Human Endothelial Cell Injury Mechanisms in Vitro

Since endothelial cell desquamation constitutes the initial event leading to acute and chronic vascular diseases, we have developed an in vitro system for studying injury mechanisms using cultured cells. The system involves the incubation of test material with confluent, 51Cr-labeled, cultured human umbilical vein endothelial cells in 100% pooled human serum; injury was measured as% endothelial cell 51Cr release (ECR) into the supernatant media. Baseline spontaneous ECR was 6.0% ± 1.5 while specific rabbit antihuman endothelial cell antibody (1:64 dilution) in the presence of fresh complement released 90% ± 3 of the total releasable 51Cr activity. Consistent with in vivo predictions, homocysteine in concentrations of 0.5-40 mM iduced dose response ECR up to 20%. Neither methionine nor homocystine increased ECR over controls. Also as expected from experimental work endotoxin (S. enteritidis) caused dose response ECR, i.e., 24% ± 2 at 10 ug/ml. Control studies with cultured human smooth muscle cells (SMC) demonstrated no ECR with homocysteine but significant release occurred with endotoxin. Specific complement dependent cytotoxic anti-endothelial cell antibody was demonstrated in the secum of two thrombotic thrombocytopenic purpura (TTP) patients at a 1:1 dilution, inducing ECR of 23.1% ± 1.4 and 21.0% ± 0.25. The antibody was also demonstrated by immunoflourescent techniques and was absorbed from the serum using human endothelial cells. One disease-free, six month survivor showed no cytotoxic activity. Serum from a patient with adult hemolytic-uremic syndrome demonstrated antibody dependent cell mediated cytotoxicity with release of 22% ± 1.1 when normal non-immune lymphocytes were added to heat inactivated seriun. Control studies with SMC showed no ECR with TTP sera. We conclude that assays of endothelial cell 51Cr release are stable, reproducible and useful in the characterization of injury mechanisms.