Endothelin-1 increased immunoreactive von Willebrand factor in endothelial cells and induced micro thrombosis in rats

This study was performed (i) to investigate the interaction between ET-1 and endothelial cells and (ii) to study the role of ET-1 in in vivo thrombosis. Fura-2AM loaded human umbilical endothelial cell cultures were incubated with 0, 25, 50 and 100 pmol of ET-1 for 24 hours (n = 6) at 37 degrees C. Fura-2 released in the media was measured by spectroflurophotometer at wavelength of 350 nm excitation and 500 nm emission. We found significant (p < 0.01) and dose dependent decrease in Fura-2 release by the cells indicating increased intracellular calcium in HUVEC. Increased calcium by ET-1 was also confirmed at single cell level by fluorescence digital image analysis using Fura-2AM. 5 ml solution of ET-1 (100 pmol/ml) was injected within the venous lumen of umbilical cords (of normal pregnancy) clumped at both ends and incubated at a temperature 37 degrees C for 3 hours (n = 7). We found intensely stained immunoreactive von Willebrand factor (vWF) on the endothelial cells of ET treated umbilical cords when compared with sham control (Umbilical cords incubated with phosphate buffer saline; n = 7). Intravenous ET-1 infusions at a rate of 1 nmol/kg/hour for 2 hours (cases, n = 7) and 5% dextrose infusions (sham control, n = 7) were performed in rats. Aorta, kidney and liver tissues were obtained to perform immunostaining with polyclonal antibody to vWF and fibrinogen. ET-1 treated rat tissues showed intense staining for vWF and fibrinogen intravascularly at hte same site.(ABSTRACT TRUNCATED AT 250 WORDS)