Aortic Endothelial Cell Monolayers Research Articles

Effects of magnesium on nitric oxide synthase activity in endothelial cells.

Magnesium modulates endothelium-dependent vasodilation in intact blood vessels. Therefore, the effects of magnesium on nitric oxide (NO) release by isolated endothelial cells and nitric oxide synthase (NOS) activity in endothelial cell homogenates were studied. Unstimulated and bradykinin-stimulated NO release by porcine aortic endothelial cell (PAEC) monolayers were unaffected by 30 min of exposure to magnesium concentrations varying from 0.010 to 10.0 mM. In contrast, when A-23187-stimulated cells were exposed to 0.01, 3.16, and 10.0 mM MgCl2, NO release was decreased by 11.3 +/- 1.8, 11.7 +/- 3.0, and 20.3 +/- 7.2%, respectively, compared with cells exposed to 1.0 mM MgCl2 (P < 0.01). These data suggested that a change in the intracellular magnesium concentration had an effect on NO release, in contrast to a change in the extracellular concentration, which did not have an effect. To further assess this possibility, crude NOS extracts were prepared from PAEC and exposed to MgCl2. NOS activity was measured via the conversion of L-[3H]arginine to L-[3H]citrulline. Increasing the concentration of MgCl2 by 1.0, 3.16, and 10.0 mM caused a 16.0 +/- 6.8, 17.1 +/- 1.7, and 38.6 +/- 5.3% decrease in citrulline formation, respectively (P < 0.05), suggesting a direct inhibition of NOS by MgCl2. No significant difference in the degree of inhibition of NOS activity was found between MgSO4 and MgCl2, thus ruling out a nonspecific chloride effect. In addition, increasing the concentration of NaCl to 15 mM had no effect on NOS activity, ruling out a nonspecific osmotic effect [101.6 +/- 10.5% of control activity, P = not significant (NS)].(ABSTRACT TRUNCATED AT 250 WORDS)

Suramin inhibits C6 glioma‐induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-micron thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-microns pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently form networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiogenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.

Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer

The macromolecular permeability of cultured bovine aortic, bovine venous, and human umbilical vein endothelial cell monolayers was decreased significantly in culture medium containing L-ascorbic acid (Asc Acid; 0.01-0.1 mM) and L-ascorbic acid 2-phosphate (Asc 2-P). Dithiothreitol, which shows reducing activity equivalent to that of Asc Acid, did not affect endothelial permeability. Asc Acid induced a sixfold increase in collagen synthesis by the endothelial cells. The coexistence of L-azetidine 2-carboxylic acid, an inhibitor of collagen synthesis, attenuated the effect of Asc 2-P in a dose-dependent manner. Another collagen synthesis inhibitor, ethyl-3,4-dihydroxybenzoate, also inhibited collagen synthesis and increased endothelial permeability. The decrease in permeability of the endothelial monolayer was dependent on a reduction of the permeability coefficient of the endothelial monolayer. These findings indicate that endothelial barrier function is stimulated by Asc Acid via an increase in collagen synthesis.

EVIDENCE OF NONINVOLVEMENT OF SWINE MHC CLASS II IN THE IN VITRO PROLIFERATIVE RESPONSE OF HUMAN LYMPHOCYTES TO PORCINE ENDOTHELIAL CELLS

