John M. Harlan

Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques. Implications for the mode of progression of advanced coronary atherosclerosis.

Endothelial attachment is the initial step in leukocyte recruitment into developing atherosclerotic lesions. To determine whether vascular cell adhesion molecule-1 (VCAM-1) expression may play a role in inflammatory cell recruitment into human atherosclerotic lesions, immunohistochemistry was performed with a polyclonal rabbit antisera, raised against recombinant human VCAM-1, on 24 atherosclerotic coronary plaques and 11 control coronary segments with nonatherosclerotic diffuse intimal thickening from 10 patients. Immunophenotyping was performed on adjacent sections to identify smooth muscle cells, macrophages, and endothelial cells. To confirm VCAM-1-expressing cell types, double immunostaining with VCAM-1 antisera and each of the cell-specific markers and in situ hybridization were performed. All atherosclerotic plaques contained some VCAM-1, compared to 45% of control segments. VCAM-1 was found infrequently on endothelial cells at the arterial lumen din both plaques (21%) and in control segments (27%), but was prevalent in areas of neovascularization and inflammatory infiltrate in the base of plaques. Double immunostaining and in situ hybridization confirmed that most VCAM-1 was expressed by subsets of plaque smooth muscle cells and macrophages. The results document the presence of VCAM-1 in human atherosclerosis, demonstrate VCAM-1 expression by human smooth muscle cells in vivo, and suggest that intimal neovasculature may be an important site of inflammatory cell recruitment into advanced coronary lesions.

Homocysteine-induced endothelial cell injury in vitro: A model for the study of vascular injury

Direct chemical injury to vascular endothelium was determined in vitro by measuring independently cell detachment and release of 51Cr from labeled human endothelial cell monolayers. Homocysteine, a sulfhydryl amino acid, induced specific 51Cr release from endothelial cells in direct proportion to its concentration between 0.1 and 10 mM. The proportion of detached cells during exposure to homocysteine was also directly related to the concentration of homocysteine. In vitro preincubation of homocysteine resulted in a progressive loss of cytotoxicity with no activity persisting after 24 hours. Mercaptoethanol, a sulfhydryl agent similar to homocysteine, also induced endothelial injury in a concentration dependent manner. Penicillamine prevented the homocysteine mediated 51Cr-release at equimolar concentrations (p< 0.001). Catalase also inhibited the sulfydryl injury (90% reduction in 51Cr release at 0.1 mM homocysteine), but superoxide dismutase had no effect, thereby suggesting a role for hydrogen peroxide in mediating injury. Neither homocystine nor methionine, produced cell injury. Human arterial smooth muscle cells were insensitive to homocysteine levels less than 25 mM. These data demonstrate homocysteine-induced endothelial injury in vitro, and suggest that this process may in part be sulphydryl-mediated.

A monoclonal antibody to the adherence-promoting leukocyte glycoprotein, CD18, reduces organ injury and improves survival from hemorrhagic shock and resuscitation in rabbits.

Leukocytes have been shown to play an important role in the development of isolated organ injury after experimental ischemia and reperfusion. To examine the role of leukocytes in generalized ischemia-reperfusion injury we used the MAb 60.3 (directed to the human leukocyte adherence glycoprotein, CD18) to block leukocyte adherence functions in a rabbit model of hemorrhagic shock and resuscitation. In control animals subjected to 1 h of shock (mean blood pressure 45 torr and mean cardiac output 30% of baseline) followed by resuscitation, only 29% survived 5 d. All had gross and histologic evidence of injury to lungs, liver, and gastrointestinal mucosa. In contrast, 100% of the MAb 60.3-treated animals survived 5 d (P less than 0.01) and organ injury was absent or markedly attenuated. The control animals also had a persistent acidosis, lost more weight, and had evidence of continued gastrointestinal bleeding in contrast to MAb 60.3-treated animals. We conclude that increased leukocyte adhesiveness plays an important role in the development of multiple organ injury and death after generalized ischemia-reperfusion and that this injury may be significantly reduced by blocking leukocyte adherence functions with the MAb 60.3.

Neutrophil-mediated endothelial injury in vitro mechanisms of cell detachment.

