Bovine Microvascular Endothelial Cells Research Articles

Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections.

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

Müller glial cells inhibit proliferation of retinal endothelial cells via TGF-β2 and Smad signaling.

Neovascularization is a sight-threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)-β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Müller) cells produce TGF-β2 under physiological and post-ischemic conditions. To characterize glial cell-derived mediators of angiogenesis regulation in glial-endothelial interactions in the retina, we co-cultured primary Müller cells and bovine microvascular retinal endothelial cells (BRECs). Müller cell-derived TGF-β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF-β receptors, and stimulated TGF-β-dependent anti-proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF-β2 as well as by the presence of Müller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti-angiogenic action of Müller cell-derived TGF-β2: (i) binding of TGF-β2 to BRECs is mediated by the type-II TGF-β receptor, leading to (ii) activation and phosphorylation of receptor-activated Smads; (iii) Müller cell-derived TGF-β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal-regulated kinase (ERK)-1/-2. Neutralizing TGF-β or TGF-β type-II receptor or blocking the activation of Smads partially abrogated the effect of Müller cell-conditioned media on BRECs. Together, our data suggest that Müller cells release TGF-β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context-dependent action of TGF-β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti-angiogenic role of TGF-β therein.

Endothelial Metabolism of Angiotensin II to Angiotensin III, not Angiotensin (1–7), Augments the Vasorelaxation Response in Adrenal Cortical Arteries

Hyperaldosteronism is linked to the development and progression of several different cardiovascular diseases. Angiotensin (Ang) II increases aldosterone secretion and adrenal blood flow. Ang II peptide fragments are produced by various peptidases, and these Angs have diverse and vital physiologic roles. Due to the uncharacteristic vasorelaxation of adrenal arteries by Ang II, we tested the hypothesis that Ang II metabolism contributes to its relaxant activity in adrenal arteries. Metabolism of Angs by bovine adrenal cortical arteries and isolated bovine adrenal vascular cells was measured by liquid chromatography-mass spectrometry. The primary Ang metabolites of adrenal arteries are Ang III and Ang (1-7), with Ang IV produced to a lesser extent. Bovine microvascular endothelial cells produced a similar metabolic profile to adrenal arteries, whereas bovine adrenal artery smooth muscle cells exhibited less metabolism. In preconstricted adrenal arteries, Ang II caused relaxation in picomolar concentrations and constrictions at 10nM. Ang-converting enzyme 2 inhibition augmented this relaxation response, whereas aminopeptidase inhibition did not. Ang III was equipotent to Ang II in relaxing adrenal arteries. Ang IV did not cause relaxation. Nitric oxide synthase inhibition enhanced Ang II-induced constriction of adrenal arteries. Aminopeptidase inhibition increased the concentration range for Ang II-induced constriction of adrenal arteries. Ang III and Ang IV did not change the basal tone but caused constriction of adrenal arteries with nitric oxide synthase inhibition. These data indicate that Ang II metabolism modulates the vascular effects of Ang II in the adrenal vasculature.

Effect of TRIF on permeability and apoptosis in bovine microvascular endothelial cells exposed to lipopolysaccharide

Bovine respiratory disease complex (BRDC) can be caused by several Gram negative bacteria. Lung endothelial cells may be damaged by the release of lipopolysaccharide (LPS) from these organisms. Toll-like receptor (TLR-4) signaling pathways include the myeloid differentiation primary response gene 88 (MyD88) and the Toll/interleukin (IL)-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF) pathways. The aim of this study was to determine which of these pathways is responsible for permeability changes, apoptosis and cytokine production in bovine lung microvascular cells exposed to LPS.Bovine lung endothelial cells were treated with a peptide to inhibit MyD88 signaling or small interfering RNA (siRNA) to inhibit TRIF signaling. Effects were measured using trans-well endothelial electrical resistance to determine cell monolayer permeability, annexin staining to estimate apoptosis and real-time PCR to measure levels of expression of IL-1β and tumor necrosis factor (TNF)-α mRNA. Inhibition of TRIF signaling reduced permeability changes and apoptosis in endothelial cells exposed to LPS. In contrast, MyD88 inhibition reduced expression of IL-1β and TNF-α mRNA in LPS treated cells, but had no effect on permeability. It was concluded that TRIF signaling in LPS-stimulated lung endothelial cells results in permeability changes and apoptosis.

