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Biomedical subjects

G M Vercellotti

Publications and source records attributed to G M Vercellotti.

At least 73 records · Page 4Linked to original sources
PubMed1991

Effects of staphylococcal toxic shock syndrome toxin 1 on aortic endothelial cells.

In staphylococcal toxic shock syndrome, hypotension and shock due to capillary leak may rapidly lead to death of the host. To investigate its pathogenesis, the cytotoxic effects of toxic shock syndrome toxin 1 (TSST-1) on porcine aortic endothelial cells (PAEC) were examined in vitro. TSST-1 killed PAEC (as measured by 51Cr release) in a dose- and time-dependent fashion and was blocked by anti-TSST-1 antibodies. Receptor-mediated endocytosis may be critical for the cytotoxic effects of TSST-1, as killing was inhibited by cold (4 degrees C) and by addition of chloroquine and methylamine. Furthermore, calcium and oxygen appeared necessary for TSST-1 effects on PAEC. Membrane receptor binding studies indicated PAEC bind TSST-1 with high affinity (Kd = 5.7 x 10(-7) M) and had 2.2 x 10(4) receptors/cell. Last, as measured by 125I-labeled albumin flux in a transendothelial permeability model, TSST-1 enhanced the permeability of PAEC monolayers in a dose- and time-dependent manner.

Animals↗
PubMed1991

Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection.

Complement plays a major role in hyperacute rejection of discordant xenografts. In immediately vascularized xenografts, such as porcine organs to humans, C activation contributes to triggering of endothelial cell activation and adhesion of leukocytes and platelets to the endothelial cells, which is followed by thrombosis and tissue necrosis. We investigated the potential utility of the membrane-associated inhibitor of C, decay accelerating factor (DAF), in the prevention of C-mediated tissue injury. We used an in vitro model of xenotransplantation consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and C. Because C inhibitors such as DAF may be relatively species-specific, we tested whether human DAF would incorporate into porcine endothelial cells and function to inhibit cytotoxicity of such cells by human C. We found that purified radiolabeled human DAF incorporated into porcine endothelial cells in a dose-dependent manner and that human DAF very significantly protected the endothelial cells from the cytotoxic effect of human C.

Animals↗
PubMed1991

Molecular mimicry in Candida albicans. Role of an integrin analogue in adhesion of the yeast to human endothelium.

Hematogenous infection with the yeast Candida albicans now occurs with increasing frequency in the neonate, the immunocompromised patient, and the hyperglycemic or hyperalimented host. Yeast-phase C. albicans expresses a protein that is antigenically and structurally related to CD11b/CD18, a member of the beta 2 integrins and a well-characterized adhesin for mammalian neutrophils. Both the neutrophil protein and its analogue in C. albicans have an identical affinity for the C3 ligand iC3b, and both proteins are significantly increased in expression at 37 degrees C. Given these several similarities, we therefore studied the role of the integrin analogue on C. albicans in the adhesion of the yeast to human umbilical vein endothelium (HUVE). After growth of C. albicans in 20 mM D-glucose, as opposed to 20 mM L-glutamate, flow cytometric analysis with monoclonal antibodies recognizing the alpha-subunit of CD11b/CD18 demonstrated a 25.0% increase in mean channel fluorescence (range 18.4-31.8%), as well as an increased percentage of yeasts fluorescing (P less than 0.02). This increased intensity of fluorescence, which corresponds to increased expression of the integrin analogue, also correlated with a significant increase of 30-80% in adhesion of glucose-grown C. albicans to HUVE (P less than 0.02). Blockade of the integrin analogue on C. albicans by monoclonal antibodies recognizing adhesive epitopes on neutrophil CD11b/CD18 inhibited glucose-enhanced adhesion of C. albicans to HUVE. Incubation of glucose-grown C. albicans with saturating concentrations of purified human iC3b, the ligand for CD11b/CD18, reduced adhesion of the yeast to HUVE by 49.7%, whereas BSA in equimolar concentration had no effect (P less than 0.001). These results identify a glucose-responsive integrin analogue on C. albicans as one of possibly several cellular structures that mediate adhesion of the yeast to human endothelium.

Antibodies, Monoclonal↗
PubMed1991

Hydrogen peroxide alters signal transduction in human endothelial cells.

