Alterations In Endothelial Cell Function Research Articles

Model for assessment of endothelial cell function and viability using the MTT dye test and [3H

To establish a reproducible in vitro model for evaluating endothelial cell function in clinical disease states. A prospective study of human umbilical vein endothelial cells (HUVEC) isolated and cultured. Department of Haematology, University of Yaounde I, Cameroon and Department of Medicine University of Newcastle-upon-Tyne, England. The optimum initial cell seeding concentration, for maximal conversion of the formazan dye was 40 x 10(3) cells/well, after 24 hours incubation. At concentrations above 40 x 103 cells/well some inhibition of dye conversion occurred. The conversion of formazan dye was directly proportional to cell numbers for the first 48 hours only, at all cell concentrations. Thereafter, cell metabolism appeared to be inhibited. Third passage endothelial cells (ECs) were exposed to a range of lipopolysaccharide (LPS) concentrations for one and 24 hours, prior to performing the MTT dye test. Dye conversion was observed after one hour at even the lowest concentration of LPS (0.1 microgram/ml), to 49.9% +/- 5.6% of unperturbed control EC, with 10 x 10(3) initial seeding numbers. After 24 hours perturbation a small but statistically significant further inhibition was observed. [3H] thymidine incorporation studies indicated that the lowest LPS concentration tested (0.1 microgram/ml) had a stimulatory effect on DNA synthesis at the higher cell concentration (20 x 10(3) cells/well). In the range of 1 to 100 micrograms/ml of the LPS tested, there was increased DNA synthesis at all cell numbers. The model may be used to monitor the effects of other agents which are known to, or could be associated with, alterations in endothelial cell function and will serve in mimicking clinical situations including hyper coagulable states.

Microvascular vasodilator response to acetylcholine is increased in women with pre-eclampsia

Objectives To evaluate in vivo microvascular responses to incremental doses of the endothelial-dependent vasodilator, acetylcholine, and the endothelial-independent vasodilator, sodium nitroprusside, in women with pre-eclampsia and gestation-matched normotensive pregnant controls. Design Prospective clinical study. Setting Southern Derbyshire Acute Hospitals Trust, and University of Nottingham Division of Vascular Medicine. Population Thirteen women with pre-eclampsia and 16 normotensive pregnant controls. Methods Cutaneous microvascular vasodilator responses to iontophoretic administration of incremental doses of acetylcholine and sodium nitroprusside (25-100μC) were evaluated under temperature-controlled conditions using laser Doppler fluximetry. Results Resting skin temperature and blood flow were similar among 13 women with pre-eclampsia [mean blood pressure 151/93 mmHg (4/3); mean gestation 35.6 weeks (1.0); and mean proteinuria 1.1 g/24h (0.2)] and 16 normotensive pregnant controls [mean blood pressure 111/63 mmHg (2/2); mean gestation 34.3 weeks (0.9)]. Peak vasodilator responses to acetylcholine were increased in women with pre-eclampsia (median flux ratio 15.1 [IQR 12.3-17.6] vs 11.7 [IQR 8.4-12.6], P<0.05), whereas sodium nitroprusside responses were not different between the two groups: 11.4 [IQR 8.6-13.4] vs 9.5 [IQR 8.0-12.3]. Conclusion In vivo microvascular vasodilator responses to acetylcholine are increased in women with pre-eclampsia, while endothelial-independent vasodilation is unchanged. Although the mechanism of acetylcholine induced vasodilation in small vessels is unclear, this study confirms previous animal data and provides in vivo evidence of altered microvascular endothelial cell function in pre-eclampsia.