Isolation of Bovine Luteal Microvascular Endothelial Cells: High Yield Method and Functional Interactions with T Lymphocytes.

Isolation of Bovine Luteal Microvascular Endothelial Cells: High Yield Method and Functional Interactions with T Lymphocytes.

Contribution of Rat Endothelial Progenitor Cells on Three-Dimensional Network FormationIn Vitro

Endothelial progenitor cell (EPC) transplantation is a promising treatment option for ischemic diseases, and understanding the mechanism mediating angiogenic effects of EPCs is essential to further improve its effectiveness as well as their application to in vitro tissue engineering. Here we investigated EPC contribution on network formation using an in vitro three-dimensional network model. Bovine microvascular endothelial cells (ECs) were seeded on collagen gel to create a three-dimensional network model. Subsequently, EPCs isolated from rat bone marrow were seeded on top of a confluent endothelial cell (EC) monolayer to create EPC+EC model. In this model, EPCs promoted EC network formation. Quantitative analyses revealed that the total length, number, and depth of networks were significantly enhanced with the addition of EPCs. EPCs tended to localize in networks formed inside collagen gel rather than on a confluent monolayer with increasing experimental duration. In addition, EPCs preferentially distributed near sprout positions in a confluent monolayer. Furthermore, EPC-conditioned medium promoted network formation, and vascular endothelial growth factor was detected in the conditioned medium. Taken together, EPCs contributed to network formation by direct incorporation with on-site growth factor secretion. The angiogenic ability of EPCs offers a possible cell source to reconstruct vascularized tissues in vitro.

Microbial Transformation of Spirolaxine, a Bioactive Undecaketide Fungal Metabolite from the Basidiomycete Sporotrichum laxum

Incubation of (+)-spirolaxine (= (3R)-5-hydroxy-7-methoxy-3-{5-[(2R,5R,7R)-2-methyl-1,6-dioxaspiro[4.5]dec-7-yl]pentyl}-2-benzofuran-1(3H)-one; 1a) with Bacillus megaterium afforded two new mono- and one new dihydroxylated metabolite(s), all OH groups being introduced on the non-activated six-membered ring. In contrast, exposure of 1a to Cunninghamella echinulata gave rise to hydroxylation on the five-membered ring of the parent structure. The structures and absolute configurations of the new products 1b-e were deduced on the basis of MS and NMR data. The metabolite 1b was investigated, in comparison to 1a, for its cytotoxicity (sulforhodamin-B test) and for its antiproliferative activity towards bovine microvascular endothelial cells (BMEC).

Elevated Glucose Attenuates Agonist- and Flow-Stimulated Endothelial Nitric Oxide Synthase Activity in Microvascular Retinal Endothelial Cells

Impaired vasoactive release of opposing vasodilator and vasoconstrictor mediators due to endothelial dysfunction is integral to the pathogenesis of diabetic retinopathy. The aim of this study was to determine the effect of hyperglycemia on the expression of endothelial nitric oxide synthase (eNOS) and the release of nitric oxide (NO) in bovine microvascular retinal endothelial cells (BRECs) under both static (basal and acetylcholine stimulated) and flow (laminar shear stress [10 dynes/cm2 and pulsatile flow 0.3 to 23 dynes/cm2) conditions using a laminar shear apparatus and an in vitro perfused transcapillary culture system. The activity and expression of eNOS, measured by nitrate levels and immunoblot, respectively, were determined following exposure of BRECs to varying concentrations of glucose and mannitol (0 to 25 mM). Under static conditions the expression of eNOS decreased significantly following exposure to increasing concentrations of glucose when compared to osmotic mannitol controls and was accompanied by a significant dose-dependent decrease in nitrate levels in conditioned medium. The acetylcholine stimulated increase in NO release (2.0 +/- 0.3-fold) was significantly reduced by 55% +/- 5% and 65% +/- 4.5% following exposure to 16 and 25 mM glucose, respectively, when compared to osmotic controls. In parallel studies, glucose significantly inhibited both laminar shear stress and pulsatile flow-induced activity when compared to mannitol. We conclude that hyperglycemia impairs agonist- and flow-dependent release of NO in retinal microvascular endothelial cells and may thus contribute to the vascular endothelial dysfunction and impaired autoregulation of diabetic retinopathy.