Successful pig-to-human xenotransplantation may expose swine endothelium to the human immune system. Since endothelial MHC class II expression is crucial in the genesis of an allogeneic lymphocyte response, the involvement of porcine MHC (SLA) class II molecules in the induction of human lymphocyte proliferation was studied. When cocultured with a confluent monolayer of irradiated porcine aortic endothelial cells (PAEC), human peripheral blood mononuclear cells (PBMC) incorporated tritiated thymidine. Monocyte depletion strongly reduced the magnitude of the lymphocyte proliferative response. Resting cultured PAEC were SLA class II-negative and an induction of these molecules during the xenogeneic mixed lymphocyte endothelial cell culture (XMLEC) was not observed. Moreover, the addition of an antibody directed against the SLA-DR molecule was without effect. Lymphocyte proliferation was also studied in response to SLA class II-positive stimulating cells--either human TNF-alpha-stimulated PAEC or porcine splenocytes. Induction of SLA class II molecules on PAEC had no effect on the human PBMC proliferative response. Moreover, human PBMC did not proliferate in response to porcine splenocytes. These results suggest (1) that SLA class II molecules are not involved in the induction of the human lymphocyte proliferative response and (2) that the endothelial nature of the stimulating cells plays a key role in this proliferation.

CGMP and Nitric Oxide Modulate Thrombin-Induced Endothelial Permeability

Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined whether nitric oxide (NO) can serve as an endogenous modulator of endothelial barrier function. Thrombin increased the passage of macromolecules through human umbilical vein and human aortic endothelial cell monolayers and concomitantly increased [Ca]2+ in vitro. Inhibition of these increases by the intracellular Ca2+ chelator BAPTA indicated that cytoplasmic Ca2+ elevation contributes to the thrombin-induced increase in endothelial permeability. The cGMP-dependent protein kinase activators 8-bromo-cGMP (8-Br-cGMP) and 8-(4-chlorophenylthio)cGMP (8-PCPT-cGMP) decreased the thrombin-induced passage of macromolecules. Two pathways accounted for this observation. Activation of cGMP-dependent protein kinase by 8-PCPT-cGMP decreased the accumulation of cytoplasmic Ca2+ in aortic endothelial cells and hence reduced the thrombin-induced increase in permeability. On the other hand, in umbilical vein endothelial cells, cGMP-inhibited phosphodiesterase (PDE III) activity was mainly responsible for the cGMP-dependent reduction of endothelial permeability. The PDE III inhibitors Indolidan (LY195115) and SKF94120 decreased the thrombin-induced increase in permeability by 50% in these cells. Thrombin treatment increased cGMP formation in the majority of, but not all, cell cultures. Inhibition of NO production by NG-nitro-L-arginine methyl ester (L-NAME) enhanced the thrombin-induced increase in permeability, which was restricted to those cell cultures that displayed an increased cGMP formation after addition of thrombin. Simultaneous elevation of the endothelial cGMP concentration by atrial natriuretic factor, sodium nitroprusside, or 8-Br-cGMP prevented the additional increase in permeability induced by L-NAME. These data indicate that cGMP reduces thrombin-induced endothelial permeability by inhibition of the thrombin-induced Ca2+ accumulation and/or by inhibition of cAMP degradation by PDE III. The relative contribution of these mechanisms differs in aortic and umbilical vein endothelial cells. NO can act in vitro as an endogenous permeability-counteracting agent by raising cGMP in endothelial cells of large vessels.

Increased capillary permeability induced by human follicular fluid: a hypothesis for an ovarian origin of the hyperstimulation syndrome

To examine the effect of follicular fluid (FF) and peritoneal fluid (PF) from patients undergoing assisted reproductive technology procedures on endothelial barrier function. This was determined in vitro by measuring the permeability of filter-grown bovine aortic endothelial cell monolayers to a permeability marker. Endothelial cells obtained from bovine thoracic arotas were treated with collagenase solution and plated on millicell filters, on which they formed confluent monolayers. Flux rate was determined at 60 minutes by measuring the radioactive tracer (3H mannitol) permeating from the apical to the basolateral part of the filter. Fifty-eight samples of FF and PF, both from stimulated and natural cycles were analyzed and grouped according to the number of eggs retrieved. Follicular fluid and PF samples from natural cycles were used as controls. There was an augmentation in the permeability rate of both FF and PF from patients undergoing controlled ovarian hyperstimulation (COH) who responded with an increasing number of eggs compared with controls (51% and 39%, respectively). When analyzing samples from patients who responded with a low number of oocytes, no significant increase was observed. It is known that in OHSS, the increase in capillary permeability is related to the administration of gonadotropins, and is believed to be mediated by a vasoactive substance of ovarian origin. In this study, FF and PF from patients undergoing COH showed a significant increase in the permeability rate through endothelial cells in vitro. Based on these findings, it could be hypothesized that if the same events took place in vivo, the isolation of this factor from ovarian source could be of significant importance to elucidate the pathogenesis of OHSS.