Glucose and Insulin Modulate the Capacity of Endothelial Cells (HUVEC) to Express P-selectin and Bind a Monocytic Cell Line (U937)

Diabetes mellitus is associated with increased prevalence of endothelial cell dysfunction and vascular diseases. Mechanisms leading to alterations in endothelial cell function are poorly understood. We report here that hyperglycaemia results in the expression of endothelial adhesion molecules involved in leukocyte adhesion and extravasation. Incubation of human umbilical cord endothelial cells (HUVEC) with 25 mM glucose induced the expression of P-selectin. This effect was reversed by the addition of 1 nM insulin. Moreover, increased ICAM-1 expression was observed upon HUVEC incubation with 25 mM glucose. Increased adhesion of U937 cells (a monocytic cell line) to endothelial cells cultured with 25 mM glucose was observed. High glucose-induced monocytes cell adhesion was inhibited by an anti-P-selectin monoclonal antibody (LYP20). These results show that high glucose concentration activates endothelial cells leading to monocytes adhesion providing further evidence that hyperglycaemia might be implicated in vessel wall lesions contributing to diabetic vascular disease.

Increased neutrophil-endothelial adhesion induced by placental factors is mediated by platelet-activating factor in preeclampsia.

Endothelial cell activation or dysfunction and neutrophil-endothelial cell adhesion have been suggested to be important in the pathophysiology of preeclampsia. However, the mechanisms that underlie the alteration of endothelial cell function in preeclampsia are unknown. Placenta from preeclamptic pregnancies produces mediators and autacoids, which may be released into the maternal circulation and modulate endothelial function. In this study, the effect of placental factor(s) on neutrophil-endothelial adhesion and the possible role of platelet-activating factor (PAF) in mediating the response have been examined. Endothelial cells were isolated from human umbilical veins (HUVECs) from normal pregnancies. Confluent primary passage HUVECs were exposed to conditioned medium derived from normal and preeclamptic placental tissue cultures, with unconditioned medium as a control. Placental-conditioned medium was prepared by incubation of placental whole villous tissue in Dulbecco's Modified Eagle's Medium (DMEM) for 48 hours. Neutrophil-endothelial adhesion assays were performed to evaluate placental factors in mediating neutrophil-endothelial adhesion, and a PAF-3H scintillation proximity assay (SPA) system was used to determine endothelial PAF production. The PAF-receptor antagonist WEB 2086 was used to block placental factor-mediated increased neutrophil-endothelial adhesion induced by conditioned medium derived from preeclamptic placenta. Neutrophils were significantly more adherent to HUVECs treated with conditioned medium from preeclamptic placentas (28.44 +/- 2.47%) than to HUVECs treated with conditioned medium from normal placentas (18.95 +/- 1.57%) or with unconditioned medium (14.60 +/- 1.29%, P < .01). Also, HUVECs exposed to preeclamptic placental-conditioned medium produced more PAF than the cells exposed to normal conditioned medium and unconditioned medium, 416.18 +/- 17.14 pg/1 x 10(7) cells versus 330.90 +/- 35.70 and 296.43 +/- 44.40 pg/1 x 10(7) cells, P < .05, respectively. The PAF receptor antagonist WEB 2086 completely blocked increased neutrophil-endothelial adhesion induced by preeclamptic placental-conditioned medium (13.24 +/- 0.81% versus 31.31 +/- 4.75%, P < .01). In preeclampsia, the placenta releases one or more factors promoting neutrophil-endothelial adhesion. The increased neutrophil-endothelial adhesion thereby induced is a PAF-mediated event. It is suggested that if preeclamptic placentas release toxic factors into the maternal circulation in vivo, these factors may contribute to the altered vascular endothelial cell function in preeclampsia.

Reduced pulmonary metabolism of endothelin-1 in canine tachycardia-induced heart failure.

