Growth factors are released by mechanically wounded endothelial cells

Growth factors may be required at sites of mechanical injury and normal wear and tear in vivo, suggesting that the direct action of mechanical forces on cells could lead to growth factor release. Scraping of cells from the tissue culture substratum at 37 degrees C was used to test this possibility. We show that scraping closely mimics in vitro both the transient plasma membrane wounds observed in cells subject to mechanical forces in vivo (McNeil, P. L., and S. Ito. 1989. Gastroenterology. 96:1238-1248) and the transient plasma membrane wounds shown here to occur in endothelial cells under normal culturing conditions. Scraping of endothelial cells from the culturing substratum released into the culture medium a potent growth-promoting activity for Swiss 3T3 fibroblasts. Growth-promoting activity was released rapidly (within 5 min) after scraping but was not subsequently degraded by the endothelial cells for at least 24 h thereafter. A greater quantity of growth-promoting activity was released by cells scraped 4 h after plating than by those scraped 4 or 7 d afterwards. Thus release is not due to scraping-induced disruption of extracellular matrix. Release was only partially cold inhibitable, was poorly correlated with the level of cell death induced by scraping, and did not occur when cells were killed with metabolic poisons. These results suggest that mechanical disruption of plasma membrane, either transient or permanent, is the essential event leading to release. A basic fibroblast growth factor-like molecule and not platelet-derived growth factor appears to be partially responsible for the growth-promoting activity. We conclude that one biologically relevant route of release of basic fibroblast growth factor, a molecule which lacks the signal peptide sequence for transport into the endoplasmic reticulum, could be directly through mechanically induced membrane disruptions of endothelial cells growing in vivo and in vitro.     

Effects of glutamine on adhesion molecule expression and leukocyte transmigration in endothelial cells exposed to arsenic

This study evaluated whether glutamine (GLN) concentration was related to endothelial surface molecule expression and the migration of polymorphonuclear neutrophils (PMNs) through endothelial cells (ECs) stimulated by arsenic. Human umbilical vein endothelial cells (HUVECs) and PMNs were treated with different GLN concentrations (0, 300, 600 and 1000 microM) for 24 h. After that, we stimulated HUVECs for 3 h with 0.5 microM arsenic, and PMNs were allowed to transmigrate to ECs for 2 h. HUVEC surface expressions of cell adhesion molecules and integrin (CD11b) and interleukin (IL)-8 receptor expressions on PMNs were measured. The transendothelial migration of PMNs was also analyzed. The results showed that cell adhesion molecule (CAM) and integrin expressions in arsenic groups were higher than in those without arsenic. Among the arsenic groups, the expression of CAMs on ECs and CD11b, and IL-8 receptor on PMNs was lowest with 0 microM compared with the other GLN concentrations. Vascular CAM-1 on ECs and CD11b on PMN expression were higher with 300 microM than with 600 and 1000 microM GLN. IL-8 secretions from ECs and PMNs were higher with 300 muM than with 600 and 1000 microM GLN, and this was consistent with the expression of the IL-8 receptor on PMNs. Polymorphonuclear neutrophil transmigration was significantly higher with 300 muM GLN than with other GLN concentrations. These results suggest that ECs and PMNs were activated after arsenic stimulation. Cell adhesion molecule expressions on ECs and PMNs were suppressed in the absence of GLN. A low GLN concentration comparable to catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. Glutamine administration at levels similar to or higher than physiological concentrations reduced IL-8 and adhesion molecule expression; PMN transmigration was also decreased after stimulation with arsenic.     

Bovine aortic endothelial cells release hydrogen peroxide.

Endothelial cells grown on microcarriers are able to release H2O2 to the extracellular environment without any added stimulus. The extracellularly released H2O2 can be detected by luminol-amplified chemiluminescence (CL) if horseradish peroxidase is added. The CL response can be reduced by catalase and blocked by superoxide dismutase, indicating that O2- could be a precursor for H2O2. The CL kinetics, i.e., a long lag time followed by a rapid shift to a new level, indicate activation of an O2(-)-producing enzyme. The cells are also able to protect themselves from H2O2 stimulation by both catalase and the glutatione system. Bradykinin stimulates the H2O2 release, but if the effect is directly stimulatory or if it acts by reduction of the protective system is at present unclear. The extracellularly released H2O2 could be a cause of injury to the endothelial cells or to the subendothelial matrix.     