Shear stress increases hydraulic conductivity of cultured endothelial monolayers.

To examine the effect of shear stress on hydraulic conductivity (Lp) of bovine aortic endothelial cell monolayers grown on polycarbonate filters, we developed a rotating disk system, which imposed a defined shear stress while Lp was measured. A 10-cmH2O pressure differential was applied to monolayers, and baseline Lp was established between 1.65 +/- 0.85 and 4.94 +/- 1.05 x 10(-7) cm.s-1.cmH2O-1. One-hour exposure to 10 dyn/cm2 shear stress caused a significant (P < 0.05) increase in Lp by 2.16-fold (+/- 0.42), and Lp remained elevated when shear stress was removed. Three-hour exposure to shear stresses between 0.1 and 20.0 dyn/cm2 revealed a threshold for shear-induced increase in Lp of 0.5 dyn/cm2. At 20 dyn/cm2, Lp initially decreased by 30% (+/- 13.4%, P < 0.05) and then increased to a level 3.76-fold (+/- 0.83, P < 0.05) greater than baseline Lp at 3 h. The shear-induced increase in Lp was reversed with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 1 mM) and could be significantly (P < 0.05) inhibited when monolayers were preincubated with 0.3 mM DBcAMP, a concentration that did not significantly affect baseline Lp. Furthermore, preincubation with a general phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (1 mM), completely blocked the shear-induced increase in Lp. On the basis of these results, we conclude that shear stress alters endothelial Lp through a cellular mechanism involving signal transduction, not by a purely physical mechanism.

Increased permeability of bovine aortic endothelial cell monolayers in response to a thromboxane A2-mimetic.

The present study was designed to investigate the ability of thromboxane to modulate the clearance rate of 125I-albumin through bovine aortic endothelial cell (BAEC) monolayer grown on polycarbonate micropore membrane. Stimulation of BAEC with the TXA2 mimetic U44069 (10(-8), 10(-7) and 10(-6) M) elicited a dose-dependent increase of labeled albumin passage across BAEC monolayers. This effect was markedly reduced by the TXA2 antagonist L655240 (10(-7) and 10(-6) M). Our results suggest that TXA2 may modulate the permeability of endothelial cells directly through activation of specific receptors.

Utilization of endothelial cell monolayers of low tightness for estimation of transcellular transport characteristics of hydrophilic compounds

The determination of transcellular transport characteristics using filter-grown brain capillary endothelial cells in conventional permeation studies requires extremely tight cell layers, exhibiting electrical resistance values of more than 500 ohm · cm 2, for avoiding interferences with the paracellular flux. The present investigations demonstrate that rate and asymmetry of transcellular transport processes can be evaluated sufficiently reliably for orientation and comparison purposes also by use of more easily obtainable leaky cell layers exhibiting electrical resistance values of about 20 ohm · cm 2 only. The studies, relying on measurements of rates for influx and efflux into and out of the cells, respectively, of the cell barrier were performed with aortic endothelial cell monolayers grown on permeable supports. Alanine, leucine, 3-(O-methyl)-D-glucose served as representative hydrophilic substrates being transported actively or by facilitated diffusion. HPLC, combined with a simple precolumn benzoylation appropriate for a simultaneous analysis of both substance groups, was used for the quantitation.

