Abha Sahni1, Sanjeev K Sahni, Charles W Francis. 1. Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY, USA. Abha_Sahni@urmc.rochester.edu
Abstract
OBJECTIVE: The purpose of this study was to investigate the receptor requirements for enhanced IL-1beta-induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen. METHODS AND RESULTS: ECs were exposed to IL-1beta with or without fibrinogen and NO was measured as nitrite. NO production by EC exposed to fibrinogen (0.3+/-0.1 micromol/L) was comparable concentration to control (0.2+/-0.1 micromol/L), but IL-1beta significantly increased NO production (0.8+/-0.1 micromol/L), and the combination of both fibrinogen and IL-1beta resulted in a further increase to 2.2+/-0.2 micromol/L (P<0.002). 7E3 or LM609, antibodies to alphavbeta3, inhibited NO production stimulated by fibrinogen-bound IL-1beta to 0.2+/-0.1 micromol/L (P<0.001) or 0.2+/-0.03 micromol/L (P<0.0001), respectively. These levels were comparable to control and significantly less than with IL-1beta (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1beta, but not vitronectin and IL-1beta, demonstrated positive Western blotting for alphavbeta3 after immunopurification with anti- IL-1R, indicating specific association between alphavbeta3 and IL-1R. Dual immunofluorescence also revealed colocalization of alphavbeta3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1beta. CONCLUSIONS: The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1beta requires the coordinated effects of colocalized alphavbeta3 and IL-1R.
OBJECTIVE: The purpose of this study was to investigate the receptor requirements for enhanced IL-1beta-induced secretion of nitric oxide (NO) by endothelial cells (ECs) in the presence of fibrinogen. METHODS AND RESULTS: ECs were exposed to IL-1beta with or without fibrinogen and NO was measured as nitrite. NO production by EC exposed to fibrinogen (0.3+/-0.1 micromol/L) was comparable concentration to control (0.2+/-0.1 micromol/L), but IL-1beta significantly increased NO production (0.8+/-0.1 micromol/L), and the combination of both fibrinogen and IL-1beta resulted in a further increase to 2.2+/-0.2 micromol/L (P<0.002). 7E3 or LM609, antibodies to alphavbeta3, inhibited NO production stimulated by fibrinogen-bound IL-1beta to 0.2+/-0.1 micromol/L (P<0.001) or 0.2+/-0.03 micromol/L (P<0.0001), respectively. These levels were comparable to control and significantly less than with IL-1beta (P<0.002). EC or fibroblasts exposed to both fibrinogen and IL-1beta, but not vitronectin and IL-1beta, demonstrated positive Western blotting for alphavbeta3 after immunopurification with anti- IL-1R, indicating specific association between alphavbeta3 and IL-1R. Dual immunofluorescence also revealed colocalization of alphavbeta3 and IL-1R only when the cells were exposed to both fibrinogen and IL-1beta. CONCLUSIONS: The enhanced NO production by ECs in the presence of fibrinogen-bound IL-1beta requires the coordinated effects of colocalized alphavbeta3 and IL-1R.
Authors: Natalie A Drucker; Amanda R Jensen; Jan P Te Winkel; Michael J Ferkowicz; Troy A Markel Journal: J Pediatr Surg Date: 2018-03-08 Impact factor: 2.545