Lytic H2O2-induced injury to human umbilical vein endothelial cells provides a model for endothelial cell damage in diverse states including acute respiratory distress and septic shock. Endothelial cell lysis is an extreme result of inflammatory cell activation. Functional alterations such as responsiveness to endothelial cell agonists and eicosanoid production might be impaired by exposure to inflammatory cell products including H2O2. Soluble mediators such as thrombin or histamine cause endothelial cell activation via a signal transduction mechanism that hydrolyzes phosphatidylinositol 4,5-bisphosphate (IP), liberating inositol trisphosphate (IP3). Accordingly, pretreatment of endothelial cells with H2O2 blocked the subsequent production of IP3 in response to thrombin and histamine. H2O2 inhibition of IP3 was time- and concentration-dependent. The endothelial cells were viable by trypan blue dye exclusion and chromium release. H2O2 inhibition of signaling was completely prevented by catalase. Iron-dependent oxidant radical formation appears critical because deferoxamine (10(-4) mol/L) pretreatment of endothelial cells prevented H2O2 inhibition of IP hydrolysis. Prostacyclin and platelet activating factor production in response to thrombin have been linked to IP hydrolysis. Pretreatment of endothelial cells with H2O2 reduced prostacyclin and platelet-activating factor production by thrombin by at least 50%. It appears H2O2 can induce defects in signaling pathways with sequelae (decreased prostacyclin and platelet-activating factor) short of endothelial cell death. The possible consequences of H2O2 interaction with endothelial cells is reviewed with the aim of presenting a hypothesis to integrate these various observations.

Catalase↗
PubMed1991

Exposure of endothelial cells to free heme potentiates damage mediated by granulocytes and toxic oxygen species.

Endothelial damage may follow exposure to toxic oxygen species generated by closely apposed ("marginated") granulocytes. Because iron markedly catalyzes oxidant damage in diverse systems, we wondered whether intercalculated heme, and/or its constituent iron, might potentiate oxidant damage of endothelium. Cultured monolayers of porcine aortic endothelial cells were exposed for brief periods to purified hemin. Uptake of heme was rapid, dose dependent, and not reversible by buffer or serum washes. Despite high levels of cell-associated heme, no direct heme-mediated cytotoxicity occurred, but heme-loaded endothelium became highly sensitive to oxidant challenge by (a) reagent H2O2, (b) enzymatically generated oxidants (xanthine/xanthine oxidase), or (c) phorbol-activated polymorphonuclear leukocytes. An increase in endothelial cell lipid peroxidation accompanied heme-augmented oxidant cytolysis, and both parameters were reduced in parallel by micromolar amounts of the hydrophobic oxygen radical scavenger/iron chelator U74500A. Endothelial uptake of heme was inhibited by a specific heme-binding protein, hemopexin. Concomitantly, hemopexin completely blocked augmented H2O2- and polymorphonuclear leukocyte-mediated cytotoxicity but only if added simultaneously and stoichiometrically with hemin. Significant loss of protection occurred if hemopexin addition was delayed 15 minutes, and protection was completely lost after a 60-minute interval. The iron moiety of heme was critical to oxidant sensitization because neither iron-free protoporphyrin IX nor tin-protoporphyrin was able to sensitize endothelial cells to H2O2 or activated polymorphonuclear leukocytes. These results may provide mechanistic insights into atherogenesis, reperfusion injury, and the organ injury accompanying hemoglobinemia or myoglobinemia.

Animals↗
PubMed1990

Tumor necrosis factor alpha/cachectin stimulates eosinophil oxidant production and toxicity towards human endothelium.

Eosinophils (EOs) participate in a variety of inflammatory states characterized by endothelial cell damage, such as vasculitis, pneumonitis, and endocarditis. We find that 100 U/ml TNF-alpha/cachectin (TNF), a concentration attainable in the blood of humans with parasitic infestations, stimulates highly purified populations of EOs to damage human umbilical vein endothelial cells (HUVEC), a model of human endothelium. This TNF-dependent EO cytotoxicity is strongly inhibited by heparin and methyprednisolone but unaffected by the platelet-activating factor antagonist BN52012 or scavengers of superoxide anion and H2O2, superoxide dismutase and catalase. However, addition of a physiologically relevant concentration of Br- (100 microM) enhances EO/TNF damage to HUVEC, implicating the possible participation of EO peroxidase (EPO) in the killing mechanism. EOs adherent to FCS-coated plastic wells more than double their production of superoxide anion and the cytotoxic EPO-derived oxidant HOBr when exposed to TNF, showing that TNF activates the respiratory burst of EOs attached to a "physiologic" surface. Unlike PMNs, EOs were not irreversibly activated to kill unopsonized endothelium by previous exposure to TNF, and did not degranulate or upregulate CR3 expression as detected by Mo1 in the presence of 100 U/ml TNF. HUVEC exposed 18 h to TNF were considerably more susceptible to lysis by PMA-activated EOs and reagent H2O2, demonstrating a direct effect of TNF upon endothelium, perhaps through inhibition of antioxidant defenses. These findings suggest that abnormally elevated serum levels of TNF may provoke EOs to damage endothelial cells and thereby play a role in the pathogenesis of tissue damage in hypereosinophilic states.