Increased Adhesion of Neutrophils to Retinal Vascular Endothelial Cells Exposed to Hyperosmolarity

Recent studies suggest that leukocytes may contribute to capillary occlusion and endothelial cell injury in diabetic retinopathy. The present study is an attempt to determine whether high glucose concentration/hyperosmolar conditions would increase neutrophil adhesion to retinal endothelial cell monolayers.Confluent monolayers of bovine retinal endothelial cells were incubated for 24 hr in 96-well microtiter plates in 5·5, 20, 50 or 100 mM glucose using 20, 50 or 100 mM mannitol as osmotic controls. After 24 hr the cells were observed by phase microscopy, washed, and then incubated with isolated human neutrophils for 20 min. Non-adherent neutrophils were washed from the monolayers, and the number of adherent neutrophils was determined using an Elisa assay for neutrophil elastase. Adhesion of neutrophils to confluent monolayers of human umbilical vein and bovine aortic endothelial cells at all levels of glucose were assayed in a similar fashion for comparison.Neutrophil adherence to bovine retinal endothelial cells was significantly increased (P < 0·05, n = 8; paired t-test) at all elevated glucose concentrations compared with adherence at control (5·5 mM) glucose concentration. The effect was also concentration dependent with 20, 50 and 100 mM glucose resulting in 35, 57 and 70% increases respectively over controls. However, mannitol at equal concentrations produced similar increases in neutrophil adherence, indicating that the increases in adhesion caused by elevated glucose concentration were due to hyperosmolarity and not some special effect of glucose. Neutrophil adherence was not significantly increased to human umbilical vein endothelial cells, at any of the elevated concentrations of glucose. Neutrophil adherence to bovine aortic endothelial cells was significantly higher for 50 and 100 mM glucose compared to control (13% and 25% increases in adhesion, respectively).These data support the hypothesis that increased adhesion of neutrophils to retinal endothelial cells exposed to a hyperosmotic environment (as is present in diabetes) may play a significant role in the pathogenesis of diabetic retinopathy.

Effect of flow on the process of endothelial cell division.

In this study we investigated the effect of flow and the associated shear stress on the process of endothelial cell division. A shear stress of 7 N/m2 (70 dyne/cm2) was applied to a monolayer of bovine aortic endothelial cells (BAECs) by using a parallel-plate flow-chamber system. Dividing cells under flow conditions were qualitatively compared with those under static-culture conditions. In addition, the duration of some phases of the cell cycle (ie, mitosis and cytokinesis) was measured for both static and flow conditions. Dividing BAECs in static cultures bowed upward; however, when cells were preconditioned by at least 6 hours of exposure to flow, they stayed relatively attached to the substrate during the entire process of cell division. Cell area significantly decreased during division for both static-culture and flow conditions. Finally, although significant changes in the duration of phases were observed in single experiments during which cells were exposed to shear stress, any differences disappeared when the data for a large number of cells were combined. Hence, on average, flow and its associated shear stress did not affect the duration of the different phases of endothelial cell division.

Fluid flow modulates vascular endothelial cytosolic calcium responses to adenine nucleotides.

To determine whether fluid flow influences the action of soluble vasoactive agonists on vascular endothelium. Confluent monolayers of bovine aortic endothelial cells (BAEC) were cultured on glass coverslips, prelabeled with the Ca(2+)-sensitive dye fura-2, and placed in a parallel-plate flow chamber designed to generate defined laminar fluid flow. Cytosolic free Ca2+ concentration ([Ca2+]i) in individual BAEC was monitored during perfusion with medium containing adenine nucleotide under defined flow conditions. Continuous perfusion with ATP (0.3-3.0 microM) or ADP (0.1-1.0 microM) evoked repetitive oscillations in [Ca2+]i in individual BAEC. The frequency of the [Ca2+]i oscillations was dependent on both nucleotide concentration and levels of applied shear stress; at constant bulk concentration of nucleotide, the frequency increased with shear stress. Stopping flow in the continuous presence of agonists immediately extinguished the oscillatory response. Elimination of extracellular Ca2+ did not inhibit the [Ca2+]i oscillations. In the presence of nonhydrolyzable nucleotide analog, ATP gamma S or ADP beta S, application of flow resulted in similar shear-dependent [Ca2+]i oscillations, suggesting that flow modulation of the [Ca2+]i response was not simply due to depletion of ATP or ADP in the vicinity of BAEC monolayers as a result of hydrolysis of nucleotides by ectonucleotidases. These findings suggest that local hemodynamic conditions may modulate the action of vasoactive agents on the vascular endothelium in vivo.

