Matrix metalloproteinase-9 regulation by nitroglycerin

Several recent studies have shown that long-term nitrate therapy may produce no benefits, or even negative outcomes, in mortality and morbidity. A possible mechanism involves the activation of matrix metalloproteinases (MMPs) by NTG, thereby destabilizing atherosclerotic plaques. This thesis was formulated to systemically examine the regulating effects of NTG on the expression of various adhesion molecules and extracellular (ECM) proteases, particularly MMP-9 and tissue inhibitor of metalloproteniases-1 (TIMP-1).Using THP-1 human macrophages, we showed that NTG exerted broad effects on the gene expression of these molecules. MMP-9 expression and activity were significantly up-regulated under both acute and repeated exposure conditions, the latter at therapeutically relevant NTG concentrations. The effects of NTG on MMP-9/TIMP-1 balance were found to be mediated significantly via the NF-KB signaling pathway. Direct chemical activation of MMP-9 by NTG and its dinitrate metabolites was observed. We found NTG to be a poorer S-nitrosylating agent of the cysteine switch (studied as a synthetic peptide) than other nitric oxide donors, but it oxidized this critical peptide sequence in peptides and proteins to form various S-oxidized species such as sulfenic, sulfinic, and sulfonic acids. The formation of a thionitrate intermediate between peptide and protein cysteine residues and NTG has been demonstrated for the first time.In vivo studies carried out in Sprague-Dawley rats implanted with NTG-loaded mini osmotic pumps showed that NTG dosing over 8 days activated plasma gelatinase activity in vivo, altered systemic MMP-9/TIMP-1 balance, and induced the expression of a complex of MMP-9 with lipocalin-2 (a NF-KB inducible protein). Pharmacokinetic analysis of NTG and dinitrate metabolite data indicated that the bioavailability of NTG released from osmotic pumps was low (about 6%).In summary, this thesis work shows for the first time that clinically relevant concentrations of NTG alter the expression and activity of matrix degrading proteases in vitro and in vivo. This action may represent a potential mechanism for the lack of long-term beneficial effects observed after nitrate therapy. It is hoped that this work will stimulate further studies that will eventually lead to further improvement of long-term nitrate therapy in future.