Plasma endothelin-1 (ET-1) increases in congestive heart failure (CHF). The pulmonary vascular bed could contribute to this increase through a reduced clearance. We evaluated the effect of tachycardia-induced CHF on pulmonary ET-1 kinetics. To discern between changes due to variations in pulmonary hemodynamics from true alterations of endothelial cell functions, we quantified ET-1 kinetics in isolated rat lungs under variable pressure and flow-rate conditions. Indicator-dilution studies were performed in anesthetized dogs (n = 14) before and 3 weeks after rapid ventricular pacing and in isolated lungs from healthy rats (n = 4). In isolated lungs, graded increases in perfusion rate from 5-25 ml/min caused gradual reductions in ET-1 extraction from 60 +/- 1.5% to 17 +/- 4.9% (mean +/- S.D.). The capacity to clear ET-1 from the circulation, as computed from the permeability-surface area product (PS), however did not vary over this range of flows. CHF increased plasma ET-1 (11.2 +/- 11.4 vs. 5.2 +/- 1.6 fmol/ml, p < 0.01), did not affect pulmonary ET-1 extraction (29.4 +/- 12.5% vs. 29.9 +/- 12.9%), but decreased the PS (8.3 +/- 5.4 cm3/s vs. 14.4 +/- 9.9 cm3/s, p = 0.038). Contrary to the invariability of the PS in normal isolated rat lungs, CHF was associated with a positive relationship between the PS and pulmonary plasma flow (r = 0.65, p < 0.01). ET-1 binding studies in lung tissues showed no significant variations in ETA and ETB receptors densities but revealed a threefold decrease in binding affinity (p < 0.01) that may explain the reduced clearance. CHF causes a reduction of pulmonary ET-1 clearance that likely contributes to the increased circulating ET-1 levels and reflects pulmonary metabolic dysfunction associated with this condition.

Oxidative stress and altered endothelial cell function in preeclampsia.

Evidence continues to accumulate that oxidative stress is a mediator of endothelial cell dysfunction and thus contributes to the cardiovascular complications of preeclampsia. The mechanisms for the interaction of oxidative stress and endothelial cell function have not been well defined. This review explores potential vasoactive pathways that may be affected by oxidative stress and have been reported to be altered in women with preeclampsia. In pathologic conditions of oxidative stress, increased production of superoxide peroxide anions and nitric oxide has been recognized to inactivate the nitric oxide as a vasorelaxant as well as produce peroxynitrite, a potent oxidant. Increase prostaglandin H (PGH) synthase activity resulting in vasoconstriction predominates in models of oxidative stress. Peroxynitrite increases PGH synthase activity in vitro, providing a potential, but as yet untested, link between oxidative stress, nitric oxide, and PGH synthase pathway, leading to reduced relaxation and increased constriction in the vasculature of women with preeclampsia. Other vasoconstrictors (such as isoprostanes and endothelin) that may be interrelated with oxidative stress and altered endothelial cell function in preeclampsia are also discussed.

Activation of endothelial cells via antibody-enhanced dengue virus infection of peripheral blood monocytes.

Although endothelial cells have been speculated to be a target in the pathogenesis of dengue hemorrhagic fever (DHF), there has been little evidence linking dengue virus infection to any alteration in endothelial cell function. In this study, we show that human umbilical vein endothelial cells become activated when exposed to culture fluids from dengue virus-infected peripheral blood monocytes. Maximum activation was achieved with culture fluids from monocytes in which virus infection was enhanced by the addition of dengue virus-immune serum, thus correlating with epidemiological evidence that prior immunity to dengue virus is a major risk factor for DHF. Activation was strongest for endothelial cell expression of VCAM-1 and ICAM-1. In contrast, activation of endothelial cell E-selectin expression appeared to be more transient, as indicated by its detection at 3 h, but not at 16 h, of treatment. Treatment of monocyte culture fluids with anti-tumor necrosis factor alpha (TNF-alpha) antibody largely abolished the activation effect (as measured by endothelial cell expression of ICAM-1), whereas treatment with IL-1beta receptor antagonist had a much smaller inhibitory effect on activation. Endothelial cells inoculated directly with dengue virus or with virus-antibody combinations were poorly infectable (compared to Vero cells or peripheral blood monocytes), and virus-inoculated endothelial cells showed no increased expression of VCAM-1, ICAM-1, or E-selectin. Taken together, the results strongly indicate that dengue virus can modulate endothelial cell function by an indirect route, in which a key intermediary is TNF-alpha released from virus-infected monocytes.