Thromboxane Modulates Endothelial Permeability

The study tests the role of thromboxane in modulating microvascular permeability in vitro. Cultured monolayers of bovine aortic endothelial cells were challenged with the thromboxane (Tx) mimic U46619. This led to disassembly of actin microfilaments, cell rounding, border retraction and interendotheHal gap formation. Pretreatment with the Tx receptor antagonist SQ 29,548 prevented the Tx mimic-induced cytoskeletal changes. The Tx mimic also altered endothelial cell barrier function. Increased permeability was indicated by the increased passage of labelled albumin across monolayers cultured on microcarriers, relative to untreated endothelial cells (p < 0.05). Furthermore, electron microscopy of endothelial cells cultured on the basement membrane of human placental amnion indicated increased permeability based on wide, interendotheHal gap formation and transit of the tracer horseradish peroxidase. Quantification of interendothelial gaps revealed an eleven-fold increase with the Tx mimic relative to untreated endothial cells (p < 0.05) and prevention by pretreatment with the Tx receptor antagonist (p < 0.05). These data indicate that Tx directly modulates the permeability of endothelial cell in vitro.

Schistosoma mansoni Egg Attachment to Cultured Human Umbilical Vein Endothelial Cells: An In Vitro Model of an Early Step of Parasite Egg Excretion

To model the attachment of Schistosoma mansoni eggs to the endothelium of the mesenteric vasculature, the interaction between S. mansoni eggs and cultured human umbilical vein endothelial cells (HUVEC) in vitro was investigated. S. mansoni eggs rapidly attached to monolayers of both HUVEC and bovine aortic endothelial cells but more slowly to monolayers of cultured fibroblasts and smooth muscle cells. While both native and glutaraldehyde-fixed eggs attached equally well to HUVEC, eggs attached only to live, metabolically active HUVEC. Attachment was enhanced by both serum and plasma factors. In addition, platelet release products increased egg attachment by 75%. Preincubation of S. mansoni eggs with soluble egg antigens promoted attachment; in contrast, preincubation of HUVEC with the antigens inhibited attachment. These results suggest that interaction of S. mansoni eggs with HUVEC is an active process that can be modulated by molecules secreted by the egg and by platelets during egg extravasation.

Endotoxin-induced arterial endothelial barrier dysfunction assessed by an in vitro model.

Using an in vitro model in which albumin transfer across monolayers of bovine aortic endothelial cells (BAEC) was measured, we have shown that lipopolysaccharide (LPS) induces a concentration-dependent increase in endothelial permeability. This increase was biphasic, having an early peak at 2 h and rising again by 24 h. Both peaks were abolished by polymixin B (PMB) but were unaffected by N omega-monomethyl-L-arginine, N omega-nitro-L-arginine methyl ester or dexamethasone. Furthermore, LPS did not stimulate nitric oxide production by BAEC following 24 h exposure. Thus, the LPS-induced increase in permeability may account for the vascular leakage of septic shock, but the L-arginine-nitric oxide system does not appear to be involved.

Retraction of cultured endothelial cell monolayer by human breast cancer cells, MCF-7.

