Endotoxin (ET) infusion into animals produces endothelial cell (EC) injury. It is not clear whether EC injury is produced by ET directly or by an ET-generated mediator, or whether ET-mediated EC injury also occurs in man and non-human primates. To answer these questions we examined the effects of ET on human umbilical vein (HUVEC) and bovine aortic (BAEC) endothelial cells in culture. ET (100 ug/ml) ± complement ± neutrophils produced no significant HUVEC Cr-release or cell detachment at 4 hours. HUVEC proliferation by direct cell count, angiotensin converting enzyme activity by labeled substrate conversion, VIII-Ag release by RIA and fibronectin release by ELISA were not affected by ET (100 ug/ml). ET alone did not induce significant release of PGI2 measured by bioassay or 6-keto-PGF. I α measured by RIA. In contrast to the lack of effect of ET on HUVEC, ET produced a time (3 hours: 22.0 ± 4.0%; 6 hours: 40.0 ± 6.0%; 24 hours: 71.0 ± 5.0%) and dose (100 pg/ml: 25.4 ± 2.5%; I ug/ml: 83.0± 4.0%; 10 ug/ml: 92.0 ± 3.0%) dependent BAEC detachment which was initially sublethal without 51 Cr-release or trypan blue dye uptake (40.6 ± 6.0% detachment, <1% Cr-release at 6 hours with ET 10 ug/ml). BAEC detachment was seen with all ET preparations including lipid A. ET-mediated BAEC detachment was inhibited by incubation at 4° but not by indomethacin, methyl prednisolone, or chi or promazine and was not dependent on serum or complement. Examination of ET-detached BAEC by SDS-PAGE after 125I- surface labeling or by immunofluorescence using anti-fibronectin antibody revealed complete loss of cell surface fibronectin. ET- mediated detachment was also seen with bovine mesenteric EC but not with bovine aortic smooth muscle cells or monkey aortic EC. These studies demonstrate the importance of studying EC from appropriate species and vascular sites. Although ET has no cytotoxic effect on HUVEC, it does induce selective, sublethal BAEC detachment associated with loss of cell surface fibronectin. If a similar effect occurs in human adult arterial or microvascular EC, it would have profound pathophysiologic effects accounting for the permeability changes and thrombosis seen in sepsis in man.
Direct Cell Surface Modification with DNA for the Capture of Primary Cells and the Investigation of Myotube Formation on Defined Patterns