Attenuation of hemolysate-induced cerebrovascular endothelial cell injury and of production of endothelin-1 and big endothelin-1 by an endothelin-converting enzyme inhibitor

Abstract

BACKGROUND Endothelin-1 (ET-1) is a potent and long-acting vasoconstrictive peptide that has been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). ET-1 has been shown to be present in the cerebrospinal fluid of patients after SAH, and substances produced during hemolysis of subarachnoid blood clots are believed to be responsible for stimulating the production of ET-1. The biosynthesis of ET-1 is a multi-step process, involving the conversion of the relatively inactive precursor big ET-1 to the mature peptide by endothelin converting enzyme (ECE), a metalloprotease. Consequently, ECE inhibitors are expected to suppress the biosynthesis of ET-1 and reduce the pathologic impact resulting from overproduction of this peptide. The purpose of the present study was to investigate the effects of an ECE inhibitor, CGS 26303, on hemolysate-induced injury of cerebral vessel endothelial cells as well as the production of ET-1 from these cells. METHODS Different doses of CGS 26303 and hemolysate were added to the culture medium for 48 hours. Cell injury was assessed by cell morphology and density, while the productions of ET-1 and big ET-1 were determined by radioimmunoassays. RESULTS Hemolysate alone increased the levels of ET-1 and big ET-1 in culture medium and caused substantial cell loss. Treatment with CGS 26303 inhibited the hemolysate-induced increases in the levels of ET-1 and big ET-1 and reduced endothelial cell injury. The protective effects of CGS 26303 were modest when this inhibitor was added simultaneously with hemolysate, but were prominent and dose-dependent when the inhibitor was given 30 minutes before the addition of hemolysate. CONCLUSION These results suggest that overproduction of ET-1 contributes significantly to hemolysate-induced damage to cerebrovascular endothelial cells.