Induction of vascular endothelial growth factor by platelet-activating factor and platelet-derived growth factor is downregulated by corticosteroids.

Vascular endothelial growth factor (VEGF) is a potent peptide growth factor specific for vascular endothelial cells, which promotes neovascularization and increases vascular permeability in vivo. Enhanced microvascular permeability and edema are common characteristics of inflammatory and neoplastic disorders. Two proinflammatory mediators, platelet-activating factor (PAF) and platelet-derived growth factor (PDGF), are known to contribute to cellular damage and tissue remodeling in a number of lung diseases. To determine whether PAF or PDGF induce VEGF gene expression in primary cultures of human pulmonary fibroblasts and pulmonary vascular smooth-muscle cells (VSMCs), we performed Northern-blot analysis and enzyme-linked immunosorbent assays (EIA). PAF and all three isoforms of PDGF (PDGF-AA, -AB, and -BB) increased VEGF mRNA in a time- and dose-dependent manner. While PAF was shown to increase VEGF mRNA at picomolar concentrations, all PDGF isoforms were effective in inducing VEGF mRNA at nanomolar concentrations. The transcriptional activation was accompanied by increased levels of VEGF protein as determined by EIA in culture medium. These results indicate that VEGF gene expression in VSMCs and fibroblasts is mediated by PAF and/or PDGF isoforms. In a paracrine mode of action, secreted VEGF may then lead to altered endothelial cell functions and vascular hyperpermeability. In the presence of the corticosteroids cortisone, hydrocortisone, dexamethasone, or prednisolone at nanomolar concentrations, this stimulus-dependent transcription of VEGF was abolished. The inhibitory effect of corticosteroids on VEGF expression could explain the clinically well-known antiedematous potency of corticosteroids on a molecular level.

Dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior.

The pathogenesis of filariasis has generally been attributed to either physical presence of the adult parasites or the host's immune response to the parasites. However, the spectrum of filariasis cannot be entirely explained by these causes, and other mechanisms must be operative. It is now evident that factors released by filarial parasites likely contribute to the pathogenesis of filarial diseases. Adult heartworms (Dirofilaria immitis) reside in the right heart and pulmonary artery, so the pulmonary artery should be exposed to the highest concentration of filarial factors. We tested the hypothesis that endothelium-dependent relaxation is altered in the in vitro pulmonary artery from heartworm-infected dogs. Relaxation responses to endothelium-dependent vasodilators (methacholine, bradykinin, substance P, and A-23187) and the nonendothelium-dependent vasodilator nitroglycerin and contractile responses were measured in rings of pulmonary artery from control and heartworm-infected dogs. Endothelium-dependent relaxation was assessed in the presence and absence of inhibitors of nitric oxide synthase, cyclooxygenase, and guanylate cyclase. Responses to methacholine, substance P, and A-23187, but not to bradykinin, nitroglycerin, norepinephrine, or KCl, were depressed in pulmonary artery from heartworm-infected dogs when compared with control, suggesting that changes in endothelial cell and not vascular smooth muscle behavior are involved in altered relaxation. The mechanism of endothelium-dependent relaxation in control pulmonary artery appears to involve nitric oxide in the case of methacholine and both nitric oxide and a cyclooxygenase product in the case of bradykinin and A-23187. The mechanism of endothelium-dependent relaxation in pulmonary artery from heartworm-infected dogs was not clearly elucidated. These data provide no evidence that heartworm infection globally influences either endothelial cell receptor function or the vascular smooth muscle guanylate cyclase guanosine 3',5'-cyclic monophosphate system, making it likely that changes in intracellular signaling are primarily responsible for the observed alteration of endothelium-mediated relaxation. Alteration of endothelial cell function by filarial parasites may be an important component in the pathology associated with filariasis.

