 degrees C. IL-6 expression was also increased with TNF-alpha exposure, but IL-6 expression was lower in 39.5 degrees C EC cultures. Transforming growth factor-beta(1) was constitutively expressed, and its expression was not influenced either by TNF-alpha or exposure to 39.5 degrees C. These data demonstrate that clinically relevant shifts in body temperature might cause important changes in the effects of proinflammatory cytokines on the endothelium.

  6. Effects of fluorides on apoptosis and activation of human umbilical vein endothelial cells.

    Science.gov (United States)

    Szczepański, M; Kamianowska, M; Kamianowski, G

    2012-04-01

    To determine the effects of fluorides on endothelial functioning. We analyzed expressions of adhesion molecules, ICAM-1 and ICAM-3, and annexin V, on the surface of human umbilical vein endothelial cells (HUVECs) exposed to various concentrations of NaF and SnF(2) . We compared the effects of fluoride-induced changes with those obtained when stimulating HUVECs with TNF-α and verified whether N-acetyl cysteine (NAC), well-known antioxidant, can prevent both fluoride- and TNF-α-induced alterations. The expressions of annexin V and ICAM-1 increased significantly after adding NaF (5.0 or 7.5mM) or Sn(2) F (0.5 or 0.75mM) to the culture medium. Pre-incubating HUVECs with NAC prevented the effects induced by 5.0 mM of NaF and 0.5 mM of Sn(2) F. Only the highest concentration of NaF (7.5mM) triggered the expression of ICAM-3. The expressions of all three molecules increased significantly upon stimulating the cultures with TNF-α (20ng ml(-1) ); these changes were not reversed by pre-incubation with NAC. Fluorides induce oxidative stress, resulting in apoptosis and activation of HUVECs, manifested by an elevated expression of ICAM-1. The oxidative stress resulting from a stimulation by the highest NaF concentration triggers ICAM-3 expression on the HUVECs' surface. © 2011 John Wiley & Sons A/S.

  7. Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities.

    Science.gov (United States)

    Aman, Jurjan; Weijers, Ester M; van Nieuw Amerongen, Geerten P; Malik, Asrar B; van Hinsbergh, Victor W M

    2016-08-01

    Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches. Copyright © 2016 the American Physiological Society.

  8. Long-Term Topical Ganciclovir and Corticosteroids Preserve Corneal Endothelial Function in Cytomegalovirus Corneal Endotheliitis.

    Science.gov (United States)

    Fan, Nai-Wen; Chung, Yu-Chien; Liu, Yao-Chung; Liu, Catherine Jui-Ling; Kuo, Yih-Shiuan; Lin, Pei-Yu

    2016-05-01

    To report the long-term outcomes of topical ganciclovir (GCV) and corticosteroids as a maintenance therapy for cytomegalovirus (CMV) corneal endotheliitis. This retrospective study included 10 eyes of 9 patients diagnosed with CMV corneal endotheliitis with a minimum 1-year follow-up at a tertiary referral hospital between 2008 and 2014. CMV corneal endotheliitis was defined by corneal edema associated with typical keratic precipitates (KPs) and a positive CMV polymerase chain reaction from aqueous humor taps. Patients receiving long-term topical 0.5% GCV and topical corticosteroids without discontinuation were included. The final corneal condition and endothelial cell density (ECD) were reported. The mean age was 45.6 ± 11.7 years. The mean follow-up duration was 48 ± 25 months. All patients exhibited typical coin-shaped and/or linear KPs. A significant resolution of corneal edema and decreased KPs were achieved within 1 month in all patients after initiating topical 0.5% GCV every 2 hours and topical corticosteroids twice a day. The dose frequency was gradually tapered to GCV 4 times and corticosteroids once or twice a day as a maintenance therapy. All 10 eyes had a clear graft or corneas at the end of this study. The mean ECD was 1630 ± 699 cells per millimeter square before treatment and 1776 ± 834 cells per millimeter square at the end of the study period. Topical 0.5% GCV and corticosteroids as a maintenance regimen without interruption effectively preserved long-term corneal endothelial function.

