Peter J. Del Vecchio

Enhancement of intracellular glutathione protects endothelial cells against oxidant damage

We studied the role of glutathione in the endothelial cell defense against H2O2 damage. Treatment of endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, depleted the cells of GSH, while L-2-oxothiazolidine-4-carboxylate, an effective intracellular cysteine delivery agent, markedly enhanced endothelial cell GSH concentration. Depletion of intracellular GSH sensitized the endothelial cells to injury by H2O2 either preformed or generated by the glucose-glucose oxidase system. In contrast, an increase of intracellular GSH protected the cells from H2O2 damage. There was an inverse, linear relationship between the intracellular GSH concentrations and killing of endothelial cells by H2O2. Our results suggest that enhancement of endothelial cell GSH may be an alternative approach toward the prevention of oxidant-induced endothelial damage such as adult respiratory distress syndrome.

Culture and characterization of pulmonary microvascular endothelial cells.

SummarySurface proteins were compared in endothelial cells (EC) obtained from bovine peripheral lung, pulmonary artery and vein, and dorsal aorta using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Galactose-containing glycoproteins [molecular weight (Mr) 160–220 and 40 kDa] binding to theRicinus communis agglutinin (RCA) and peanut agglutinin (PNA) were selectively observed on pulmonary microvessel EC as compared to EC from pulmonary artery, pulmonary vein, and dorsal aorta. The unique RCA- and PNA-binding profiles of EC from the pulmonary artery and microvessels may be important in characterizing EC from different sites in the pulmonary circulation. The pulmonary microvessel EC monolayer was also 15-fold more restrictive to transendothelial flux of [14C]sucrose (Mr=342 Da) than the pulmonary artery EC monolayer. In contrast, the microvessel EC were only six- and twofold more restrictive to the flux of larger tracer molecules, ovalbumin (Mr 43 kDa) and albumin (Mr=69 kDa) than pulmonary artery EC. The greater restrictiveness of pulmonary microvessel EC monolayer indicates a major phenotypic difference in the cultured pulmonary microvessel EC barrier function.

Expression of bovine and mouse endothelial cell antioxidant enzymes following TNF-α exposure

Abstract Endothelial cells are primary targets for injury by reactive oxygen species. Endothelial catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganous superoxide dismutase (MnSOD) provide potential antioxidant enzymatic defenses against oxidant-induced cellular damage. Previous studies in vivo and in vitro have demonstrated that in certain cell types exposure to oxidants may increase the expression of one or more of these antioxidant enzymes, thus providing greater intracellular potential to withstand oxidant-induced cells stress. To test whether endothelial antioxidant enzyme expression is influenced by similar oxidant-induced stresses in vitro, we have exposed endothelial cells to tumor necrosis factor-alpha (TNF-α) and have measured levels of catalase, CuZnSOD and MnSOD mRNA, and protein. Our results demonstrate a selective increase of MnSOD mRNA, with coordinate increases of both MnSOD protein and enzyme activity in endothelial cells treated for 24/h with TNF-α. In contrast, levels of catalase and CuZnSOD mRNA and protein remained unchanged in these cells after TNF-α treatment. These observations were made in microvessel endothelial cells derived from murine and bovine sources. Our results indicate that TNF-α can act specifically to increase enzymatic antioxidant potential in endothelial cells by induction of a particular antioxidant enzyme encoding mRNA species. These data demonstrate the capacity of endothelial cells to mount an antioxidant defense in response to exposure to an inducer of oxidative damage.

Association of α-crystallin with actin in cultured lens cells

The nature of the beaded filaments in the lens fiber cell has been debated for some time. One explanation is that beaded filaments represent an association of α-crystallin with actin filaments. By using a double labelling technique that allowed us to view actin filaments and α-crystallin in the same cell we have demonstrated that some of the α-crystallin in lens cells is indeed associated with actin.

Thrombin induced platelet adhesion to endothelium is modified by endothelial derived relaxing factor (EDRF)

We investigated the hypothesis that thrombin-induced attachment of platelets to endothelial cells is modulated by EDRF. Thrombin significantly increased binding of radiolabelled platelets to cultured endothelium and to an intact pulmonary vasculature under flow conditions. These increases in binding were potentiated with hemoglobin (HB) and inhibited by superoxide dismutase (SOD) in both systems. We suggest that thrombin, in addition to enhancing platelet adhesion, elicits EDRF release from endothelial cells and that EDRF serves an antithrombotic function in the down regulation of platelet adhesion.

Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice

We examined the effect of lipopolysaccharide (LPS) treatment on the expression of manganese and copper/zinc superoxide dismutase (MnSOD and Cu/ZnSOD) mRNA and protein in resident peritoneal macrophages and lung endothelial cells derived from LPS-sensitive (LPS-s) and LPS-resistant (LPS-r) mice. Macrophages from both LPS-s and LPS-r mice treated with LPS for 24 h produced increased levels of MnSOD mRNA and protein. In contrast, levels of lung endothelial cell MnSOD mRNA and protein from LPS-s mice were increased by LPS treatment, while no increases in these parameters were observed in endothelial cells from LPS-r mice. Tumor necrosis factor-alpha (TNF alpha) treatment, however, did increase levels of MnSOD mRNA in both LPS-s and LPS-r endothelial cells to an equal extent. Both macrophage and endothelial cell Cu/ZnSOD mRNA and protein levels were not significantly affected by LPS treatment. These results demonstrate that the mutation that affects susceptibility to LPS in LPS-r mice exerts a differential influence on MnSOD inducibility in a cell specific manner.