Tumor necrosis factor decreases thrombin receptor expression in endothelial cells.

We examined the effects of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF alpha) on the expression of proteolytically activated thrombin receptor (PATR) in human umbilical vein endothelial cells (HUVEC). PATR mRNA and protein levels were measured in confluent HUVEC monolayers after challenge with TNF alpha. Northern analysis indicated that TNF alpha treatment resulted in 2- to 3-fold decrease in PATR mRNA in a time- and dose-dependent manner. PATR mRNA level returned to the control level within 6 hr. The nuclear run-on assay indicated that the decreased mRNA signal was due to reduction in the transcription rate. Immunoblotting experiments indicated that the decrease in expression of PATR protein followed in time the decrease in mRNA; the lowest level of protein expression was achieved at 22 hr after TNF alpha treatment. PATR protein returned to basal value within 40 hr after TNA alpha challenge. To assess alterations in endothelial cell function after TNF alpha treatment, we measured thrombin-induced increase in cytosolic Ca2+ ([Ca2+]i) and the cell shape change (measured by decrease in electrical impedance of endothelial cell monolayer). In HUVEC treated with TNF alpha (100 U/ml for 22 hr), the rise in [Ca2+]i after thrombin challenge was approximately 2-fold less than in control thrombin-treated cells. The decrease in electrical impedance of HUVEC monolayers in response to thrombin after TNF alpha treatment was also significantly reduced. However, the rise in [Ca2+]i in response to histamine was not altered by TNF alpha pretreatment. In conclusion, TNF alpha exposure of endothelial cells decreased both mRNA and protein expression of PATR, which explain the decreased activation of thrombin generated signals after the TNF alpha exposure.

Tumor necrosis factor decreases thrombin receptor expression in endothelial cells

We examined the effects of the proinflammatory cytokine, tumor necrosis factor-α (TNFα), on the expression of proteolytically activated thrombin receptor (PATR) in human umbilical vein endothelial cells (HUVEC). PATR mRNA and protein levels were measured in confluent HUVEC monolayers after challenge with TNFα. Northern analysis indicated that TNFα treatment resulted in 2- to 3-fold decrease in PATR mRNA in a time- and dose-dependent manner. PATR mRNA level returned to the control level within 6 hr. The nuclear run-on assay indicated that the decreased mRNA signal was due to reduction in the transcription rate. Immunoblotting experiments indicated that the decrease in expression of PATR protein followed in time the decrease in mRNA; the lowest level of protein expression was achieved at 22 hr after TNFα treatment. PATR protein returned to basal value within 40 hr after TNFα challenge. To assess alterations in endothelial cell function after TNFα treatment, we measured thrombin-induced increase in cytosolic Ca2+ ([Ca2+]i) and the cell shape change (measured by decrease in electrical impedance of endothelial cell monolayer). In HUVEC treated with TNFα (100 U/ml for 22 hr), the rise in [Ca2+]i after thrombin challenge was ∼2-fold less than in control thrombin-treated cells. The decrease in electrical impedance of HUVEC monolayers in response to thrombin after TNFα treatment was also significantly reduced. However, the rise in [Ca2+]i in response to histamine was not altered by TNFα pretreatment. In conclusion, TNFα exposure of endothelial cells decreased both mRNA and protein expression of PATR, which explain the decreased activation of thrombin generated signals after the TNFα exposure. © 1996 Wiley-Liss, Inc.