  9. Copeptin under glucagon stimulation.

    Science.gov (United States)

    Lewandowski, Krzysztof C; Lewiński, Andrzej; Skowrońska-Jóźwiak, Elżbieta; Stasiak, Magdalena; Horzelski, Wojciech; Brabant, Georg

    2016-05-01

    Stimulation of growth hormone (GH) and adrenocorticotropic hormone (ACTH) secretion by glucagon is a standard procedure to assess pituitary dysfunction but the pathomechanism of glucagon action remains unclear. As arginine vasopressin (AVP) may act on the release of both, GH and ACTH, we tested here the role of AVP in GST by measuring a stable precursor fragment, copeptin, which is stoichiometrically secreted with AVP in a 1:1 ratio. ACTH, cortisol, GH, and copeptin were measured at 0, 60, 90, 120, 150, and 180 min during GST in 79 subjects: healthy controls (Group 1, n = 32), subjects with pituitary disease, but with adequate cortisol and GH responses during GST (Group 2, n = 29), and those with overt hypopituitarism (Group 3, n = 18). Copeptin concentrations significantly increased over baseline 150 and 180 min following glucagon stimulation in controls and patients with intact pituitary function but not in hypopituitarism. Copeptin concentrations were stimulated over time and the maximal increment correlated with ACTH, while correlations between copeptin and GH were weaker. Interestingly, copeptin as well as GH secretion was significantly attenuated when comparing subjects within the highest to those in the lowest BMI quartile (p Copeptin is significantly released following glucagon stimulation. As this release is BMI-dependent, the time-dependent relation between copeptin and GH may be obscured, whereas the close relation to ACTH suggests that AVP/copeptin release might be linked to the activation of the adrenal axis.

  10. Strategies for Stimulating Discussion

    Science.gov (United States)

    2017-06-01

    practices describe ways to facilitate communication during discussion. Use constructive questioning strategies . Facilitators serve to ensure the...lecture and group discussion teaching strategies in developing critical thinking skills. Communication Education, 45, 212-227. Gilmore, T. N., & Schall, E...U.S. Army Research Institute for the Behavioral and Social Sciences Research Report 2008 Strategies for Stimulating Discussion

  11. Stimulant Medication for ADHD

    OpenAIRE

    J Gordon Millichap

    1990-01-01

    The use of stimulant medication by primary care physicians in treatment of attention deficit hyperactivity disorder was evaluated by a random national survey of family practitioners and direct screening of 457 patients in midwestern cities and is reported from the Division of Developmental Disabilities, Departments of Pediatrics and Preventive Medicine University of Iowa, Iowa City and Department of Psychology, University of Kentucky, Lexington.

  12. Development Block or Stimulant

    DEFF Research Database (Denmark)

    Katznelson, Ron D; Howells, John

    subsequent to the courts upholding the validity of Edison's claim 2. We argue that Edison's patent had the effect of stimulating downstream invention. This analysis is contrary to several recent papers that have been published in the student-reviewed US law literature that assume Edison's patent was used...

  13. E-selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide-binding oligomerization domain-like receptors and Toll-like receptors.

    Science.gov (United States)

    Wan, M; Liu, J R; Wu, D; Chi, X P; Ouyang, X Y

    2015-10-01

    Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and Toll-like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E-selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E-selectin expression. Porphyromonas gingivalis also induced nuclear factor-κB (NF-κB) and P38 mitogen-activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF-κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF-κB and P38 MAPK significantly attenuated E-selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non-redundant roles in endothelial cell activation following P. gingivalis infection. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. C1q/TNF-Related Protein-9 Ameliorates Ox-LDL-Induced Endothelial Dysfunction via PGC-1α/AMPK-Mediated Antioxidant Enzyme Induction

    Directory of Open Access Journals (Sweden)