Antigens, Differentiation↗
PubMed1990

Release of heparan sulfate from endothelial cells. Implications for pathogenesis of hyperacute rejection.

Heparan sulfate proteoglycan associated with endothelial cells in normal blood vessels inhibits intravascular coagulation and egress of blood cells and plasma proteins, key features of hyperacute rejection. It was shown herein that exposure of cultured porcine endothelium to human serum as a source of natural antibodies and complement caused cleavage and release of 5% of endothelial cell proteoglycans within 4 min and greater than 50% within 1 h. Proteoglycan release depended on activation of the classical complement pathway and preceded irreversible cell injury. These findings suggest that loss of endothelial cell proteoglycan may be a critical step in the pathogenesis of hyperacute rejection and in diseases involving humoral injury to endothelial cells.

Animals↗
PubMed1990

Evidence for a role of platelet activating factor in the pathogenesis of irreversible but not reversible myocardial injury after reperfusion in dogs.

The role of platelet activating factor (PAF) in myocardial injury after either brief (15 minutes, stunned myocardium) or prolonged (90 minutes, infarcted myocardium) coronary artery occlusion and 3 hours of reperfusion of the left anterior descending coronary artery was investigated in barbital-anesthetized dogs. Regional myocardial blood flow was measured by radioactive microspheres, regional segment shortening by sonomicrometry, and infarct size by the triphenyltetrazolium chloride stain. Infarct size expressed as a percentage of the area at risk was significantly reduced by the intravenous administration of two structurally unrelated PAF antagonists, BN 52021 (10 mg/kg and 1 mg/kg/hr) and CV-3988 (3 mg/kg and 0.3 mg/kg/hr). Infarct size was 38% +/- 5% in the saline (control) group, (n = 7), 22% +/- 5% in the BN 52021 group (n = 7), and 19% +/- 5% in the CV-3988 group (n = 8). However, the intravenous administration of BN 52021 (5 and 10 mg/kg) and CV-3988 (5 mg/kg) had no effect on functional recovery (regional segment shortening) in the stunned myocardium after brief occlusion and reperfusion. Regional myocardial blood flow, hemodynamic data, and the incidence of cardiac arrhythmias were not significantly affected by PAF antagonists in both series of experiments at any time. These data suggest that PAF may play an important role in the pathogenesis of an evolving myocardial infarction that follows a prolonged coronary artery occlusion and reperfusion. Furthermore, PAF antagonists may have a beneficial role in reduction of the injury produced during an acute infarction. Finally, these data indicate that PAF does not appear to be an important mediator of myocardial stunning.

Animals↗
PubMed1990

Transplantation of discordant xenografts: a review of progress.

Hyperacute rejection, apparently initiated by natural antibodies and complement, has been viewed as an absolute barrier to the xenotransplantation of vascularized grafts between different species. Until recently, little was known about the molecular and physiological basis for this barrier nor was there evidence that the barrier might be more than transiently breached. In this paper Jeffrey Platt, Fritz Bach and colleagues describe a model of hyperacute rejection and propose that, if hyperacute rejection can be averted for a period after transplantation, prolonged xenograft survival will be possible.

Animals↗
PubMed1990

Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression.

