Vascular endothelial growth factor enhances the expression of urokinase receptor in human endothelial cells via protein kinase C activation.

Among other proteolytic enzymes, the urokinase-type plasminogen activator (u-PA)/plasmin cascade contributes to cell migration and the formation of capillary-like structures in a fibrinous exudate. The u-PA receptor (u-PAR) focuses proteolytical activity on the cell surface of the endothelial cell and hereby accelerates the pericellular matrix degradation. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 enhance u-PA receptor expression in human endothelial cells. In this paper we show that the protein kinase C (PKC) inhibitors Ro31-8220 and GF109203X inhibit VEGF165-induced u-PAR antigen expression in human endothelial cells, whereas PKC inhibition had no effect on FGF-2-induced u-PAR antigen enhancement. In addition, inhibition of PKC activity had no effect on VEGF165- or FGF-2-induced proliferation in human endothelial cells. We conclude that VEGF165 induces u-PAR via a PKC-dependent pathway, whereas proliferation is induced via a different pathway probably involving tyrosine phosphorylation of proteins downstream of the VEGF receptors.

Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction.

In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca(2+)](i) was not elevated by LPA. Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca(2+)](i) and requires activation of RhoA and Rho kinase.

Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.

Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca(2+)](i) and activation of the Ca(2+)/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca(2+)](i) by BAPTA. These data indicate that RhoA/Rho kinase and Ca(2+) represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin-induced endothelial permeability without affecting activation of RhoA by thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways.

Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome.

The hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children. The role of a verocytotoxin (VT)-producing Escherichia coli has been strongly implicated in the epidemic form of HUS. Although direct toxicity of VT on glomerular endothelial cells has been demonstrated, it remained still unclear how the VT is transported from the intestine to the target organs. In this study we demonstrate that VT, when incubated in whole blood, binds rapidly and completely to human polymorphonuclear leukocytes (PMNs) and not to other components of blood. Binding studies with (125)I-VT-1 showed a single class of binding sites on freshly isolated, nonstimulated human PMNs. The K(d) of VT-binding to PMNs was 10(-8) mol/L, 100-fold less than that of the VT-receptor globotriaosylceramide. On incubation of VT-preloaded PMNs with human glomerular microvascular endothelial cells (GMVECs), transfer of VT-1 to the endothelial cells occurred. Incubation of nonstimulated GMVECs with VT-preloaded PMNs, but not with PMNs or VT-1 alone, caused inhibition of protein synthesis and cell death. Our data are in concert with a role of PMNs in the transfer of VT from the intestine to the kidney endothelium. This transfer occurs by selective binding to a specific receptor on PMNs and subsequent passing of the ligand VT to the VT-receptor on GMVECs, which causes cell damage. This new mechanism further underpins the important role of PMNs in HUS.

Simvastatin improves disturbed endothelial barrier function.

BACKGROUND: Recent clinical trials have established that inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) reduce the risk of acute coronary events. These effects of statins cannot be fully explained by their lipid-lowering potential. Improved endothelial function may contribute to the positive effects of statin treatment. METHODS AND RESULTS: In the present study, we report that simvastatin reduces endothelial barrier dysfunction, which is associated with the development of atherosclerosis. Treatment of human umbilical vein endothelial cells for 24 hours with 5 micromol/L simvastatin reduced the thrombin-induced endothelial barrier dysfunction in vitro by 55+/-3%, as assessed by the passage of peroxidase through human umbilical vein endothelial cell monolayers. Similar effects were found on the thrombin-induced passage of (125)I-LDL through human aortic endothelial cell monolayers. This reduction in barrier dysfunction by simvastatin was both dose and time dependent and was accompanied by a reduction in the thrombin-induced formation of stress fibers and focal adhesions and membrane association of RhoA. Simvastatin treatment had no effect on intracellular cAMP levels. In Watanabe heritable hyperlipidemic rabbits, treatment for 1 month with 15 mg/kg simvastatin reduced vascular leakage in both the thoracic and abdominal part of the aorta, as evidenced by the Evans blue dye exclusion test. The decreased permeability was not accompanied by a reduction of oil red O-stainable atherosclerotic lesions. CONCLUSIONS: These data show that simvastatin, in a relatively high concentration, improves disturbed endothelial barrier function both in vitro and in vivo. The data also support the beneficial effects of simvastatin in acute coronary events by mechanisms other than its lipid-lowering effect.

Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: role of protein kinases, calcium, and RhoA.

In the present study, we differentiated between short- and long-term effects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (<3 minutes) decrease in barrier function, as evidenced by a decreased transendothelial electrical resistance and an increased passage of 22Na ions. This increase in permeability was inhibited completely by chelation of intracellular calcium ions by BAPTA-AM and inhibition of calmodulin activity and myosin light chain (MLC) phosphorylation. The presence of serum factors prolonged the barrier dysfunction induced by histamine. Thrombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increase in permeability induced by thrombin. Genistein and BAPTA-AM inhibited the thrombin-induced permeability in an additive way, causing together an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F-actin fiber and focal attachment formation. Inhibition of RhoA by C3 transferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not elevate the cellular cAMP levels. No evidence was found for a significant role of protein kinase C in the thrombin-induced increase in permeability or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a physiological way, thrombin induces a prolonged increase in permeability by "calcium sensitization," which involves protein tyrosine phosphorylation and RhoA activation.

