Accumulation of pepstatin in cultured endothelial cells and its effect on endothelial processing

Aspartic acid proteases have been implicated in the processing of ET-1(1-39) to ET-1(1-21). To further understand the role of this class of enzymes in ET-1 synthesis, cultured vascular endothelial cells were incubated with pepstatin, and the accumulation of the inhibitor and its effect on the processing of ET-1(1-39) was examined. Pepstatin accumulated in the cells in a time-dependent manner, to a concentration (greater than 10(-7) M) sufficient to inhibit aspartic acid proteases. Pepstatin did not alter the ratio of ET-1(1-21) to ET-1(1-39), nor did it affect the rate of secretion of either peptide. When endothelial cells were incubated with phosphoramidon under identical conditions, the secretion of ET-1(1-21) was significantly reduced with a concomitant increase in the secretion of ET-1(1-39). These results suggest that the processing of ET-1(1-39) does not involve a pepstatin-sensitive aspartic acid protease, and that the enzyme responsible for generating ET-1(1-21) is sensitive to phosphoramidon.     

Methylmercury-injury effect on tube formation by cultured human vascular endothelial cells

The effect of methylmercury chloride (MeHg) on growth and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were collected by enzymatic digestion with collagenase. Precultivation of HUVECs with MeHg at concentrations of 1.0–50.0 mol/L exerted negligible effects on the viable cell number, while the viable cell number was slightly reduced at 100 mol/L and fell to zero at concentrations exceeding 500.0 mol/L MeHg. The viable cell number was depressed in a concentration-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 mol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal bovine serum (FBS) increased tube formation in a dose-dependent manner, with half-maximum stimulation of tube formation at approximately 3.4% FBS. The length of tube formation decreased time-dependently at concentrations of 0.1 and 1.0 mol/L MeHg. Pretreatment of Matrigel with 1 mol/L MeHg before the cell seeding reduced the tube formation by HUVECs. These results suggest that the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function.Abbreviations MeHg methylmercury chloride - HUVECs human umbilical vein endothelial cells     

Cytochemical study of the effect of aluminium on cultured brain microvascular endothelial cells

Summary The cytotoxic effect of aluminium was studied on cultured goat brain microvascular endothelial cells used as an in vitro model of the blood—brain barrier. Confluent monolayers of these cells were exposed for 4 days to aluminium maltol and, for control purposes, to maltol alone, and also to cadmium chloride as a known cytotoxic substance. The localization of plasmalemma-bound enzymatic activities of 5-nucleotidase and Ca²⁺-ATPase and the distribution of sialic acid residues were studied at the ultrastructural level.It was observed that the reaction for 5-nucleotidase activity was only insignificantly affected, indicating its resistance to the cytotoxic action of both substances used. On the contrary, the activity of Ca²⁺-ATPase was evidently suppressed, especially in the interendothelial clefts where junctional complexes are presumably to be formed. Aluminium also affects the density of sialic acid residues, as shown by their redistribution, leading to the appearance of relatively long segments of unlabelled apical cell surface.The data obtained suggest that observed changes in the localization of Ca²⁺-ATPase and sialic acid residues can lead ultimately to impairment of the formation and maintenance of intercellular junctions and to disturbances in the negatively charged domains of the endothelial cell surface. Whether these alterations, induced in vitro, contribute to in vivo disturbances of blood—brain barrier function requires further experimental study.     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells.     

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

Anti-angiogenic effects of homocysteine on cultured endothelial cells

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Protein phosphorylation in cultured endothelial cells

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP-dependent protein kinase, three calcium/calmodulin-dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP-dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP-dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin-dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.     