    Haijian Sun

    2017-05-01

    Full Text Available Oxidized low-density lipoprotein (ox-LDL accumulation is one of the critical determinants in endothelial dysfunction in many cardiovascular diseases such as atherosclerosis. C1q/TNF-related protein 9 (CTRP9 is identified to be an adipocytokine with cardioprotective properties. However, the potential roles of CTRP9 in endothelial function remain largely elusive. In the present study, the effects of CTRP9 on the proliferation, apoptosis, migration, angiogenesis, nitric oxide (NO production and oxidative stress in human umbilical vein endothelial cells (HUVECs exposed to ox-LDL were investigated. We observed that treatment with ox-LDL inhibited the proliferation, migration, angiogenesis and the generation of NO, while stimulated the apoptosis and reactive oxygen species (ROS production in HUVECs. Incubation of HUVECs with CTRP9 rescued ox-LDL-induced endothelial injury. CTRP9 treatment reversed ox-LDL-evoked decreases in antioxidant enzymes including heme oxygenase-1 (HO-1, nicotinamide adenine dinucleotide phosphate (NAD(PH dehydrogenase quinone 1, and glutamate-cysteine ligase (GCL, as well as endothelial nitric oxide synthase (eNOS. Furthermore, CTRP9 induced activation of peroxisome proliferator-activated receptor γ co-activator 1α (PGC1-α and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK. Of interest, AMPK inhibition or PGC1-α silencing abolished CTRP9-mediated antioxidant enzymes levels, eNOS expressions, and endothelial protective effects. Collectively, we provided the first evidence that CTRP9 attenuated ox-LDL-induced endothelial injury by antioxidant enzyme inductions dependent on PGC-1α/AMPK activation.

  15. Desflurane inhibits endothelium-dependent vasodilation more than sevoflurane with inhibition of endothelial nitric oxide synthase by different mechanisms.

    Science.gov (United States)

    Kazuma, Satoshi; Tokinaga, Yasuyuki; Takada, Yukimasa; Azumaguchi, Ryu; Kimizuka, Motonobu; Hayashi, Shunsuke; Yamakage, Michiaki

    2018-01-01

    The effects of desflurane on endothelium-dependent vasodilation remain uncertain, whereas sevoflurane is known to inhibit it. Endothelium-dependent vasodilation is mainly mediated by endothelial nitric oxide synthase. The effects of desflurane on endothelium-dependent vasodilation were compared with those of sevoflurane, and inhibition mechanisms, including phosphorylation of endothelial nitric oxide synthase and the calcium pathway, were evaluated for the two anesthetics. We hypothesized that desflurane would inhibit endothelium-dependent vasodilation in a concentration-dependent manner more than sevoflurane, with inhibition of a calcium pathway. Isolated rat aortic rings were randomly assigned to treatment with desflurane or sevoflurane for measurements of the vasodilation ratio. To determine NO production with desflurane and sevoflurane, an in vitro assay was performed with cultured bovine aortic endothelial cells. These cells were also used for measurement of intracellular calcium or Western blotting. For endothelium-dependent vasodilation, the ratio of vasodilation was more significantly inhibited by 11.4% desflurane than by 4.8% sevoflurane. Inhibition did not between 5.7% desflurane and 2.4% sevoflurane. No inhibitory effect of desflurane or sevoflurane was observed in endothelium-denuded aorta. Desflurane inhibited nitric oxide production caused by stimulation of bradykinin significantly more than sevoflurane. Desflurane had a greater suppressive effect on the bradykinin-induced increase in intracellular calcium concentration than did sevoflurane. Sevoflurane, but not desflurane, inhibited phosphorylation of the serine 1177 residue by bradykinin stimulation. Desflurane inhibited endothelium-dependent vasodilation more than sevoflurane through inhibition of a calcium pathway. Sevoflurane inhibited endothelium-dependent vasodilation by inhibition of phosphorylation of the serine 1177 residue of endothelial nitric oxide synthase. Copyright © 2017 Elsevier

  16. S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

    Science.gov (United States)

    Most, Patrick; Lerchenmüller, Carolin; Rengo, Giuseppe; Mahlmann, Adrian; Ritterhoff, Julia; Rohde, David; Goodman, Chelain; Busch, Cornelius J; Laube, Felix; Heissenberg, Julian; Pleger, Sven T; Weiss, Norbert; Katus, Hugo A; Koch, Walter J; Peppel, Karsten