Neutrophil capture by selectins on endothelial cells exposed to cigarette smoke

We used a novel perfusion system to expose cultured human umbilical vein endothelial cells (HUVEC) to water-soluble components of cigarette smoke and study subsequent adhesion of flowing neutrophils. Neutrophils did not bind to HUVEC immediately after it had been exposed to cigarette smoke, but many adhered 90-150 min after exposure. The effect was reduced if the exposed medium was made serum-free, but this reduction was partially reversed if low density lipoprotein was added. Treatment of smoke-exposed HUVEC with antibodies against E-selectin or P-selectin reduced adhesion by approximately 50% or 75%, respectively; a combination of both antibodies essentially abolished adhesion. Enzyme-linked immunosorbent assay confirmed that exposure to smoke caused HUVEC to upregulate surface expression of E- and P-selectin. Thus, water-soluble constituent(s) of cigarette smoke cause efficient selectin-mediated capture of flowing neutrophils. This pro-inflammatory response may contribute to pathology associated with smoking, especially in tissues remote from the lung.     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

Cerebromicrovascular endothelial cells are resistant to L-glutamate

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by l-glutamate (l-glut) or N-methyl-d-aspartate (NMDA) in vitro. However, high levels of l-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to l-glut or NMDA. Viability was unaffected by 8-h exposure to 1-10 mM l-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM l-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM l-glut or NMDA. The Ca(2+) ionophore calcimycin (5 microM), but not l-glut (1 mM), increased intracellular Ca(2+) levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of l-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.     

Intracellular calcium 'signatures' evoked by different agonists in isolated bovine aortic endothelial cells.

Agonist induced increases in intracellular free calcium, [Ca2+]i, were measured in single Fura-2 loaded bovine aortic endothelial (BAE) cells by dual wavelength microspectrofluorimetry. Low doses of ATP (less than 10 microM) induced complex changes in [Ca2+]i. These changes usually consisted of a large initial transient peak with subsequent fluctuations superimposed upon a maintained rise in [Ca2+]i. Higher doses of ATP (greater than 50 microM) produced much simpler biphasic increases in [Ca2+]i in individual cells. Acetylcholine and bradykinin also elicited increases in [Ca2+]i in single cells in confluent monolayers of endothelial cells. However, only acetylcholine produced complex fluctuations. High doses of acetylcholine evoked simple rises in [Ca2+]i similar to those seen with high doses of ATP. In contrast, bradykinin evoked relatively simple rises in [Ca2+]i at all doses used. These results indicate that the mechanisms responsible for generating agonist induced increases in [Ca2+]i in BAE cells are not homogeneous. ATP and acetylcholine produced more complex Ca2+ changes or 'signatures' in single confluent bovine aortic endothelial cells than bradykinin. All three agonists appeared to release Ca2+ from intracellular stores as well as stimulating Ca2+ influx. The possible mechanisms underlying these phenomena are discussed.     

T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1.

The action of human rIL-1 beta on confluent, quiescent monolayers of human umbilical vein endothelial cells (HUVEC) has been studied for the induction of new membrane proteins. Two approaches have been taken. The first is a quantitative two-dimensional gel analysis of [35S]cysteine-labeled membrane proteins of HUVEC with and without cytokine treatment. This analysis indicates that there are a restricted number of new membrane proteins synthesized in the first 6 h of IL-1 treatment, on the order of 19 out of a total of over 600 detectable proteins. Second, we have prepared two mAb (1E7 and 2G7) to different epitopes of a major inducible sialoglycoprotein with molecular mass of 114 kDa and an isoelectric point of 4.6 to 4.8. These antibodies were compared with two additional antibodies, 3B7 and 7A9, which were shown to react with the endothelial leukocyte adhesion molecule-1 (ELAM-1) protein as expressed in COS cells. The 1E7/2G7 protein is distinct from ELAM-1, based upon biochemical comparisons as well as the inability of the 1E7 and 2G7 antibodies to react with ELAM-1-transfected COS cells. The protein defined as 1E7/2G7 is neither expressed constitutively nor in an inducible manner on PBMC, granulocytes, platelets, fibroblasts, or keratinocytes. The 7A9 and 3B7 antibodies are shown to block granulocyte binding to IL-1-activated HUVEC. The 2G7 antibody is effective at inhibiting the binding of T cells but not granulocytes to IL-1-activated endothelium, suggesting this new protein is an adhesion protein that may be active in vivo in T cell-endothelial cell adhesion-related events such as inflammation or lymphocyte recirculation. In addition, T cells were shown to utilize the ELAM-1 protein in binding to cytokine-activated HUVEC. Antibodies directed to both proteins had additive effects on inhibition of T cell adhesion.     

Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury.