Cytotoxicity of polypropylenimine dendrimer conjugates on cultured endothelial cells

The cytotoxicity and time-dependent membrane disruption by polypropylenimine dendrimer conjugates on cultured human umbilical vein endothelial cells (HUVEC) is reported. Fluorescently labeled derivatives of generation 5 polypropylenimine dendrimers were prepared via conversion of amines to acetamides or through the covalent attachment of high molecular weight poly(ethylene glycol) (PEG) chains. Direct interactions between the fluorescent dendrimer conjugates and HUVEC were monitored using confocal fluorescence microscopy to track dendrimer movement across the plasma membrane and the fluorescent staining of cell nuclei. Propidium iodide and lactate dehydrogenase cytotoxicity assays confirmed that chemical modification of the surface amines of the parental dendrimer to neutral acetamide or PEG functionalities eliminated their acute cytotoxicity. Cationic primary-amine-containing dendrimers demonstrated drastic time-dependent changes in the plasma membrane permeability and prominent cytotoxicity. However, complete removal of the primary amines or masking of the cationic surface via PEGylation decreased dendrimer cytotoxicity. Thus, preventing electrostatic interactions of dendrimers with cellular membranes apparently is a necessary step toward minimizing the toxicity of delivery vehicles to the endothelium.     

The inhibitory effect of simvastatin on the ADMA-induced inflammatory reaction is mediated by MAPK pathways in endothelial cells.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is emerging as a key contributor for endothelial dysfunction associated with inflammation. Statins can inhibit vascular inflammatory reaction and improve endothelial function. The aim of this study was to investigate in human endothelial cells the signaling pathways of ADMA-induced inflammatory reaction and potential inhibitory effects of simvastatin. Endothelial cells were cultured and used for all of the studies. Tumor necrosis factor-alpha(TNF-alpha) and soluble intercellular adhesion molecule-1 (sICAM-1) were determined by enzyme-linked immunosorbent assay. Nuclear factor-kappaB (NF-kappaB) was assayed by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinases (MAPKs), including p38 MAPK and extracellular signal-related kinase (ERK(1/2)), were characterized by Western blot analysis. Treatment with ADMA (3-30 micromol/L) increased the concentration of sICAM-1 in a dose-dependent manner. ADMA (30 micromol/L) significantly enhanced the concentrations of TNF-alpha and sICAM-1, the activity of NF-kappaB and the phosphorylation of p38 MAPK and ERK(1/2). The increased secretion of TNF-alpha and sICAM-1 and the increased activity of NF-kappaB by ADMA were altered by SB203580 (5 micromol/L) or PD98059 (20 micromol/L), but not by LY294002 (20 micromol/L). Simvastatin (0.1, 0.5, or 2.5 micromol/L) markedly inhibited the elevated concentrations of TNF-alpha and sICAM-1, the activity of NF-kappaB, and the phosphorylation of p38 MAPK and ERK(1/2) induced by ADMA. Simvastatin inhibited ADMA-induced inflammatory reaction by p38 MAPK and ERK(1/2) pathways in cultured endothelial cells.     

Calcium influx induced by activation of tyrosine kinase receptors in cultured bovine aortic endothelial cells

We studied the ionic currents activated by basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) in cultured bovine aortic endothelial cells (BAE-1) by using patch-clamp and single-cell fluorimetric calcium measurements. In whole-cell, voltage-clamp experiments at V(h) = -50 mV, the addition of either bFGF (20 ng/ml) or IGF-I (50 ng/ml) induced an inward current with similar amplitude, time course, and permeation properties. The response was dependent on receptor occupancy and showed a desensitisation in the continued presence of the factors. Ionic substitutions in whole-cell experiments indicated that the current barely discriminated among Na(+), Ca(+), and K(+) ions. Accordingly, stimulation with bFGF or IGF-I induced a dose-dependent [Ca(2+)](i) elevation completely due to entry from the extracellular medium, whereas no detectable release from internal stores was observed. Calcium influx was dependent on protein tyrosine kinase (PTK) activity; it was significantly inhibited by treatment with genistein or tyrphostin 47, two PTK inhibitors, and not affected by inactive analogues, daidzein, and tyrphostin 1. Moreover, addition of 200 microM Na(3)VO(4), an inhibitor of protein tyrosine phosphatase (PTP) activity, evoked the responses to the factors both in patch-clamp and in fluorimetric measurements. Cell-attached recordings using 100 mM CaCl(2) in the pipette showed that bFGF and IGF-I activate calcium-permeable channels with similar properties. These results provide evidence for a calcium influx induced by two factors that bind to tyrosine kinase receptors (RTK) in endothelial cells.     