    2013-01-04

    Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the

  17. Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Izuagie Attairu Ikhapoh

    2015-01-01

    Full Text Available Coronary artery stenting or angioplasty procedures frequently result in long-term endothelial dysfunction or loss and complications including arterial thrombosis and myocardial infarction. Stem cell-based therapies have been proposed to support endothelial regeneration. Mesenchymal stem cells (MSCs differentiate into endothelial cells (ECs in the presence of VEGF-A in vitro. Application of VEGF-A and MSC-derived ECs at the interventional site is a complex clinical challenge. In this study, we examined the effect of atherogenic cytokines (IL-6, TNFα, and Ang II on EC differentiation and function. MSCs (CD44+, CD73+, CD90+, CD14−, and CD45− were isolated from the bone marrow of Yucatan microswine. Naïve MSCs cultured in differentiation media containing VEGF-A (50 ng/mL demonstrated increased expression of EC-specific markers (vWF, PECAM-1, and VE-cadherin, VEGFR-2 and Sox18, and enhanced endothelial tube formation. IL-6 or TNFα caused a dose-dependent attenuation of EC marker expression in VEGF-A-stimulated MSCs. In contrast, Ang II enhanced EC marker expression in VEGF-A-stimulated MSCs. Addition of Ang II to VEGF-A and IL-6 or TNFα was sufficient to rescue the EC phenotype. Thus, Ang II promotes but IL-6 and TNFα inhibit VEGF-A-induced differentiation of MSCs into ECs. These findings have important clinical implications for therapies intended to increase cardiac vascularity and reendothelialize coronary arteries following intervention.

  18. Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

    Directory of Open Access Journals (Sweden)

    Simona F Spampinato

    Full Text Available The ability of the Blood Brain Barrier (BBB to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS: fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

  19. Low-energy laser irradiation increases endothelial cell proliferation, migration, and eNOS gene expression possibly via PI3K signal pathway.

    Science.gov (United States)

    Chen, Chung-Huang; Hung, Huey-Shan; Hsu, Shan-Hui

    2008-01-01

    The purpose of this study, therefore, was to determine the mechanisms by which low-energy laser irradiation (LELI) may exert some of its angiogenic effects via the PI3 kinase/eNOS signaling pathway and induce endothelial cell migration and neovascularization, an important and necessary part of wound healing. The possible molecular mechanism of helium-neon (He-Ne) laser irradiation on endothelial cells was proposed. He-Ne laser at 632.5 nm was used to stimulate human umbilical vein endothelial cell (HUVEC), and its effect on cell proliferation, nitric oxide secretion, and cell migration was determined. Irradiation enhanced endothelial nitric oxidase synthase (eNOS) protein expression, and irradiation of less than 0.26 J/cm(2) enhanced eNOS gene expression in HUVEC. The cell migration ability was promoted for HUVEC irradiated with 0.26 J/cm(2). This agreed with the vinculin protein expression induced by irradiation. In addition, the angiogenesis was promoted. The induced eNOS expression was inhibited by LY294002, indicating that the effect of laser on EC could be attributed to the up-regulation of eNOS expression through PI3K pathway at the cellular and molecular levels as a result of the He-Ne laser. The study has shown that LELI increased endothelial cell proliferation, migration, NO secretion, and identified that activation of PI3K/Akt pathway was a critical step for the elevated for eNOS expression upon LELI.

  20. Vitamin E isoforms differentially regulate intercellular adhesion molecule-1 activation of PKCα in human microvascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Hiam Abdala-Valencia