A human breast cancer cell line (MCF-7), when sealed on confluent bovine pulmonary aortic endothelial cell (CPAE) monolayers, induced morphological changes (retraction) in CPAE cells. The area of retraction depended on the incubation time and the number of MCF-7 cells, suggesting that MCF-7 cells had the capacity to retract CPAE cells. This capacity was reduced by 60% by pretreatment of MCF-7 cells with 17β-estradiol (E) and progesterone (Pg). The extent of retraction was not affected by the addition of various protease inhibitors. CPAE retraction was induced also by adding conditioned medium (CM) from the culture of MCF-7 cells. Considerably less activity was detected in the CM obtained from MCF-7 cells cultured in the presence of E and Pg. The retraction was reversed in 24 h by culturing the monolayer in fresh medium without CM and was not induced by trypsin treatment of the CM.

Oxygen tension regulates neutrophil adhesion to human endothelial cells via an LFA-1-dependent mechanism.

Extravasation of leukocytes at the sites of ischemia-reperfusion is thought to exacerbate the tissue injury. It has been proposed that leukocyte accumulation is a secondary effect of the ischemic damage, mediated by inflammatory cytokines. We have recently demonstrated that physiologically low levels of oxygen tension alone can have a direct effect on the adhesive characteristics of mesenchymal cells for lymphocytes. We now report that decrease of oxygen tension in the environment induces the adhesion of neutrophils to human endothelial cells in culture. Adhesion of human neutrophils to human umbilical vein, bovine aortic, and mouse microvascular endothelial cell monolayers, which had been incubated at pO2 of 50 torr for 3 hours, increased 2.5-fold, 2-, and 1.5-fold, respectively. The effects of decreased oxygen concentration on adhesion were not mediated by a soluble factor elaborated by the hypoxic cells. Low oxygen tension upregulates a saturable, endothelial cell-associated adhesion mechanism, capable of withstanding centrifugation forces greater than 160g. Hypoxia-induced adhesion was inhibited by LFA-1-specific (CD11a/CD18 integrin) antibodies, but not by antibodies directed against the ICAM-1 ligand for the LFA-1 receptor. These studies demonstrate that decreases in oxygen tension alone increase the adhesive properties of endothelial cells for leukocytes. In addition, they provide evidence for the existence of a new ligand for the LFA-1 molecule on endothelial cells which can be affected by hypoxic environments.

Effect of elevated glucose on cyclic GMP and eicosanoids produced by porcine aortic endothelium.

The short-term effects of elevated glucose on cyclic GMP (cGMP) and eicosanoid production in pig aortic endothelial cell monolayers was determined by incubating cells in 5.5 mM or 44 mM glucose for 6 hours. Bradykinin- or A23187-stimulated cGMP production was significantly reduced in cells incubated in 44 mM glucose compared with 5.5 mM glucose. Stimulation of cGMP levels with exogenously added nitric oxide (NO) was also decreased to a similar extent in cells exposed to 44 mM glucose. These data suggest that NO production stimulated by bradykinin or A23187 was unchanged by elevated glucose. Assayed eicosanoids, including 6-ketoprostaglandin (PG) F1 alpha, PGE2 alpha, and 15(S)-hydroxy-(5Z, 8Z, 11Z, 13E)-eicosatetraenoic acid, stimulated by bradykinin or A23187, were increased in cells exposed to 44 mM glucose. These eicosanoid products formed from exogenously added arachidonic acid did not differ between cells incubated in 5.5 mM or 44 mM glucose. Hyperosmolar concentrations of mannose or sucrose had no effect on cGMP levels but did mimic the effect of elevated glucose on eicosanoid production. These data suggest that hyperglycemia in diabetes may interfere with NO-induced guanylate cyclase activation but not NO production in the endothelium and that increased phospholipase activity, secondary to hyperosmolarity, may account for elevated eicosanoid levels.

Supernatant of endothelial cells exposed to laminar flow inhibits mesangial cell proliferation.