Lack of evidence for the direct activation of endothelial cells by adult female and microfilarial excretory-secretory products

Lymphangiectasia (dilation of the lymphatic vessel (LV)) is pathognomonic for lymphatic filariasis. In both infected humans and animal models of infection, lymphangiectasia is not restricted to the site of the worm nest, but is found along the infected vessel. These observations argue that soluble products secreted by the worm could be mediating this effect by activating the lymphatic endothelial cells (LEC) lining the vessel. We tested the ability of filarial Excretory-Secretory products to activate LECs, but were unable to detect a direct effect of the Excretory-Secretory products on the activation of LEC as assessed by a variety of approaches including cellular proliferation, cell surface molecule expression and cytokine and growth factor production (although other mediators used as positive controls did induce these effects). Collectively, these results do not support the hypothesis that Excretory-Secretory products directly activate LECs.     

The effect of dexamethasone on the cytotoxic and enzymatic response of cultured endothelial cells to radiation

Experiments were conducted to determine (1) whether glucocorticoids directly protected endothelial cells (EC) from radiation and (2) if angiotensin converting enzyme (ACE) activity, known to be increased by glucocorticoid, played a role in the EC response to radiation. Confluent monolayers of EC cultured from bovine aorta EC were treated with dexamethasone (10(-6) M); after irradiation (5.0 Gy, 60Co gamma), ACE and lactate dehydrogenase (LDH) activities, DNA and protein contents, and nuclei number were measured. Twenty-four hours after 5 Gy, there was increased cell loss (-40%, P less than 0.001), greater LDH release (greater than 100%, P less than 0.001), more LDH activity per cell (+40%, P less than 0.001), and unchanged ACE activity compared to sham-irradiated control EC. However, 48 hr after 5 Gy, ACE activity per cell was decreased (-24%, P less than 0.005). A 48-hr exposure to dexamethasone alone was accompanied by a slight cell loss (-10%, P less than 0.001) and increased cellular ACE activity (+40-140%, P less than 0.001), but a 24-hr dexamethasone exposure was not cytotoxic and did not change ACE activity. Dexamethasone exposure for 48 hr before and after irradiation did not attenuate cell loss or LDH release. However, combined dexamethasone treatment and radiation increased cellular ACE activity at a time when neither agent alone had an effect (24-hr dexamethasone exposure before 5 Gy and assayed 24 hr after 5 Gy). This interaction between radiation and dexamethasone treatment suggests that the glucocorticoid modifies the cell's response to injury. Although this interaction does not ameliorate radiation cytotoxicity, maintenance of ACE levels in injured vessels by hormones may have physiological significance in the hemodynamics of irradiated tissues.     

The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells

Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca²⁺-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca²⁺ but independent of intracellular cAMP.     

The effect of ionophore A23187 on albumin internalization in cultured human umbilical vein endothelial cells

The effects of calcium and the calcium ionophore A23187 on endocytosis were studied in cultured human umbilical vein endothelial cells using iodinated human albumin to measure bulk phase endocytosis. In the absence of the ionophore, varying the levels of extracellular calcium did not affect endocytosis. In the presence of 10 μM A23187, the endocytic clearance of albumin decreased approx. 50% when exposed to physiological concentrations of extracellular calcium, but increased approx. 50% at lower calcium concentrations. Since the ionophore is known to alter cellular calcium levels, these results are compatible with a role for intracellular calcium in the modulation of endothelial cell endocytosis.     

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.