Neutrophil-mediated endothelial injury was assessed in vitro using assays of cell lysis and cell detachment. Activation of human peripheral blood neutrophils adherent to human umbilical vein endothelial cell monolayers by serum-treated zymosan produced dose-dependent endothelial cell detachment without concomitant cell lysis. This injury was inhibited by neutral protease inhibitors, but not by catalase or superoxide dismutase. Neutrophils from a patient with chronic granulomatous disease also produced endothelial cell detachment when activated by serum-treated zymosan similar to normal neutrophils. Endothelial detachment was also produced by cell-free postsecretory media from activated neutrophils or by partially purified human neutrophil granule fraction and was inhibitable by tryptic, elastase, and serine protease inhibitors, but not by an acid protease inhibitor. Analysis of iodinated endothelial cell surface proteins that had been exposed to partially purified neutrophil granule fraction showed complete loss of proteins migrating in the region of fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This result was prevented in the presence of neutral protease inhibitors. We conclude that neutrophil-derived neutral proteases mediate endothelial cell detachment in vitro through digestion of endothelial cell surface proteins including fibronectin.

Outcome after hemorrhagic shock in trauma patients.

BACKGROUND It is essential to identify patients at high risk of death and complications for future studies of interventions to decrease reperfusion injury. METHODS We conducted an inception cohort study at a Level I trauma center to determine the rates and predictors of death, organ failure, and infection in trauma patients with systolic blood pressure < or = 90 mm Hg in the field or in the emergency department. RESULTS Among the 208 patients with hemorrhagic shock (blood pressure < or = 90 mm Hg), 31% died within 2 hours of emergency department arrival, 12% died between 2 and 24 hours, 11% died after 24 hours, and 46% survived. Among those who survived > or = 24 hours, 39% developed infection and 24% developed organ failure. Increasing volume of crystalloid in the first 24 hours was strongly associated with increased mortality (p = 0.00001). CONCLUSION Hemorrhage-induced hypotension in trauma patients is predictive of high mortality (54%) and morbidity. The requirement for large volumes of crystalloid was associated with increased mortality.

Glutathione redox cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide.

We have examined the role of the glutathione redox cycle as an antioxidant defense mechanism in cultured bovine and human endothelial cells by disrupting the glutathione redox cycle at several points. Endothelial glutathione reductase was selectively inhibited with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU). Cellular stores of reduced glutathione were depleted by reaction with diethylmaleate (DEM) or 1-chloro-2,4-dinitrobenzene (CDNB) or by inhibition of glutathione synthesis with buthionine sulfoximine (BSO). Whereas several strains of untreated bovine and human endothelial cells were resistant to lysis by enzymatically generated hydrogen peroxide, BCNU-treated cells were readily lysed in a time- and dose-dependent manner. Glucose-glucose oxidase-mediated lysis of BCNU-treated bovine endothelial cells was catalase-inhibitable and directly related to BCNU concentration and endogenous glutathione reductase activity. Pretreatment of bovine endothelial cells with BCNU did not potentiate lysis by distilled water, calcium ionophore, lipopolysaccharide, or hypochlorous acid. Depletion of cellular reduced glutathione by reaction with DEM or CDNB or by inhibition of glutathione synthesis by BSO also potentiated endothelial lysis by enzymatically generated hydrogen peroxide. Inhibition of endothelial glutathione reductase by BCNU or depletion of reduced glutathione by BSO increased endothelial susceptibility to lysis by hydrogen peroxide generated by phorbol myristate acetate-activated neutrophils. We conclude that the glutathione redox cycle plays an important role as an endogenous antioxidant defense mechanism in cultured endothelial cells.

Selectins: a family of adhesion receptors.

Recent data have shown that a group of cell surface proteins, originally studied independently as lymphocyte homing receptors or as activation-induced surface proteins of platelets and/or endothelial cells (Stoolman, 1989) are structurally related. Each is an integral membrane protein with an N-terminal, C-type lectin domain followed by an EGF-like module, multiple copies of the consensus repeat units characteristic of complement-binding proteins, a transmembrane segment, and a short cytoplasmic domain. The three known proteins having this structure are encoded by closely linked genes on the long arm of human and mouse chromosome 1 (Watson et al., 1990). The gene structures are related, and the genes clearly arose by gene duplication. These proteins are all involved in cell-cell adhesion events and constitute a new family of cell adhesion receptors. A wide variety of names are used to designate these proteins, owing to their independent discovery by different laboratories working in several fields. This diversity of nomenclature interferes with the dissemination of information about these proteins. After consultation among the researchers working on these proteins and other scientists, we propose that this family of proteins be named selectins to reflect the involvement of carbohydrate recognition in their functions. Individual members of the family will be designated by a prefix capital letter, as is done for the cadherins (e. g., E-, N-, P-). Letters can be chosen based on the source of the original discovery but are not intended to imply cell type specificity. The three known selectins are: L-selectin

Endothelial cell death induced by tumor necrosis factor-alpha is inhibited by the Bcl-2 family member, A1.

