Complement-Mediated Loss of Endothelium-Dependent Relaxation of Porcine Coronary Arteries

Abstract

Abstract Reperfusion of the ischemic myocardium results in the loss of endothelium-dependent relaxation. We have shown recently that the alternate complement pathway is activated immediately on reperfusion of the ischemic porcine myocardium. We hypothesized that complement activation directly attenuates endothelium-dependent relaxation of porcine coronary arteries. Bradykinin (BK) or substance P concentration-dependently relaxed precontracted (U46619, 50 nmol/L) left anterior descending coronary artery (LAD) rings in vitro. Addition of zymosan to human (10%) or porcine (10%) serum for 30 minutes significantly ( P <0.05) increased the EC 50 of BK-induced LAD relaxation from 4±1 to 418±159 nmol/L (n=8) and from 9±3 to 281±132 nmol/L (n=7), respectively. Similarly, addition of zymosan to 10% human serum (HS) for 30 minutes increased the EC 50 of substance P–induced LAD relaxation from 0.4±0.1 to 30±14 nmol/L (n=9, P <.05). Basal release of nitric oxide was reduced significantly in LAD rings exposed to zymosan-activated HS compared with HS alone. Addition of soluble CR1 (sCR1, 10 nmol/L) to zymosan-activated HS preserved BK-induced relaxation (EC 50 ) of the LAD rings (control, 4±1 nmol/L; sCR1+zymosan+serum, 2±1 nmol/L; n=6). Zymosan-activated C8-depleted HS (10%) did not attenuate the EC 50 of BK-induced coronary artery relaxation (3±1 to 3±1 nmol/L, n=7, P =NS). Zymosan-activated C8-depleted HS plus C8 (6 μg/mL) increased the EC 50 of BK-induced coronary artery relaxation from 4±1 to 423±141 nmol/L (n=12, P <.05). We have further demonstrated that C5b-9 complexes can be found on the luminal surface of LAD endothelial cells after 5 minutes of exposure to zymosan-activated HS by using C5b-9 reactive monoclonal antibody fluorescent immunohistochemistry and confocal microscopy. We conclude that complement activation directly attenuates endothelium-dependent relaxation through the formation of the terminal membrane attack complex (C5b-9).