Bovine microvascular endothelial cells immortalized with human telomerase

Primary cultures of bovine microvascular endothelial cells (BME) isolated from the adrenal cortex, are commonly used to study vascular endothelium, but have a limited life span. To circumvent these limitations, we have immortalized BME cells with either simian virus 40 (SV40) or with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have investigated whether the clonal populations obtained, maintain differentiated properties characteristic of microvascular endothelium. Immortalized cells were characterized for maintenance of typical endothelial morphology, marker expression, and functional characteristics including uptake of Acetylated low-density lipoprotein (Ac-LDL), capillary-like tube formation in three-dimensional collagen gels, as well as metalloproteinase (MMP) and plasminogen activator (PA)-mediated extracellular proteolysis. Whilst immortalization of BME cells with SV40 was associated with loss of endothelial-specific properties, hTERT-BME exhibited an endothelial phenotype similar to that of wild-type endothelial cells. Specifically, they showed a typical cobblestone morphology, were contact-inhibited, expressed endothelial cell-specific markers (e.g., CD31, vWF) and both fibroblast growth factor receptor 1 (FGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, they expressed receptors for LDL. Importantly, when grown on collagen gels, hTERT-BME cells underwent MMP-dependent tube-like structure formation in response to VEGFR-2 activation. In a collagen gel sandwich assay, hTERT-BME formed tubular structures in the absence of exogenously added angiogenic cytokines. Sustained tube formation was induced by VEGF-A alone or in combination with FGF-2. From 17 sub-clones that displayed a non-transformed phenotype, a high proliferative capacity and tubulogenic properties in three-dimensional collagen gels, we isolated two distinct subpopulations that display a highly specific response to VEGF-A or to FGF-2. We have generated hTERT-BME cells that maintain endothelial-specific properties and function and have isolated clones that respond differentially to VEGF-A or FGF-2. These immortalized cell lines will facilitate the study of endothelial cell biology.

New Concepts for the Functional Maturation of the Bovine Claw: Endothelial Biology, Angiogenesis and Ageing

Introduction: Endothelial dysfunction and impaired angiogenesis are connected to ageing. However, the mechanisms involved in age‐related alterations are not fully elucidated, whereby reduced VEGF‐expression and decreased mobilization of circulating endothelial progenitor cells are suggested as possible causes. When cultured in vitro, endothelial cells often enter a growth arrest phase called ‘cellular senescence’ that is consistent with many changes in age‐related vascular disease. Based on the decreasing lifespan observed in high yielding dairy cows, a hypothesis has been formulated that these cows, due to their enormous metabolic performance, show an accelerated process of ageing, resulting in typical production‐related diseases, particularly lameness. As a basis for further research into the role of endothelial cell biology in high producing cattle, the aim of this study was to set up a model of endothelial ageing. As a first step, the role of angiogenesis in the functional adaptation of the microvasculature of the bovine pododerma was examined.Material and methods: Scanning electron microscopy of vascular corrosion casts of healthy juvenile and adult claws as well as pathologically altered claws, and routine light microscopy of pododermal serial sections was compared to an in vitro model of angiogenesis based on cultured bovine microvascular endothelial cells. For detection of angiogenic molecular control mechanisms effective in both, the pododermal system and our cell culture model, lectin‐ and immunohistochemistry as well as RT‐PCR were carried out.Results and discussion: In the adolescent and adult claw, sprouting and intussusceptive angioadaptation was demonstrated. Evidence of angiogenic processes was also detected in the pathologically altered claws. Immunolocalization of angiogenic factors within the dermo‐epidermal system of the claw was established. The detected angioadaptation may be visible expression of the increased metabolic demands of the claw caused by the growing body‐weight‐load. Increase of the pododermal vascular density via angiogenesis was also the connectional reparative principle in pathologically altered claws.