Latent infection of vascular cells with herpes-viruses may play a pathogenic role in the development of human atherosclerosis. In a previous study, we found that cultured human umbilical vein endothelial cells (HUVECs) infected with herpes simplex virus 1 (HSV-1) became procoagulant, exemplified both by their enhanced assembly of the prothrombinase complex and by their inability to reduce adhesion of platelets. We now report two further procoagulant consequences of endothelial HSV infection: loss of surface thrombomodulin (TM) activity and induction of synthesis of tissue factor. Within 4 hr of infection of HUVECs, TM activity measured by thrombin-dependent protein C activation declined 21 +/- 3% (P less than 0.05) and by 18 hr, 48 +/- 5% (P less than 0.001). Similar significant TM decrements accompanied infection of bovine aortic endothelial cells. Identical TM loss was induced with HSV-2 infection but not with adenovirus infection. Decreased surface expression of TM antigen (measured by the specific binding of a polyclonal antibody to bovine TM) closely paralleled the loss of TM activity. As examined by Northern blotting, these losses apparently reflected rapid onset (within 4 hr of HSV infection) loss of mRNA for TM. In contrast, HSV infection induced a viral-dose-dependent increase in synthesis of tissue factor protein, adding to the procoagulant state. The results indicate that loss of endothelial protein-synthetic capacity is not a universal effect of HSV infection. We suggest that the procoagulant state induced by reduction in TM activity and amplified tissue factor activity accompanying HSV infection of endothelium could contribute to deposition of thrombi on atherosclerotic plaques and to the "coagulant-necrosis" state that characterizes HSV-infected mucocutaneous lesions.

Cell Transformation, Viral↗
PubMed1990

Herpes simplex virus-infected cells disarm killer lymphocytes.

Human endothelial cells or human foreskin fibroblasts infected with herpes simplex viruses (HSVs) potently inhibit the lytic activity of natural killer cells and interleukin 2-activated killer cells. The inhibition occurs after as little as 8 hr of viral infection and requires contact between effector cells and HSV-infected targets. Inhibition evidently stems from direct blockade of killer cell function because killer cells placed atop HSV-infected targets rapidly become incapable of lysing subsequently added HL-60 or K-562 cells. The impairment of killer cell function is prevented when protein glycosylation in HSV-infected cells is blocked with tunicamycin. These studies may be relevant for understanding the persistence of herpes simplex virus infections and, further, suggest a mechanism for failed immune surveillance.

Adenoviruses, Human↗
PubMed1990

Endothelial cell platelet-activating factor primes neutrophil responses: amplification of endothelial activation by neutrophil products.

We have shown that platelet-activating factor (PAF) primes neutrophil (PMN) responses and enhances their ability to damage endothelial cells. Furthermore, thrombin-stimulated endothelial cells which produce PAF can augment and prime PMN superoxide production, elastase release and adhesion to endothelium. We wondered whether these marginated neutrophils (PMN) themselves, or their release products, might feedback and further amplify endothelial cell activation. To measure cellular activation, we assessed changes in endothelial cell intracellular calcium [( Ca2+]i) in endothelial monolayers loaded with Fura-2, and PAF production by [3H]acetate incorporation into phospholipid. Resting PMNs induced no change in [Ca2+]i, while N-formyl-L-methionine-L-leucyl-L-phenylalanine stimulated PMN and their lysosomal products caused a 25% increase in endothelial cell calcium. Sonicates of PMN produced a much larger increase in [Ca2+]i than activated PMN; the effect of PMN sonicates could in part be reduced by the serine protease inhibitor, alpha 1 antitrypsin. In contrast, purified neutrophil elastase did not alter endothelial cell [Ca2+]i. Despite hydrogen peroxide's ability to increase [Ca2+]i, catalase failed to inhibit the PMN-induced rise in [Ca2+]i. Since polyanionic heparin inhibited the PMN sonicate rise in calcium, a cationic protein released by PMN was thought to be responsible. The cationic primary granule enzyme, cathepsin G, duplicates the rise in [Ca2+]i seen with PMN sonicates. Furthermore, PAF production increased threefold in response to neutrophil sonicates. Thus, during inflammation, when coagulation and inflammatory cells are activated the endothelium responds by priming PMNs and promoting margination.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1990

Iron loading of endothelial cells augments oxidant damage.