Selectivity of the endothelial monolayer: effects of increased permeability.

We investigated the mechanism of thrombin-induced increases in endothelial monolayer permeability by examining the effect of thrombin on the molecular sieving characteristics of the endothelial monolayer and comparing the responses of arterial- and venous-derived endothelial cell lines. Bovine pulmonary artery (BPA) and pulmonary vein (BPV) endothelial cells were similarly harvested and cultured. The endothelial cells were grown to confluence on gelatinized polycarbonate filters and the permeabilities to sucrose, albumin, and IgG were measured and corrected for effects of unstirred layers. The control permeabilities of BPA and BPV were similar with both monolayers, demonstrating selectivity to different sized tracer molecules. alpha-Thrombin (10(-6) M) increased the permeability of both BPA and BPV to albumin and sucrose. The permeability of BPA was increased to a greater extent than BPV, perhaps due to phenotypic differences. In both cell lines, the permeability increase was most pronounced for albumin, which by pore theory is best described by an increase in the radius of the small pore pathway for diffusion.

Matrix fibronectin disruption in association with altered endothelial cell adhesion induced by activated polymorphonuclear leukocytes

Sequestration of activated polymorphonuclear leukocytes (PMN) within the lung microcirculation may contribute to pulmonary vascular injury following trauma, sepsis, or disseminated intravascular coagulation. In this study cultured rat endothelial cells were utilized to evaluate the effect of PMN activation on endothelial cell attachment. The concept that disruption of the extracellular fibronectin matrix is associated with altered endothelial cell adhesion was also tested. Rat endothelial cells were grown in culture and identified by morphological techniques as well as immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with 51Cr in order to establish a cell injury assay based on release of free 51Cr or cell-associated 51Cr. PMN activation was verified microscopically and by chemiluminescence activity following phorbol myristate acetate (PMA) or opsonized zymosan exposure. Following incubation with PMA, the leukocytes aggregated, chemiluminesced vigorously, and caused endothelial cell injury and detachment as determined by release of 51Cr-labeled endothelial cells. PMNs exposed to serum-treated zymosan exhibited a more modest chemiluminescence burst which was consistent with their decreased activity to injure the endothelial monolayer. With PMA activation the degree of endothelial detachment from the monolayer increased as a function of time with a plateau observed by 3 hr. Microscopic immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin after incubation with PMA-activated neutrophils in association with endothelial cell disadhesion. Thus, exposure of activated rat PMN to rat endothelial cells in culture induces endothelial damage and an associated disruption of the fibronectin matrix which may contribute to endothelial cell detachment.

Release of fibronectin fragments from endothelial cell monolayers exposed to activated leukocytes: Relationship to plasma fibronectin levels after particle infusion☆

Fibronectin is found in a soluble form in plasma as well as in an insoluble form in tissues. It is produced by cultured endothelial cells and can be localized in vitro and in vivo between adjacent endothelial cells as well as underneath endothelial cells in association with their collagenous matrix, where it is believed to influence cell adhesion. Fibronectin is susceptible to proteolytic cleavage by enzymes released from activated leukocytes. In the present study, cultured rat endothelial cells developed a fibrillar fibronectin network in their extracellular matrices in addition to releasing soluble, intact fibronectin (440 kDa) into their culture medium. Exposure of monolayers of cultured endothelial cells to activated polymorphonuclear leukocytes (PMNs) results in disruption of the fibrillar matrix fibronectin, damage to the endothelial cell monolayer, and presence of fibronectin fragments in the culture medium. In addition, acute leukocyte activation and peripheral leukopenia in vivo as induced by the intravenous infusion of foreign test particles also resulted in the appearance of low-molecular-weight fibronectin fragments in plasma. In the in vivo studies, the appearance of fibronectin fragments preceded the release of intact 440-kDa fibronectin in the plasma after its acute depletion by particle injection. Thus, activated leukocytes, adherent to an endothelial surface in vitro and in vivo, may result in degradation of matrix fibronectin and the release of fibronectin fragments into the extracellular environment. In vivo fibronectin fragments in blood may serve as a stimulus for the subsequent synthesis and/or release of intact plasma fibronectin.

Regulation of antioxidant enzyme expression in LPS-treated bovine retinal pigment epithelial and corneal endothelial cells

Retinal pigment epithelial (RPE) and corneal endothelial (CE) cells, because of their locations and functions, are continuously exposed to toxic oxidants. Protection from these toxic materials may be due, in part, to the action of endogenous antioxidant enzymes. We have established the presence of mRNAs that encode antioxidant enzymes in bovine RPE and CE cells and have determined the effect of bacterial lipopolysaccharide (LPS) on their expression. The most striking change in antioxidant enzyme expression is an increase in the level of mitochondrial manganous superoxide dismutase (MnSOD) mRNA in the LPS-treated RPE and CE cells. This increase in mRNA expression is accompanied by a slight increase in MnSOD activity as determined by SOD activity gels.