Pododermal angiogenesis and angioadaptation in the bovine claw

Pododermal microvascularization has been suggested to play a key role in the physiological function of the bovine claw and in the pathogenesis of claw diseases. According to our working hypothesis, angiogenesis plays a central role in the physiological and pathological function of the claw and is induced by the pro-angiogenic vascular endothelial growth factor (VEGF). As a basis for further research, the aim of the present study was to examine the mechanisms of pododermal angiogenesis in the functional adaptation of the microvasculature of the claw in histological serial sections and microcorrosion casts of healthy juvenile and adult claws as well as pathologically altered claws. Scanning electron microscopy of microcorrosion casts allowed assessment of the 3D aspect of pododermal angiogenesis and angioadaptation, and was substantiated by a concomitant examination of a 3D in vitro model of angiogenesis based on cultured bovine microvascular endothelial cells. Particularly in the juvenile, but also in the adult claw, sprouting and intussusceptive angioadaptation was demonstrated and resembled the respective stages of in vitro angiogenesis. Evidence of angiogenic processes was also detected in the pathologically altered claws displaying symptoms of subclinical laminitis and/or the digital dermatitis complex. The detected angioadaptation was visible expression of the increased metabolic demands of the claw caused by the growing body weight load. Angiogenic remodeling of the pododermal angioarchitecture was also the connectional reparative principle in pathologically altered claws. Related research perspectives for prophylaxis and therapy of claw diseases are discussed.

Estrogen induced changes in Akt-dependent activation of endothelial nitric oxide synthase and vasodilation

Objectives: Acute administration of estrogen results in vasodilation and increased nitric oxide (NO) production. We examined the hypothesis that this is due to activation of Akt/PKB which subsequently increases eNOS activity. Methods and results: Treatment of bovine microvascular and human umbilical endothelial cells (HUVEC) with 17-β-estradiol (E2) (10 −9 to 10 −5 M) increased phosphorylation of Akt within 1 min and this was followed by phosphorylation of eNOS. These effects were blocked by wortmannin, a PI(3)K inhibitor and the upstream activator of Akt. The estrogen receptor antagonist, ICI 182,780, inhibited eNOS phosphorylation. E2 increased calcium dependent NOS activity and nitrite production and this was inhibited by wortmannin and ICI 182,780. E2 increased the vasodilatory response of aortic rings to acetylcholine and wortmannin blocked the effect. E2 (10 −9 M) dilated cerebral microvascular vessels under conditions of no flow, constant flow and increasing flow and this was blocked by wortmannin. Tamoxifen, a partial estrogen receptor antagonist, also dilated the microvessels. Conclusions: E2 increases NO production through an Akt/PKB dependent pathway. This is associated with increased sensitivity to endothelial dependent dilation. In cerebral microvessels, E2 and tamoxifen produce significant dilation at low concentrations with and without acetylcholine induced stimulation of endothelial vasodilation.

Chlorpyrifos Alters Functional Integrity and Structure of an In Vitro BBB Model: Co-cultures of Bovine Endothelial Cells and Neonatal Rat Astrocytes

The blood–brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9–13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1–10 μM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43–100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 μM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 μM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB integrity and structure.

Novel function of ascorbic acid as an angiostatic factor.

Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4-6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 micro mol/CAM with half-maximal inhibition at 2.5 micro mol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H(2)O(2)) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.