We investigated the effects of culture medium conditioned with endothelial cells exposed to hemodynamic shear forces on modulation of mesangial cell (MC) growth. Confluent monolayers of bovine aortic endothelial cells, grown in medium containing 10% fetal calf serum, were exposed to static or to laminar flow conditions for 24 h using a cone-and-plate device. Endothelial cell-conditioned medium was used to study the growth of bovine MC by [3H]thymidine uptake. The proliferative response of MC to fresh medium (containing 10% fetal calf serum) and to culture medium from endothelial cells under static flow [66.7 +/- 34.1 vs. 73.9 +/- 30.0 counts/min (cpm) x 10(-3)] was comparable. In contrast, medium conditioned with endothelial cells exposed to laminar shear stress of 8 dyn/cm2 almost completely abolished MC proliferation (5.8 +/- 6.9 cpm x 10(-3), P < 0.01). To establish whether this effect is due to endothelial cell production of a substance that inhibits MC proliferation or simply to metabolization of serum growth factors in the culture medium, we performed shear stress experiments using serum free medium and we added 10% fetal calf serum after shear exposure just before the proliferation assay. In this condition a significant antiproliferative effect of endothelial cell supernatant under laminar flow was obtained (27.7 +/- 23.4 vs. 68.8 +/- 45.8 cpm x 10(-3), laminar vs. static, P < 0.05), suggesting that endothelial cells under shear stress effectively produce a factor that inhibits MC proliferation. These results would suggest that local glomerular capillary blood flow could play a role in the regulation of MC mitogenesis.

Arterial endothelial barrier dysfunction: actions of homocysteine and the hypoxanthine‐xanthine oxidase free radical generating system

1. Endothelial barrier function was assessed by use of an in vitro model in which transfer of trypan blue-labelled albumin was measured across monolayers of bovine aortic endothelial cells grown on polycarbonate membranes. 2. Addition of either hypoxanthine (0.2 mM) or xanthine oxidase (20 mu ml-1) alone during a 90 min incubation did not affect albumin transfer across endothelial cell monolayers, but a combination of both increased transfer. 3. The increase in albumin transfer induced by hypoxanthine and xanthine oxidase was abolished by catalase (3 u ml-1), reduced by allopurinol (4 mM), but unaffected by superoxide dismutase (6000 u ml-1), the hydroxyl radical scavengers, mannitol (15 mM), dimethylthiourea (10 mM) and N-(2-mercaptopropionyl)-glycine (1 mM), the iron chelator, deferoxamine (0.5 mM), ferric chloride (50 microM), an inhibitor of nitric oxide synthase, NG-nitro-L-arginine (30 microM), or the antioxidant, dithiothreitol (3 mM). 4. Hydrogen peroxide (0.1-30 mM) itself increased albumin transfer across endothelial cell monolayers, exhibiting a biphasic concentration-response curve. The increase induced by 0.1 mM hydrogen peroxide was abolished in the presence of 0.3 u ml-1 catalase whilst that induced by 10 mM hydrogen peroxide was abolished by 3000 u ml-1 catalase. 5. Homocysteine (0.5-1.5 mM) did not affect albumin transfer across endothelial monolayers when added alone, but when added in combination with copper sulphate (50 microM), which catalyses its oxidation, a significant increase in albumin transfer was observed. 6. The increase in albumin transfer induced by the combination of homocysteine (1.5 mM) and copper sulphate was abolished by catalase (1 u ml-1), but was unaffected by superoxide dismutase (6000 u ml-1), mannitol (15 mM), dimethylthiourea (1 mM) or deferoxamine (0.5 mM).7. The data suggest that the endothelial barrier dysfunction induced by the combination of hypoxanthine and xanthine oxidase is mediated solely by the action of hydrogen peroxide and not by superoxide anion, hydroxyl radical, peroxynitrite anion or hypochlorous acid. The copper-catalysed oxidation of homocysteine also induces endothelial barrier dysfunction through the generation of hydrogen peroxide.These findings may have relevance to the endothelial barrier dysfunction associated with ischaemia reperfusion injury and the atherogenic actions of homocysteine.