Pharmacological Potentiation of Natriuretic Peptide Limits Polymorphonuclear Neutrophil-Vascular Cell Interactions

Abstract

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

Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus

“…It has been recently shown that elastase released by activated PMNs is able to degrade fibronectin 23,39 and provoke SMC anoikis. 23,39 In the present Preincubation of the fibrin matrix with activated PMN releasates (n ϭ 5), luminal extracts (n ϭ 5), or purified elastase impaired BMSCs adhesion, spreading, and growth on the fibrin gel. Cell detachment was prevented by elastase inhibitors ␣ 1 -antitrypsin and SLPI.…”

“…23 Cells were maintained in HBSS. The viability, measured by the release of lactate dehydrogenase (LDH) 29 and purity of the final preparation were greater than 99 and 95%, respectively.…”

“…Because leukocyte elastase released from activated PMNs is able to degrade fibronectin, 23,39 Figure 2E 2, and 3) showed the presence of proteolytic degradation products of fibronectin: column 4, purified fibronectin; column 5, purified fibronectin preincubated with activated PMN-conditioned medium (500 l of PMN-conditioned medium (10 6 PMN/ml for 1 g of fibronectin, 1 hour of incubation at 37°C)) serve as non-degraded and degraded fibronectin controls, respectively.…”

“…As we have previously demonstrated that leukocyte elastase released by activated PMNs induced anoikis of SMCs, 23 the hypothesis that extracts from luminal thrombus containing leukocyte elastase can induce a similar phenomenon was tested. Incubation of cultured SMCs with extracts of luminal thrombus induced cell retraction and detachment ( Figure 3B) which did not occur with extracts of the abluminal part of the thrombus or with control buffer.…”

“…22 In parallel, it has been reported that elastase released by PMN degranulation is able to induce loss of cell anchorage and apoptosis (anoikis) of cultured adherent cells. 19,23 It thus appears probable that elastase released by PMNs trapped within the fibrin matrix may play a role in preventing the colonization of the mural thrombus by mesenchymal cells. To test this hypothesis, we analyzed first the release and storage of neutrophil proteases by human aneurysmal thrombi ex vivo.…”