Human thrombospondin 2 inhibits proliferation of microvascular endothelial cells

This study was performed to characterize human thrombospondin 2 (TSP2). TSP2 has recently attracted attention as an endogenous negative regulator of angiogenesis in tumorigenesis. We cloned and transfected human TSP2 cDNA into the human colon cancer cell line SW-480. Stable transfectants (TSP2-1, TSP2-6) overexpressing TSP2 were established. Growth characteristics of TSP2-transfectants were investigated in vitro and in vivo. TSP2-transfectants showed similar growth properties to vector-transfectants and wild-type SW-480 cells. The overexpression of transfected human TSP2 cDNA did not affect proliferation of SW-480 cells. When the conditioned media of TSP2-transfectants were added to cultures of bovine pulmonary microvascular endothelial cells (BPMEC), the BPMEC proliferation was significantly inhibited. These results suggested that human TSP2 is a potential inhibitor of angiogenesis.

Synergistic Induction of t-PA by Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor and Localization of t-PA to Weibel-Palade Bodies in Bovine Microvascular Endothelial Cells

Endothelial cell migration is stimulated by members of the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) families, and is dependent on extracellular proteolytic activity provided by enzymes of the plasminogen activator (PA) system. Here we report that in bovine microvascular endothelial cells (BME cells), bFGF principally increased urokinase-type PA (u-PA) while tissue-type PA (t-PA) was increased mainly by VEGF. In bovine aortic endothelial cells (BAE cells), bFGF increased u-PA, whereas VEGF had no effect. Co-added bFGF and VEGF increased t-PA mRNA levels and enzyme activity in both cell types in a synergistic manner. Tissue-type plasminogen activator (t-PA) immunoreactivity colocalized with von Willebrand factor, a marker for Weibel-Palade bodies. Co-added bFGF and VEGF increased the number of t-PA-positive cells as well as the number of t-PA-positive granules per cell. Localization of t-PA in regulated storage granules endows endothelial cells with the potential to rapidly increase proteolytic activity in the pericellular environment.

Hypoxia-Inducible Angiopoietin-2 Expression Is Mimicked by Iodonium Compounds and Occurs in the Rat Brain and Skin in Response to Systemic Hypoxia and Tissue Ischemia

Angiopoietins are ligands for the endothelial cell tyrosine kinase receptor Tie-2. Ang-1, the major physiological activator of Tie-2, promotes blood vessel maturation and stability. Ang-2 counteracts this effect by competitively inhibiting the binding of Ang-1 to Tie-2. Using a combined RNase protection/semiquantitative reverse transcriptase-polymerase chain reaction approach, we demonstrate that hypoxia up-regulates Ang-2 mRNA levels by up to 3.3-fold in two human endothelial cell lines. In bovine microvascular endothelial (BME) cells, the flavoprotein oxidoreductase inhibitor diphenylene iodonium (DPI) and the related compound iodonium diphenyl mimic induction of Ang-2 but not vascular endothelial growth factor (VEGF) by hypoxia; in combination with hypoxia, DPI further increases Ang-2 expression but has no effect on the induction of VEGF by hypoxia. Neither Ang-2 or VEGF was increased by cyanide or rotenone, suggesting that failure in mitochondrial electron transport is not involved in the oxygen-sensing system that controls their expression. In ischemic rat dorsal skin flaps or in the brain of rats maintained for 12 hours under conditions of hypoxia, Ang-2 mRNA was up-regulated 7.5- or 17.6- fold, respectively. VEGF was concomitantly increased, whereas expression of Ang-1, Tie-2, and the related receptor Tie-1 was unaltered. In situ hybridization localized Ang-2 mRNA to endothelial cells in hypoxic skin. These findings 1) show that up-regulation of Ang-2 by hypoxia occurs widely in endothelial cells in vitro and in vivo; 2) suggest that induction of Ang-2, but not VEGF, by hypoxia in BME cells is controlled by a flavoprotein oxidoreductase that is sensitive to iodonium compounds; and 3) point to Ang-2 and VEGF as independently regulated and selective effectors of hypoxia-induced vascular sprouting.

Effect of adenosine receptor agonists and antagonists on transport of adenosine in bovine heart microvascular endothelial cells.