Endothelial cells in culture: a model to study in vitro vascular toxicity

This review discusses the importance of cultured endothelial cells in the evaluation of the potential toxicity of a drug and for understanding the toxic effects of some compounds on the vascular system. Vascular toxicity is observed when subjects are exposed to chemicals present in the air or after ingestion of xenobiotics or drugs. Furthermore, some drugs can lead to side-effects owing to an alteration of endothelial cell function. Endothelial cells of human and animal origin can be cultured and several of their properties can be studied using different experimental systems. Cyclosporin and penicillamine have been shown to reduce angiogenesis in vitro, as has also been reported for monocrotaline pyrrole. Other components, such as pyrrolizidine alkaloid, were found to be cytotoxic, as demonstrated by chromium-51 or lactate dehydrogenase release. More subtle changes can be detected in peroxidation, phospholipase activity and prostacyclin production. Endothelial cells cultured to confluency can be used to measure in vitro permeability to radiolabelled inulin or albumin. Tunicamycin, an inhibitor of glycosylation, increases permeability. Xenobiotics such as lead inhibit the production of plasminogen activator (t-PA) or by disrupting the thromboxane-A 2/prostacyclin balance, which promotes a thrombotic process.

Shear stress and cyclic strain modulation of gene expression in vascular endothelial cells.

Vascular endothelial cells are exposed to a complex humoral and hemodynamic environment. Investigations over the last two decades have demonstrated that hemodynamic forces cause alterations in endothelial cell function. More specifically, blood flow-induced wall shear stress and cyclic strain modulate gene expression of various bioactive molecules synthesized and secreted by endothelial cells. Although there currently is not a known unifying mechanism linking mechanical agonists to changes in gene expression, various second messengers, transcription factors, mechanically sensitive promoter elements, and cytoskeletal architecture have been proposed as mediators in the signal transduction pathway. Knowledge of the mechanisms by which hemodynamic forces modulate endothelial cell gene expression will lead to novel gene therapy applications and allow more effective management of cardiovascular diseases.

Prolonged hypercholesterolemia induces reversible alterations in venous vasomotor function.

Hypercholesterolemia is associated with altered arterial endothelial and smooth muscle cell function. This study examines the influence of hypercholesterolemia on external jugular venous endothelial and smooth muscle cell vasoreactivity. Eighteen New Zealand White rabbits received a 1% cholesterol diet: in nine animals, this diet was continued until harvest at 8 weeks (hypercholesterolemic group), but in the other nine animals, the diet was changed to standard rabbit chow after 4 weeks and continued for a further 4 weeks (cholesterol reduction group). The change in the diet resulted in a 70% decrease in serum cholesterol concentration. Eight animals received standard rabbit chow for 8 weeks. Hypercholesterolemia induced hypersensitivity and increased maximal contractions to norepinephrine and endothelin-1. In addition, the maximal response to bradykinin increased, and a contraction to serotonin was induced in the veins from the hypercholesterolemic animals. Cholesterol reduction induced bradykinin hypersensitivity but had no effect on endothelin-1 sensitivity. Norepinephrine hypersensitivity returned to normal and the serotonin response disappeared. A decrease in the maximal contractile responses to these agonists was also observed. Hypercholesterolemia interfered with dose-dependent, EDRF (endothelium derived relaxing factor)-mediated relaxation induced by acetylcholine but, following the reduction of serum cholesterol, normal acetylcholine-induced, endothelium-dependent relaxation returned. Non-endothelium-dependent relaxation to sodium nitroprusside of precontracted veins was unaffected by the presence of high cholesterol concentrations. There were no morphological changes apparent in the veins of either the hypercholesterolemic or the cholesterol reduction groups. In conclusion, this study suggests that hypercholesterolemia induces reversible functional abnormalities in venous tissue and this ability of the jugular veins to recover may be, in part, linked to the lack of morphological changes.