Effect of methylglyoxal on the physico-chemical and biological properties of low-density lipoprotein.

In patients with diabetes, non-enzymatic glycation of low-density lipoprotein (LDL) has been suggested to be involved in the development of atherosclerosis. alpha-Dicarbonyl compounds were identified as intermediates in the non-enzymatic glycation and increased levels were reported in patients with diabetes. We studied the effect of the alpha-dicarbonyl compound methylglyoxal (MG) on the physicochemical and biological properties of LDL. MG dose-dependently modifies LDL, as indicated by the formation of fluorescent products and the increase of a net negative charge. MG (10 mmol/l) induced major modifications of arginine residues (up to 85%) and minor lysine modifications (less than 6%). MG-LDL preparations generated small amounts of superoxide anion radicals as measured by the reduction of cytochrome c, but this was not accompanied by peroxidation of the polyunsaturated fatty acids of MG-LDL. MG-LDL showed diminished recognition and uptake by the human LDL receptor in cultured cells and a markedly increased plasma clearance rate in vivo in rats. The reduced association and degradation of 125I-oxidised LDL by murine macrophages indicates recognition of MG-LDL by a scavenger receptor. Surprisingly, MG-LDL caused significantly less cholesteryl ester synthesis in murine macrophages, as compared to native LDL and oxidised or acetylated LDL. Highly modified MG-LDL did not induce activation of human endothelial cells, as measured by the expression of monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1.

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity.

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.

Cholesterol content of the rat lens is lowered by administration of simvastatin, but not by pravastatin.

The influence of the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors pravastatin and simvastatin on lens cholesterol metabolism was investigated in the rat. Short-term organ culture experiments with explanted lenses from 21-day-old Wistar rats showed that simvastatin was at least 35 times more effective than pravastatin in inhibiting cholesterol synthesis. In vivo the cholesterol content of the rat lens increased linearly with age. Experiments were designed to answer the question whether simvastatin and pravastatin inhibit lens cholesterol synthesis in vivo, which would result in a reduced cholesterol accumulation in the lens with age. Young Wistar rats were weaned at an age of 21 days and had ad libitum access to a chow supplemented with 10-100 mg vastatin kg-1 (drug consumption: 1.5-15 mg vastatin kg-1 body weight day-1, respectively) or no additions for 3 weeks. Both drugs induced the HMG-CoA reductase activity in rat liver microsomes (isolated after 1, 2 and 3 weeks of treatment) to a similar extent. This indicates that the two drugs inhibited hepatic cholesterol synthesis to a comparable extent. During the whole treatment period no significant differences between control and drug-treated animals could be observed when the wet weight and protein content of the lenses were considered. However, a striking difference between the control group and pravastatin group (50 mg drug kg-1 diet) on the one hand and the simvastatin group (50 mg drug kg-1 diet) on the other was observed when the cholesterol content of the lenses were compared as a function of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Pravastatin and simvastatin differently inhibit cholesterol biosynthesis in human lens.

PURPOSE: In the current study, the hypocholesterolemic drugs pravastatin and simvastatin were compared for their influence on cholesterol biosynthesis in the human lens. METHODS: For measurements of cholesterol and fatty acid synthesis rates, human lenses were incubated for 20 hr in the presence of [14C]-acetate, and pravastatin or simvastatin. Radiolabeled [14C]-cholesterol and [14C]-fatty acids were determined. To avoid the influence of individual differences, one lens from each donor was incubated without drug (control) and the other lens was incubated in the presence of drug. For each lens pair, the percentage inhibition of the cholesterol synthesis caused by the drug was calculated. Fatty acid synthesis was not influenced by the drugs. By comparing the fatty acid synthesis rate of the drug-incubated with the control lens of a pair, a predefined exclusion criterion was used to eliminate lens pairs in which the lenses had no comparable biosynthetic capacities. RESULTS: Using various concentrations of the drugs, a dose-response curve was constructed for the inhibition of the cholesterol synthesis. The IC50 values (drug concentration give 50% inhibition) were 0.5 mumol/l and 0.004 mumol/l for pravastatin and simvastatin, respectively. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in microsomal membranes from human lens cortex was inhibited by simvastatin and pravastatin to the same extent. CONCLUSIONS: Under the conditions used in this study, cholesterol synthesis in human lenses is inhibited by simvastatin 100-fold more effectively than by pravastatin. This difference was likely due to differences in the intracellular exposure of the reductase to the drugs in intact human lenses.

Biosynthetic capacity of the human lens upon aging.