Adenosine produces a variety of effects through binding to adenosine receptors. It has been found that the adenosine receptors: A1, A2A, A2B and A3, are involved in the cardioprotection and cerebroprotection, in regulation of sleep and pain, in immunological and inflammatory responses, in respiratory regulation and in kidney function, the processes known to be regulated by adenosine . Since the attempts to activate Ado receptors for clinical benefits through pharmacologically elevated adenosine concentrations were not successful , a number of synthetic adenosine receptor agonists and antagonists were developed to manipulate Ado receptor function . Although adenosine receptor agonists/antagonists binding was studied in detail, relatively little is known about other biochemical effects of these compounds. In our experiments a number of agonists and antagonists of adenosine receptors (A1, A2, A3) was studied for effects on adenosine transport and adenosine release in bovine heart microvascular endothelial cells (BHMEC). In the BHMEC cells, in which the coronary responses to adenosine agonists in many cases seem to be endothelium mediated 7,8 , adenosine receptor agonists, particularly of A3 and A1 type, inhibited adenosine influx (IC50 0.766 μM) (Table 1). The most potent inhibition was observed with I-ABA, ABA, CHA, and N6-phenyladenosine. Contrary, the A2 receptor

The effects of vascular endothelial growth factor on endothelial cells: A potential role in preeclampsia

Objectives: Preeclampsia is primarily a disorder of the maternal endothelium. An as yet unidentified circulating factor causes widespread alteration in endothelial function, and levels of vascular endothelial growth factor are elevated in preeclampsia. We hypothesized that vascular endothelial growth factor is involved in the alteration of endothelial function and set out to find further evidence for this contention. Study Design: Bovine microvascular endothelial cells (B-88) were cultured in vitro. These cultured cells were then stimulated with vascular endothelial growth factor and with plasma from women with preeclampsia in the presence and absence of anti–vascular endothelial growth factor antibody. Prostacyclin, nitric oxide, and lactate dehydrogenase levels were measured. Results: Vascular endothelial growth factor induced a significant concentration-dependent increase in prostacyclin production but not nitric oxide production. Cells stimulated with plasma from women with preeclampsia showed increases in production of both prostacyclin and nitric oxide. Vascular endothelial growth factor concentration in plasma was correlated with prostacyclin production by stimulated cells. The increase in prostacyclin production that usually followed the addition of plasma did not occur when anti–vascular endothelial growth factor antibody was present. Conclusions: Vascular endothelial growth factor has the ability to alter endothelial cell function in a manner analogous to that of plasma from women with preeclampsia. (Am J Obstet Gynecol 2000;182:176-83.)

Effect of shear stress on the hydraulic conductivity of cultured bovine retinal microvascular endothelial cell monolayers

The shear stress of flowing blood on endothelial cells increases water transport (hydraulic conductivity, Lp) in several vascular beds in vivo and has been hypothesized to play a role in elevating vascular transport in ocular diseases such as diabetic retinopathy. The purpose of this study is to determine the response of Lp to varying levels of shear stress using an in vitro model of the blood-retinal barrier: bovine retinal endothelial cells (BRECs) grown on polycarbonate filters. The study also addresses the role of nitric oxide (NO) and other downstream effectors in mediating shear-induced changes in water transport. A step change in shear stress of 10 dyn/cm 2 did not produce a significant change in Lp over 3 hours, whereas a 20 dyn/cm 2 step change elevated Lp by 14.6-fold relative to stationary controls at the end of 3h of shear exposure. 20 dyn/cm 2 of shear stress stimulated the endothelial monolayers to release nitric oxide in a biphasic manner and incubation of the BRECs with a nitric oxide synthase (NOS) inhibitor, L-NMMA, significantly attenuated the shear-induced Lp response. These experiments demonstrate that NO is a key signaling molecule in the pathway linking shear stress and Lp in BRECs. A widely studied pathway downstream of NO involves the activation of guanylate cyclase (GC), guanosine 3', 5'-cyclic monophosphate (cGMP) and protein kinase G (PKG). It was observed that incubation of BRECs with the GC inhibitor, LY83583 (10 µM) or the PKG inhibitor, KT5823 (1 µM) did not significantly alter the shear-induced Lp response. Also the cGMP analogue, 8-br-cGMP (1mM), did not affect the baseline Lp over 4h. These results demonstrate that shear stress elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.