Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

Abstract

Several studies suggested that long-term nitrate therapy may produce negative outcomes in patient mortality and morbidity. A possible mechanism may involve nitrate-mediated activation of various extracellular matrix (ECM) proteases, particularly matrix metalloproteinase-9 (MMP-9), and adhesion molecules in human macrophages, leading to the destabilization of atherosclerotic plaques. We examined the gene and protein regulating effects on THP-1 human macrophages by repeated exposure to therapeutically relevant concentrations of nitroglycerin (NTG) and possible involvement of nuclear factor (NF)-κB signaling mechanism in mediating some of these observed effects. THP-1 human macrophages repeatedly exposed to NTG (at 10 nM, added on days 1, 4 and 7) exhibited extensive alterations in the expression of multiple genes encoding ECM proteases and adhesion molecules. These effects were dissimilar to those produced by a direct nitric oxide donor, diethylenetriamine NONOate. NTG exposure significantly up-regulated NF-κB DNA nuclear binding activity and MMP-9 protein expression, and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression; these effects were abrogated in the presence of the NF-κB inhibitor parthenolide (a chemical inhibitor derived from the feverfew plant). Further, we examined whether our in vitro findings (an elevated MMP-9/TIMP-1 ratio and gelatinase activity) can be translated to in vivo effects, in a rat model. Sprague-Dawley rats exposed continuously to NTG subcutaneously for 8 days via mini-osmotic pumps showed significant induction of plasma MMP-9 dimer concentrations and the expression of a complex of MMP-9 with lipocalin-2 or neutrophil gelatinase associated lipocalin (NGAL). Plasma gelatinase activity was significantly increased by NTG over the entire study period, attaining peak elevation at day 6. Plasma TIMP-1 protein was down-regulated significantly by day 2 and days 4 to 7 in the NTG-treated rats. Pharmacokinetic monitoring of NTG and its dinitrate metabolites indicated that concentrations were well within therapeutic levels observed in humans. Our studies indicate that clinically relevant concentrations of NTG not only altered ECM matrix by changing the expression of multiple genes that govern cellular integrity, affecting cellular MMP-9/TIMP-1 balance in THP-1 human macrophages possibly via NF-κB activation, but also led to systemic changes in MMP-9/ TIMP-1 expression and gelatinase activity in rats. These effects may contribute to extracellular matrix degradation and possible atherosclerotic plaque destabilization.

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

“…These same authors also showed that the NO donor DETA/NO promoted sulfonic acid modifications of the cysteine‐switch of pro‐MMP‐9 (Krishnatry et al . ,b). Collectively, these reports support a primary role for NO in regulation of MMP‐9 proteolytic activity.…”

“…; Krishnatry et al . ). To better understand the relationship between NO, MMP activity and amyloid degradation, we have used both in vitro and in vivo approaches.…”

“…Our laboratories and others have demonstrated that nitric oxide (NO) regulates MMP-9 activity and the activity of its endogenous inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 at both the mRNA and protein levels. At low concentrations, NO increased MMP-9 activity in a guanylyl cyclase-dependent manner through TIMP-1 protein suppression, while at high concentrations, NO directly activated and inactivated the enzyme, which was reactive nitrogen species dependent (Death et al 2002;Ridnour et al 2007;Weiss et al 2010;Krishnatry et al 2011a). To better understand the relationship between NO, MMP activity and amyloid degradation, we have used both in vitro and in vivo approaches.…”

Organic Nitrate Metabolism and Action: Toward a Unifying Hypothesis and the Future—A Dedication to Professor Leslie Z. Benet

“…In cultured endothelial cells, it was demonstrated that NTG exposure increased S‐glutathionylation and activity of p21ras, which is integral to the signaling of the AKT and extracellular signal‐regulated kinase 1/2 (ERK 1/2) pathways, which play a part in the regulation of smooth muscle cell proliferation 100. NTG incubation led to a significant increase in the nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB p50/p65) activity in differentiated human monocytic leukemia cells,101 possibly through nuclear factor kappa‐B kinase subunit alpha (IKK‐α) activation via its cysteine‐179 residue.…”

“…This cap can degrade and weaken, ultimately leading to rupture of the plaque, platelet adhesion, and thrombus and ultimately resulting in infarction. We have shown that NTG exposure, both in vitro and in vivo , can activate matrix metalloproteinases (MMPs), which degrade components of the extracellular matrix, leading to weakening of the fibrous cap 101,116. The mechanism of the activation of an MMP by NTG involves the oxidation of the “cysteine switch,”117 which converts the inactive pro‐MMP to its active protein.…”