Objective-Activated polymorphonuclear neutrophils (PMNs) are the main source of circulating neutral endopeptidase (NEP). We tested the hypothesis that NEP inhibition could potentiate the effect of atrial natriuretic peptide (ANP) on PMN-vascular cell interactions in vitro. Methods and Results-ANP alone and its potentiation by retrothiorphan, the NEP inhibitor, significantly inhibited superoxide, lysozyme, and matrix metalloproteinase (MMP)-9 release by N-formyl-Met-Leu-Phe-stimulated PMNs. Activated PMNs degraded exogenous ANP, which was prevented by NEP inhibition. Hypoxia significantly increased the adhesion of PMNs to endothelial cells and their subsequent MMP-9 release by 60% and 150%, respectively (PϽ0.01). ANP and its potentiation by retrothiorphan limited PMN adhesion to hypoxic endothelial cells and thus decreased their MMP-9 release (PϽ0.01). Smooth muscle cells (SMCs) incubated with conditioned medium of N-formyl-Met-Leu-Phe-stimulated PMNs exhibited morphological and biochemical changes characteristic of apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling positivity, nuclear condensation/fragmentation, poly ADP-ribose polymerase cleavage, and DNA laddering). SMC detachment and subsequent apoptosis could be related to leukocyte elastase-induced pericellular proteolysis, inasmuch as both events are inhibited by elastase inhibitors. ANP and its potentiation by retrothiorphan were able to limit elastase release, fibronectin degradation, and SMC apoptosis. with the use of leukotriene B4 as a trigger. In contrast, Matsumura et al 8 showed that ANP and its potentiation by NEP inhibition increased intracellular cGMP, which limited protease release by PMNs.Therefore, we hypothesized that the potentiation of ANP by NEP inhibition could limit PMN-vascular cell interactions by acting predominantly on PMNs. For this purpose, we have developed an in vitro approach testing the ability of ANP potentiation by NEP inhibition (1) to inhibit PMN protease release, (2) to limit PMN-endothelial cell interactions in hypoxic conditions, and (3) to limit the pericellular proteolysis induced by activated PMN secretions.
Methods
Cell CulturesHuman umbilical vein endothelial cells (HUVECs) were harvested and cultured as described. 12 Human smooth muscle cells (SMCs) were obtained from venous wall explants after removal of the adventitia and were cultured. 13