Effect of Lead Acetate on Nitrite Production by Murine Brain Endothelial Cell Cultures

One of the mechanisms by which lead may cause a perturbation in the nervous system is the alteration of endothelial cell function. This study investigated the effect of lead acetate on constitutive and cytokine-induced production of nitrite, a marker of nitric oxide, in brain microvascular endothelial cells. Nitric oxide synthase may be a target for lead and changes in its function can result in a cascade of physiological effects seen in vivo. Concentrations of 10, 100, and 1000 nM lead acetate, in the presence or absence of 100 ng lipopolysaccharide/ml, 400 U interferon-γ/ml and 100 U tumor necrosis factor-α/ml, were added to confluent cultures of brain microvascular endothelial cells. Concentrations of lead acetate as low as 10 nM decreased constitutive levels of nitrite by 50% without inhibiting the inducible levels. Addition of 1 μM lead acetate had no effect on [ 3H]L-leucine incorporation, lactate dehydrogenase release, or cellular morphology, indicating that the effect was selective. Increasing the concentration of extracellular calcium to 2 mM abolished the inhibitory effect of lead acetate on the constitutive production of nitrite. These studies suggest that low concentrations of lead are capable of inhibiting nitrite produced by the calcium-dependent constitutive form of nitric oxide synthase while the calcium-independent, inducible form of nitric oxide synthase is not affected. These data provide another testable hypothesis for the as yet undetermined mechanisms of lead neurotoxicity.

Free Radical-Induced Alterations in Endothelial Cell Function

Free radical-induced injury to the arterial wall has been implicated in the pathogenesis and progression of atherosclerosis. To model the in vitro effects of free radicals on endothelial cell function, protein and lipid synthesis were measured after exposing cells to a superoxide generating system of xanthine (X = 100 μM) and xanthine oxidase (XO = 0.2 units). Total protein synthesis, measured by [ 35S]methionine uptake, decreased by 87.65 ± 2.04% over 4 hr compared to controls ( P < 0.05). Examination of lipid synthesis by high-performance liquid chromatography in cells prelabeled with either [ 3H]oleic acid or [ 3H]sodium acetate revealed alterations in all lipid classes. Phospholipid and neutral glyceride synthesis significantly decreased in a time- and dose-dependent fashion compared to controls (two-way ANOVA). In contrast, cholesterol synthesis and lipid peroxidation increased in a time- and dose-dependent fashion. When X = 200 μM and XO = 0.3 units, there was a statistically significant increase in cholesterol synthesis and lipid peroxidation within 24 hr (Tukey's HSD). We conclude that there is evidence of endothelial cell injury as measured by decreases in protein, glyceride, and phospholipid synthesis. The concurrent increases in lipid peroxidation and cholesterol synthesis may explain the relationship between free radical injury and the pathogenesis of atherosclerosis.

The Effect of Diabetes on the Proliferation of Aortic Endothelial Cells

Diabetic vascular disease is more severe, diffuse, and accelerated when compared to nondiabetic vascular disease. Endothelial cell injury or alteration in endothelial cell function is hypothesized to be the initial cellular event in the pathophysiology of diabetic vascular disease. We examined the effect of insulin-treated and untreated alloxan diabetes on the proliferation of rabbit aortic endothelial cells in vitro by growing thoracic aortic endothelial cells from alloxan diabetic rabbits in serum obtained from alloxan diabetic rabbits. Diabetes adversely affected the proliferation of aortic endothelial cells; the most significant decrease in cell proliferation was noted in untreated diabetic cells. Crossover studies between endothelial cells and serum from different groups revealed that diabetes slows endothelial proliferation by not only a serum effect but also an intrinsic cellular effect. These observations suggest that diabetes adversely affects the proliferation of aortic endothelial cells by changing cell and serum functions.

Sera from preeclamptic women specifically activate human umbilical vein endothelial cells in vitro: morphological and biochemical evidence.