Lipid and protein biosynthesis were investigated in organ culture of human lenses from subjects ranging from 8 to 95 years old. A marked increase of protein and cholesterol content of the human lens with age was found. No significant linear correlation of age with either cholesterol or fatty acid synthesis was observed. Moreover, there are no indications that either synthesis declines with increasing age of the subject. Notwithstanding the large variety in age of the subjects, cholesterol and fatty acid synthesis of the individual lenses were correlated with each other in a positive linear way. Non-cataractous lenses as old as 80 years revealed a normal protein synthesis pattern as determined by incorporation of [35S]methionine into protein and analysis by 2D-gel electrophoresis. In addition, they maintained a high protein biosynthetic capacity.

Eicosanoid profile of healing colon anastomosis and peritoneal macrophages in the rat.

Because intraperitoneal administration of prostaglandin E2 (PGE2) has a negative influence on the healing of colonic anastomosis, the production of eicosanoid products in the healing rat colon after resection and anastomosis was studied using high performance liquid chromatography. Normal colonic tissue metabolizes small amounts of arachidonic acid into cyclo-oxygenase and lipoxygenase products. After construction of an anastomosis, however, there is increased production of lipoxygenase products, while cyclooxygenase activity remains low. Increased amounts of PGE2 and other cyclo-oxygenase products are not produced after anastomosis of the colon and probably do not play a major role in uncomplicated healing of the large intestine in the rat. During the first eight days of repair in the anastomosed colonic tissue, a statistically significant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) production was found compared with control colon tissue (p = 0.001). At the same time peritoneal macrophages from these rats showed increased 12-HETE production. Eicosanoid synthesis of peritoneal macrophages resembled eicosanoid synthesis of anastomosed colon taken from the same rat indicating that 12-HETE, in particular, may be of macrophage origin.

The formation of thromboxane B2, leukotriene B4 and 12-hydroxyeicosatetraenoic acid by alveolar macrophages after activation during tumor growth in the rat.

Pieces of tumor tissue were implanted subcutaneously in the right flank of BN female rats. After 3, 7, 10, 12, 14 and 17 days the lungs were lavaged and the alveolar macrophages collected. The cells were activated with the calcium ionophore A23187 and the formation of thromboxane B2 (TxB2), leukotriene B4 (LTB4) and 12-hydroxyeicosatetraenoic acid (12-HETE) determined. The formation of TxB2 decreased considerably until day 7. Thereafter, no changes occurred. The formation of LTB4 increased after the tumor implantation until day 10 and remained stable for the rest of the period, 12-HETE formation was approximately similar, with a decrease at day 12 but continued to increase after day 14. These results suggest that during tumor growth an inhibition of the cyclo-oxygenase or thromboxane synthase occurs and an activation of the C5- and C12-lipoxygenases of the alveolar macrophages.

Differential effects of malotilate on 5-, 12- and 15-lipoxygenase in human ascites cells.

Macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B4 and 5-monohydroxyeicosatetraenoic acid. The cyclooxygenase products 6-keto-PGF1 alpha and thromboxane B2 were the most important prostaglandin-like substances. Malotilate, an anti-fibrotic substance, selectively inhibited the 5-lipoxygenase, whereas both the 12- and the 15-lipoxygenase pathways were stimulated. The effects of malotilate on eicosanoid production differ from those of known lipoxygenase inhibitors. Such differential effects have not been reported previously.

Endotoxin protection against pulmonary oxygen toxicity and its reversal by acetyl salicylic acid: role of eicosanoid production by broncho-alveolar lavage cells.

Small doses of endotoxin markedly increase the survival rate of adult rats exposed to 98% oxygen for periods that are normally lethal. The lysine salt of acetyl salicylic acid (L-ASA) partially reverses this protective effect of endotoxin. In this pilot study we investigated the level of eicosanoid production by broncho-alveolar lavage (BAL) cells and found that BAL cells of endotoxin protected rats, present in abundance, have an equal or increased capacity of HHT, 15-HETE, 12-HETE, LTB4 and 5-HETE production. These data suggest that production of the lipoxygenase products by BAL cells does not seem to play an important role in the pathogenesis of pulmonary oxygen toxicity. We did not find any indication for the occurrence of shunting of arachidonic acid metabolism to the lipoxygenase pathway as an explanation for the reversal of endotoxin's protective action by L-ASA.

Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid.

Incubation of bovine polymorphonuclear leukocytes (PMNs) with arachidonic acid leads to the formation of four lipoxins. The same lipoxins are also formed upon incubation of bovine PMNs with 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5(S)-hydroperoxy, 15(S)-hydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid or 5(S),15(S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid. A 5,6-epoxide as intermediate in lipoxin formation in the bovine PMN is highly improbable because the 5-hydroxy compounds are as good substrates as the 5-hydroperoxy compounds. Moreover, the two main lipoxins were found to coelute with the two lipoxins produced via a triple dioxygenation of arachidonic acid by soybean lipoxygenase-1. Hence the bovine PMN is the first cell for which evidence is presented that the formation of lipoxins proceeds mainly via triple dioxygenation and not via 15-hydroxy-leukotriene A4 as is proposed for human and porcine PMNs.

Demonstration of a 12-lipoxygenase activity in bovine polymorphonuclear leukocytes.

In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.