Effect of Metalloprotease Inhibition on Age-Associated Arterial Remodeling

National Institute On Aging

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Abstract

Age-associated arterial remodeling involves changes to the arterial wall including collagen deposition, elastin fragmentation, amyloidosis as well as an increase in arterial pressure. This adverse arterial remodeling is linked to proinflammatory signaling, including milk fat globule EGF-8 (MFG-E8) and its fragment medin, transforming growth factor-beta1 (TGF-1), monocyte chemoattractant protein 1 (MCP-1), and proendothelin 1 (pro-ET1), activated by extracellular MMPs and orchestrated, in part, by the transcriptional factor, ETS-1. We tested the hypothesis that inhibition of MMP activation can decelerate age-associated arterial adverse remodeling and its increase in arterial pressure. Indeed, chronic administration (8 months) of the broad-spectrum MMP inhibitor, PD166793, via a daily gavage, to 16-month-old FXBN rats markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) inhibition of age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of MCP-1 and TGF- 1 activity; (5) a diminution in the phosphorylation activity of the profibrogenic signaling molecule SMAD-2/3; (6) inhibition of pro-ET1 activation; and (7) a downregulation of the expression of ETS-1. In addition, our in vitro study shows that an exposure of cultured vascular smooth muscle cells (VSMC)to pro-ET1 increased both the transcription and translation levels of ETS-1, and these effects were markedly reduced with MMP inhibition. Furthermore, infection of VSMCs with an adenovirus harboring a full-length ETS-1 cDNA increased levels of activated forms of both TGF-1 and MCP-1 proteins. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by preservation of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure. Caloric restriction (CR), mimicking the effects of MMP inhibition, significantly improves arterial health. Immunostaining of the aortic wallsshows that the number of intimal VSMCs was increased in old rats when compared to young rats fed ad libitum (AL), but was substantially reduced in the CR rat with aging. The intima-medial collagen deposition was increased, and the elastin fraction was decreased in the aortic walls of old AL rats. Impressively, aortic collagen and elastin fibers did not significantly change in the CR rat during aging. We found that age enhanced in situ MMP-2 and MCP-1 activation within the aortic wall of AL rats, but these were blunted in the aorta of the old CR rats. Additionally, TGF-1, a potent pro-fibrogenic cytokine, and a product of MMP-2 cleavage, and its downstream signaling molecule, p-SMAD-2, was enhanced in old rats when compared to young AL rats, but CR reduced this effect. The protein milk fat globule EGF 8 (MFG-E8) and its fragment, medin, were also increased in old AL rats determined by immunoblotting when compared to young AL rats, but this was attenuated in the CR rats. The intima-media gradient of a potent chemo-attractant, platelet derived growth factor (PDGF) and its receptor beta (PDGFR-) was increased in old compared to young AL rats, but was attenuated in CR rats. In addition, CR decreased early passage VSMCs invasive capability in vitro in response to platelet derived platelet factor -BB (PDGF-BB), both in young (26%) and old (15%) compared with VSMCs from AL rats. Interestingly, CR substantially decreased MCP-1 expression in early passage VSMCs compared to cells from AL rats; and medin treatment up-regulated PDGFR- in both young and old AL rat VSMCs. In summary, CR, like an MMP inhibitor, retards age-associated arterial restructuring in rats, at least in part, via a (1) reduction of MFG-E8, MMP2, MCP-1, and TGF-1 activation, (2) the intima-media PDGF gradient, and (3) VSMCs invasive capability. We recently found that the vasorin gene encoding a 170-190 kDa protein markedly decreases in both the arterial wall and in VSMCs with aging. Vasorin has a high affinity for TGF-1 and strongly inhibits interactions between TGF-1 and its type II receptor while markedly affecting the down-regulation of MMP-2 activation, SMAD-2/3 phosphorylation, and collagen I in VSMCs. In vivo, transcription and translation of vasorin are markedly decreased within the aortic walls of old (30 mo) vs. young (8 mo) FXBN rats. In vitro, levels of vasorin protein both intracellular and secreted forms by primary cultured early passage VSMC from old aortae are substantially reduced compared to those from young aortae. Furthermore, co-immunoprecipitation reveals that the interactions of vasorin and TGF-1in VSMCs are significantly decreased with aging. Treating young VSMCs with Angiotensin (Ang II) mimics increased levels of p-SMAD-2/3 and collagen type I production seen in old cells or in young VSMCs treated with siRNA of vasorin. These effects were abolished by the Ang II type I receptor (AT1) antagonist, Losartan, or by overexpressing the vasorin gene in old VSMCs. Old VSMCs exposed to Losartan show a reduction in the production of p-SMAD2/3 and collagen. Furthermore, overexpressing vasorin in young VSMCs inactivates and substantially inhibits their invasive proliferative capacity with aging with an inactivation of MMP-2. In addition, compared to those from age-matched wild type mice, the deficiency of the TGF-1 signal enhances phosphorylation of p-SMAD2/3 and collagen deposition in vasorin -/- mice. This is accompanied by an increase in proliferation cell nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4) observed in the adult (5-6 mo) vasorin knockout mouse arterial wall and myocardial interstitial space. These findings suggest that vasorin is a potential novel molecule to retard arterial VSMC inflammation with aging.