Endothelial cell dysfunction could explain many of the pathophysiological changes observed in preeclampsia. Markers of endothelial cell activation including increased circulating Von Willebrand factor (VWF) and cellular fibronectin (cFN) antedate clinically evident disease. We have therefore proposed that alteration of endothelial cell function by circulating agent(s) produced by the placenta initiates the clinical syndrome. This hypothesis predicts that there are a factor(s) in the blood of women with preeclampsia that are capable of altering endothelial cell function. We and others have examined in vitro interactions of maternal serum and endothelial cells to test this hypothesis. Our initial report indicating increased release of [51Cr]chromate from human umbilical vein endothelial cells (HUVE) suggested a lethal, lytic effect of serum from preeclamptic women. However, more specific indicators of endothelial cell structure and function do not support such a nonspecific effect. The morphology of HUVE was minimally altered after exposure to sera of preeclamptic women, and the entry of propidium iodide entry into cells, a sensitive indicator of membrane integrity, also was not increased. These findings, in combination with the increased expression of mRNA for platelet-derived growth factor (PDGF), suggest endothelial cell activation rather than cell death in response to sera from preeclamptic women. Comparison of the effects of endotoxin and sera from preeclamptic women also supports the specificity of this response. Whereas endotoxin strikingly increased VWF on the surface of HUVE and tissue factor activity in conditioned media while minimally increasing cFN, preeclamptic sera increased cFN but had no demonstrable effect on VWF or tissue factor activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Depression of endothelium-dependent relaxation in aorta from rats with Brugia pahangi lymphatic filariasis.

A role for altered endothelial cell function is emerging in the pathogenesis of disease. We have previously demonstrated that Dirofilaria immitis, the canine heartworm, depresses endothelium-dependent responses and alters the mechanism of relaxation in the in vivo femoral artery of infected dogs. Exposure of rat aorta to the parasite or parasite-conditioned medium selectively depresses endothelium-dependent relaxation. D. immitis is closely related to the major human filarial pathogens. This study was designed to examine the effect of chronic infection with the filarial nematode Brugia pahangi on endothelium-mediated responses of the rat aorta in vitro. We tested the hypothesis that endothelium-dependent responses are depressed in the aorta from rats infected with B. pahangi. Rings of thoracic and abdominal aorta were suspended in muscle baths for measurement of isometric tension. Dose-response relations to norepinephrine, endothelium-dependent dilators (acetylcholine, histamine, and A23187), and nitroglycerin were done. In some experiments, inhibitors of cyclooxygenase (indomethacin and aspirin), guanylate cyclase (methylene blue), and nitric oxide formation (N-nitro-L-arginine methyl ester; L-NOARG) were used. No differences in vascular reactivity were detected in the thoracic aorta. In contrast, endothelium-dependent responses in abdominal aorta of Brugia-infected rats were significantly depressed when compared with control aorta from noninfected rats. Acetylcholine relaxation was further depressed by indomethacin and aspirin. After L-NOARG, acetylcholine relaxation in control abdominal aorta was completely abolished; however, in abdominal aorta of Brugia-infected rats, acetylcholine still caused relaxation. Methylene blue inhibited acetylcholine relaxation in both control and Brugia-infected abdominal aorta; however, relaxation in Brugia-infected aorta was significantly greater than control. This study demonstrates that endothelium-dependent relaxation can be altered by chronic experimental filarial infection in the absence of direct contact between the blood vessel and the parasite. The mechanism of relaxation in the Brugia-infected abdominal aorta appears to be altered when compared with control, suggesting that parasites are capable of modulating vascular reactivity by inducing changes in endothelial cell behavior. The mechanism may involve parasite-induced local inflammation or alterations in endothelial cell metabolism. Understanding how chronic experimental filarial infection alters vascular reactivity may enhance our understanding of the pathogenesis of human filariasis.

Modulation of membrane cholesterol levels: Effects on endothelial cell function

The endothelial cell lining of blood vessels is now recognized as an active interface between blood and the underlying tissue. Modulation of cholesterol levels in several cell types has resulted in altered cell function. We have removed cholesterol from the endothelial cell membrane and have observed corresponding alterations in endothelial cell function. Following depletion of cholesterol from the endothelial cells, polymorphonuclear leukocyte adhesion to the cells was decreased. Angiotensin-converting enzyme activity of the endothelial cells was increased following removal of cholesterol from the endothelial cell membranes. The results of fluorescence polarization measurements suggest that these changes may be partially explained by altered membrane order.