The effect of hypoxia on subsequent susceptibility of porcine pulmonary artery endothelial cells (PAEC) to hydrogen peroxide (H2O2) injury was studied. Preexposure of PAEC to hypoxia for 3 or more h significantly increased susceptibility to subsequent H2O2 challenge. Analysis of the activities of antioxidant enzymes and xanthine oxidase/dehydrogenase suggested that changes in these enzymes in hypoxic PAEC were not responsible for the increased susceptibility. However, hypoxia resulted in significant time-dependent decreases in total glutathione at 12 h or more. The rate of glutathione regeneration in diethylmaleate-treated PAEC and the rate of uptake of cystine and glycine were significantly lower during hypoxia. Hypoxia also caused depletion of ATP and NADPH levels in PAEC, but these did not occur until well after hypoxia-enhanced susceptibility to H2O2 injury was demonstrable. Alterations in glutathione levels and enhanced susceptibility were reversible when hypoxic PAEC were returned to normoxia. These results indicate that hypoxia increased the susceptibility to H2O2 injury by decreasing the ability of PAEC to maintain and regenerate cellular glutathione content in response to H2O2 challenge.     

Altered plasma proteins released from platelets and endothelial cells are associated with human patent ductus arteriosus

Patent ductus arteriosus is the third most common congenital heart disease and resulted from the persistence of ductal patency after birth. Ductus arteriosus closure involves functional and structural remodeling, controlled by many factors. The changes in plasma protein levels associated with PDA closure are not known. Here we for the first time demonstrate six key differential plasma proteins in human patent ductus arteriosus patients using proteomic technology and present a model to illustrate the constriction and closure of ductus arteriosus. Differentially expressed proteins were analyzed by using isobaric tags for relative and absolute quantification and validated by enzyme-linked immunosorbent assay in new samples. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD008568. We found 74 upregulated and 98 downregulated proteins in the plasma of patients with PDA. Five decreased proteins (platelet factor 4, fibrinogen, von Willebrand factor, collagen, and mannose binding lectin-associated serine protease-2) and one increased protein (fibronectin) may increase the risk of patent ductus arteriosus. Those proteins are closely related to platelet activation and coagulation cascades, complement mannan-binding-lectin, and other systemic signaling pathways. Our findings for the first time indicate that the differential proteins involved in different pathways may play key roles in the nonclosure of the ductus arteriosus in humans and may be developed as biomarkers for diagnosis. All those findings may be served as the basis of understanding the etiology and pathogenesis of patent ductus arteriosus.     

A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase‐dependent manner by serum‐starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho‐associated, coiled‐coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.     

Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.     

Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide

Recent studies have suggested that hydrogen peroxide (H2O2), a reactive compound formed endogenously in the breakdown of superoxide, may mediate the induction of apoptosis in various cell types in response to external stimuli. However, the role of H2O2 in the apoptotic pathway has not been clearly established. The purpose of this study was to determine if H2O2 treatment could induce apoptosis through the activation of caspases. Doses of H2O2 ranging from 10 microM to 100 microM, when added to HL-60 cells, resulted in the cleavage of poly(ADP-ribose) polymerase (PARP) from its native 113 Kd form to a processed 89 Kd fragment, indicative of cells undergoing apoptosis. PARP was predominantly in the fragmented form when doses of 20 microM and greater were used. A time course study of changes in PARP processing in H2O2-treated cells revealed that 10 and 50 microM H2O2 required 6 and 3 h, respectively, to specifically degrade PARP, suggesting that the H2O2-induced PARP cleavage is both time and concentration dependent. Since PARP is cleaved by CPP32 (caspase-3), we next determined if H2O2 was capable of effecting changes in CPP32 activity. The caspase activity was assayed using a colorimetric substrate, DEVD-pNa. Results of these experiments showed that H2O2 increased caspase activity at 3 h, corresponding to the time of appearance of fragmented PARP. Also, CPP32 activity and PARP processing were both significantly suppressed by caspase-3 inhibitors. Taken together, these results suggest that H2O2 mediates specific cleavage of PARP and possibly apoptosis by activating caspase 3.     

Mitogen-activated protein phosphorylation in endothelial cells exposed to hyperosmolar conditions

The effect of hyperosmolarity on the induction of the mitogen-activated protein kinases (MAPK) was studied in bovine aortic endothelial cell (EC). Different types of agents were used to differentiate the effects of osmolarity from other variables. Hypertonic treatment with physiologically relevant levels of NaCl (350 mOsm/kg H(2)O) significantly increased the level of expression of p38 within 2 min, and ERK-1/2 and JNK after 10 min. The inductions peaked between 30 and 60 min and returned to baseline levels within 2 h. A similar pattern of induction occurred with ionic contrast agent. p38 induction by glucose and mannitol showed a similar pattern, although the level of ERK-1/2 phosphorylation was not as robust, and JNK was not induced by glucose. Urea did not affect the level of induction of the MAPK isoforms. It is concluded that MAPK plays an important role in hyperosmolality-induced signal transduction. Different osmotic agents induce MAPK expression differently. No MAPK induction with urea implies that cell shrinkage may be an important component of hyperosmolality-induced MAPK phosphorylation.