    Full Text Available ICAM-1-dependent leukocyte recruitment in vivo is inhibited by the vitamin E isoform d-α-tocopherol and elevated by d-γ-tocopherol. ICAM-1 is reported to activate endothelial cell signals including protein kinase C (PKC, but the PKC isoform and the mechanism for ICAM-1 activation of PKC are not known. It is also not known whether ICAM-1 signaling in endothelial cells is regulated by tocopherol isoforms. We hypothesized that d-α-tocopherol and d-γ-tocopherol differentially regulate ICAM-1 activation of endothelial cell PKC.ICAM-1 crosslinking activated the PKC isoform PKCα but not PKCβ in TNFα-pretreated human microvascular endothelial cells. ICAM-1 activation of PKCα was blocked by the PLC inhibitor U73122, ERK1/2 inhibitor PD98059, and xanthine oxidase inhibitor allopurinol. ERK1/2 activation was blocked by inhibition of XO and PLC but not by inhibition of PKCα, indicating that ERK1/2 is downstream of XO and upstream of PKCα during ICAM-1 signaling. During ICAM-1 activation of PKCα, the XO-generated ROS did not oxidize PKCα. Interestingly, d-α-tocopherol inhibited ICAM-1 activation of PKCα but not the upstream signal ERK1/2. The d-α-tocopherol inhibition of PKCα was ablated by the addition of d-γ-tocopherol.Crosslinking ICAM-1 stimulated XO/ROS which activated ERK1/2 that then activated PKCα. ICAM-1 activation of PKCα was inhibited by d-α-tocopherol and this inhibition was ablated by the addition of d-γ-tocopherol. These tocopherols regulated ICAM-1 activation of PKCα without altering the upstream signal ERK1/2. Thus, we identified a mechanism for ICAM-1 activation of PKC and determined that d-α-tocopherol and d-γ-tocopherol have opposing regulatory functions for ICAM-1-activated PKCα in endothelial cells.

  1. Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

    Directory of Open Access Journals (Sweden)

    Haroldo A Toque

    Full Text Available Elevated arginase (Arg activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO synthase (NOS and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC from Akita mice.Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser(1177 (in aorta and CC and nNOS expression (in CC were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks.Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.

  2. Blood Outgrowth Endothelial Cells Increase Tumor Growth Rates and Modify Tumor Physiology: Relevance for Therapeutic Targeting

    Directory of Open Access Journals (Sweden)

    Kalpna Gupta

    2013-02-01

    Full Text Available Endothelial cell precursors from human peripheral blood have been shown to home to areas of neovascularization and may assist tumor growth by increasing or fortifying blood vessel growth. In the present study, the influence of these cells on tumor growth and physiology was investigated and the role of these cells as a therapeutic target or in determining treatment sensitivity was tested. After isolation from human blood and expansion in vitro, actively growing cells with verified endothelial phenotype (Blood Outgrowth Endothelial Cell, BOEC were injected i.v. into tumor bearing mice for three consecutive days. The growth rate was significantly enhanced in relatively small RERF human lung tumors (i.e., less than 150 mm3 grown in immunocompromised mice by an average of 1.5-fold while it had no effect when injections were given to animals bearing larger tumors. There were no signs of toxicity or unwanted systemic effects. We also observed evidence of increased perfusion, vessel number, response to 15 Gy radiation and oxygenation in RERF tumors of animals injected with BOECs compared to control tumors. In addition, FSaII murine fibrosarcoma tumors were found to grow faster upon injection of BOECs. When FSaII tumors were subjected to a partial thermal ablation treatment using high intensity focused ultrasound (HIFU there was consistently elevated detection of fluorescently labeled and i.v. injected endothelial precursors in the tumor when analyzed with optical imaging and/or histological preparations. Importantly, we also observed that BOECs treated with the novel anti-angiogenic peptide anginex in-vitro, show decreased proliferation and increased sensitivity to radiation. In vivo, the normal increase in FSaII tumor growth induced by injected BOECs was blunted by the addition of anginex treatment. It appears that endothelial precursors may significantly contribute to tumor vessel growth, tumor progression and/or repair of tumor damage and may improve

  3. Microvesicles Derived from Inflammation-Challenged Endothelial Cells Modulate Vascular Smooth Muscle Cell Functions.

    Science.gov (United States)

    Pan, Qunwen; Liu, Hua; Zheng, Chunyan; Zhao, Yuhui; Liao, Xiaorong; Wang, Yan; Chen, Yanfang; Zhao, Bin; Lazartigues, Eric; Yang, Yi; Ma, Xiaotang