Transition metals, particularly iron, will potentiate oxidant damage to isolated cell organelles, plasma membranes, and DNA when added to in vitro incubation systems. However, similar studies of intact cells have been hampered by the relative impermeability of whole cells to iron. We have iron loaded cultured endothelial cells by using the iron-chelating fungistat 8-hydroxyquinoline (8HQ). 8HQ forms lipophilic chelates with iron and rapidly transfers the metal across the intact plasma membrane of endothelial cells. After brief exposure to 8HQ and subsequent thorough washing of endothelial cells, the cell-associated iron cannot be removed by the powerful chelator deferoxamine, clearly indicating the intracellular location of 8HQ-transported iron. Iron-loaded cells (but not cells exposed to high concentrations of 8HQ or iron separately) are extremely sensitive to oxidants (1) produced externally by phorbol-stimulated granulocytes, (2) generated intracellularly by menadione, or (3) added as H2O2. In the latter instance, as little as 7 mumol/L H2O2 provokes destruction of approximately 50% of iron-loaded endothelial cells, whereas untreated endothelium readily survives exposure to H2O2 concentrations as high as 2 mmol/L. Cytotoxicity is accompanied by membrane lipid peroxidation (formation of thiobarbituric acid-reactive substances). Both cytotoxicity and lipid peroxidation are inhibited by the lipophilic 21-aminosteroid U74500A ("lazaroid") (50% inhibitory concentration = approximately 0.5 mumol/L), whereas deferoxamine (250 mumol/L) is ineffective (suggesting iron intercalation into hydrophobic domains of the cell). We conclude that this pharmacologic model for iron loading of intact cells may yield valuable insights into the pathogenic importance of intracellular iron in iron overload states, inflammation, and cellular injury.

Animals↗
PubMed1990

Proinflammatory and procoagulant effects of herpes simplex infection on human endothelium.

Atherosclerotic lesions have been reported to contain herpes simplex virus (HSV) genomic material. This and other evidence suggests that latent viral infection may be an atherogenic trigger. Moreover, active HSV lesions manifest histologically marked fibrin deposition in microvessels. Our laboratory tested in vitro whether HSV infection would cause human umbilical vein endothelial cells to become procoagulant and attract inflammatory cells. Early infection of human endothelial cells with HSV-1 alters the surface conformation as detected by merocyanine 540 staining. The efficiency of prothrombinase complex assembly increases, resulting in a two- to threefold accelerated rate of thrombin generation on the cell surface of virally infected endothelium. HSV infection of endothelium results in a marked increase in thrombin-induced platelet adhesion with a concomitant decrease in prostacyclin secretion in response to thrombin. Viral infection enhances coagulation by decreasing endothelial thrombomodulin expression and subsequent activation of protein C. Viral infection also induces tissue factor in human endothelial cells within 4 hours of infection. Not only does the endothelial monolayer become procoagulant when infected with HSV, it also becomes a more adherent surface for granulocytes. Resting and stimulated granulocyte adherence is enhanced twofold on virally infected endothelium. Enhanced adhesion is accompanied by excessive granulocyte-mediated lysis of 51Cr-labeled HSV-infected endothelium and endothelial cell detachment from its substrate. Exaggerated endothelial detachment correlated with poor binding of infected endothelial cells to substratum matrix proteins. Resuspended virus-infected cells bound significantly less well to tissue culture containers coated with fibronectin, laminin, and type IV collagen. HSV-infected endothelium alters the anticoagulant properties of the endothelium causing it to become procoagulant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1989

Thrombin-treated endothelium primes neutrophil functions: inhibition by platelet-activating factor receptor antagonists.

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or "prime" polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginated PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10(-7) M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021, when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF--a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial/PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.

Endothelium, Vascular↗
PubMed1989

Impermeant stilbene disulfonic acids block chemotactic peptide receptor function on human granulocytes.

Anion transport is important in a variety of cell functions. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) are two impermeant agents that have been reported to specifically block the anion channel in erythrocytes. These agents block several responses of human neutrophils to stimulation by immune complexes, the synthetic chemotaxin N-formyl-met-leu-phe (FMLP), and a calcium ionophore. They also alter the function of C3b receptors on the neutrophil surface. We studied the effects of DIDS and SITS on the aggregation of human neutrophils, a process that has been implicated in a number of diverse clinical syndromes. Both DIDS and SITS inhibited granulocyte aggregation induced by FMLP, zymosan-activated plasma, 12-O-tetra-decanoylphorbolmyristate acetate (TPA), and the calcium ionophore A23187. To further study the mechanism of inhibition the effects of DIDS and SITS on FMLP-receptor function were tested. Similar concentrations of the anion channel blockers also inhibited binding of radiolabeled FMLP to its specific receptor on the neutrophil surface. Inhibition of binding was due to a decrease in both the number and affinity of the surface receptors available for FMLP; DIDS did not inactivate FMLP. The effects of stilbene disulfonic acid on cell function may be due to effects of these agents on other cell-surface structures in addition to the anion channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