Endothelial cells play a central role in the inflammatory process. Tumor necrosis factor-α (TNF) is a multifunctional cytokine which elicits many of the inflammatory responses of endothelial cells. While TNF directly causes apoptosis of tumor cells and virally infected cells, normal cells are generally resistant. However, most resistant cells, including human endothelial cells, can be rendered susceptible to TNF by inhibiting RNA or protein synthesis. This finding suggests that TNF provides a cell survival signal in addition to a death signal. We have previously cloned a human Bcl-2 homologue, A1, and shown that it is specifically induced by proinflammatory cytokines but not by endothelial growth factors. In this study, we show that retroviral-mediated transfer of the A1 cDNA to a human microvascular endothelial cell line provides protection against cell death initiated by TNF in the presence of actinomycin D. The induction of A1 by TNF in this system is mediated via a protein kinase C pathway. Since TNF signaling has also been shown to proceed via ceramides, we tested whether exogenous ceramides could induce A1. Our findings indicate that ceramides do not induce A1 but do up-regulate c-jun and induce endothelial death. Ceramide-activated endothelial death is also inhibited by A1, suggesting that TNF may initiate divergent survival and death pathways via separate lipid second messengers.

Effect of sulfinpyrazone on homocysteine-induced endothelial injury and arteriosclerosis in baboons.

The effect of sulfinpyrazone on endothelial injury induced by homocysteine has been studied both in vitro, using cultured human umbilical vein endothelial cells, and in vivo, using a primate model of homocysteine-induced arteriosclerosis. Oral sulfinpyrazone (250 mumol/kg body weight per day in three divided doses) in eight chronically homocystinemic baboons (0.14 +/- 0.04 mM plasma homocystine) decreased the extent of aortic endothelial injury as measured morphometrically by silver staining techniques, compared with six untreated comparably homocystinemic animals (denuded surface averaged 0.5% with range 0-2.1 vs 7.7 +/- 1.6%, respectively; P less than 0.001). Sulfinpyrazone therapy to homocystinemic baboons also normalized platelet survival and turnover measurements (5.1 +/- 0.4 days and 70,000 +/- 11,000 platelets/microliter per day vs. 2.8 +/- 0.6 days and 179,000 +/- 19,000 platelets/microliter per day in untreated homocystinemic controls; P less than 0.001). Sulfinpyrazone therapy also reduced the size and frequency of homocysteine-induced intimal lesion formation (P less than 0.001). Although sulfinpyrazone reduced the amount of specific 51Cr release from cultured human umbilical vein endothelial cells induced by 10 mM homocysteine after 24 hours of co-incubation, no effect was observed in assays of endothelial cell detachment when sulfinpyrazone (10(-5) M) or its thioether metabolite were pre- or co-incubated during 24 hours with homocysteine (2.5-10 mM). These data suggest that sulfinpyrazone may protect endothelial cells from injury in vivo by some apparently indirect mechanism.

Leukocyte-endothelial interactions: clinical trials of anti-adhesion therapy.

ObjectiveThis review describes efforts to develop therapies directed at leukocyte and endothelial adhesion molecules for the treatment of acute and chronic inflammatory diseases, including hemorrhagic shock. Data Sources and Study SelectionPublished research and review articles and Web sites relating to the clinical use of drugs directed to leukocyte or endothelial cell adhesion molecules. Data Extraction and SynthesisThe results of relevant studies of adhesion blockade are reviewed. Trials in putative clinical ischemia-reperfusion disorders, particularly traumatic shock, are emphasized. Trials are designated as positive or negative, depending on whether the primary end points established by the trial investigators were met. ConclusionsBlockade of leukocyte adhesion to endothelium by monoclonal antibodies or other antagonists has been demonstrated to reduce vascular and tissue injury in a wide variety of animal models of inflammatory and immune disease. Anti-adhesion therapy directed at lymphocyte trafficking has shown efficacy in several phase 2 and 3 clinical trials in inflammatory bowel disease, multiple sclerosis, and psoriasis. Despite strong preclinical data, results of phase 2 and 3 trials of neutrophil adhesion blockade in putative ischemia-reperfusion disorders—stroke, myocardial infarction, and hemorrhagic shock—have been disappointing.