    2016-01-01

    Purpose: Microvesicles (MV) can modulate the function of recipient cells by transferring their contents. Our previous study highlighted that MV released from tumor necrosis factor-α (TNF-α) plus serum deprivation (SD)-stimulated endothelial progenitor cells, induce detrimental effects on endothelial cells. In this study, we investigated the potential effects of endothelial MV (EMV) on proliferation, migration, and apoptosis of human brain vascular smooth cells (HBVSMC). Methods: EMV were prepared from human brain microvascular endothelial cells (HBMEC) cultured in a TNF-α plus SD medium. RNase-EMV were made by treating EMV with RNase A for RNA depletion. The proliferation, apoptosis and migration abilities of HBVSMC were determined after co-culture with EMV or RNase-EMV. The Mek1/2 inhibitor, PD0325901, was used for pathway analysis. Western blot was used for analyzing the proteins of Mek1/2, Erk1/2, phosphorylation Erk1/2, activated caspase-3 and Bcl-2. The level of miR-146a-5p was measured by qRT-PCR. Results: (1) EMV significantly promoted the proliferation and migration of HBVSMC. The effects were accompanied by an increase in Mek1/2 and p-Erk1/2, which could be abolished by PD0325901; (2) EMV decreased the apoptotic rate of HBVSMC by approximately 35%, which was accompanied by cleaved caspase-3 down-regulation and Bcl-2 up-regulation; (3) EMV increased miR-146a-5p level in HBVSMC by about 2-folds; (4) RNase-treated EMV were less effective than EMV on HBVSMC activities and miR-146a-5p expression. Conclusion: EMV generated under inflammation challenge can modulate HBVSMC function and fate via their carried RNA. This is associated with activation of theMek1/2/Erk1/2 pathway and caspase-3/Bcl-2 regulation, during which miR-146a-5p may play an important role. The data suggest that EMV derived from inflammation-challenged endothelial cells are detrimental to HBVSMC homeostatic functions, highlighting potential novel therapeutic targets for vascular diseases.

  4. Blood Outgrowth Endothelial Cells Increase Tumor Growth Rates and Modify Tumor Physiology: Relevance for Therapeutic Targeting

    Energy Technology Data Exchange (ETDEWEB)

    Pagan, Jonathan, E-mail: jdpagan@uams.edu; Przybyla, Beata; Jamshidi-Parsian, Azemat [Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Gupta, Kalpna [Vascular Biology Center and Division of Hematology-Oncology Transplantation, Department of Medicine, University of Minnesota Medical School, MN 72223 (United States); Griffin, Robert J. [Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

    2013-02-18

    Endothelial cell precursors from human peripheral blood have been shown to home to areas of neovascularization and may assist tumor growth by increasing or fortifying blood vessel growth. In the present study, the influence of these cells on tumor growth and physiology was investigated and the role of these cells as a therapeutic target or in determining treatment sensitivity was tested. After isolation from human blood and expansion in vitro, actively growing cells with verified endothelial phenotype (Blood Outgrowth Endothelial Cell, BOEC) were injected i.v. into tumor bearing mice for three consecutive days. The growth rate was significantly enhanced in relatively small RERF human lung tumors (i.e., less than 150 mm{sup 3}) grown in immunocompromised mice by an average of 1.5-fold while it had no effect when injections were given to animals bearing larger tumors. There were no signs of toxicity or unwanted systemic effects. We also observed evidence of increased perfusion, vessel number, response to 15 Gy radiation and oxygenation in RERF tumors of animals injected with BOECs compared to control tumors. In addition, FSaII murine fibrosarcoma tumors were found to grow faster upon injection of BOECs. When FSaII tumors were subjected to a partial thermal ablation treatment using high intensity focused ultrasound (HIFU) there was consistently elevated detection of fluorescently labeled and i.v. injected endothelial precursors in the tumor when analyzed with optical imaging and/or histological preparations. Importantly, we also observed that BOECs treated with the novel anti-angiogenic peptide anginex in-vitro, show decreased proliferation and increased sensitivity to radiation. In vivo, the normal increase in FSaII tumor growth induced by injected BOECs was blunted by the addition of anginex treatment. It appears that endothelial precursors may significantly contribute to tumor vessel growth, tumor progression and/or repair of tumor damage and may improve the

  5. The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Ramasri Sathanoori

    Full Text Available Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen

  6. Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts.

    Science.gov (United States)

    Zakharova, I S; Zhiven', M K; Saaya, Sh B; Shevchenko, A I; Smirnova, A M; Strunov, A; Karpenko, A A; Pokushalov, E A; Ivanova, L N; Makarevich, P I; Parfyonova, Y V; Aboian, E; Zakian, S M

    2017-03-03

    Endothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source-post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium. Heterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endoth