Effect of Metalloprotease Inhibition on Age-Associated Arterial Remodeling

National Institute On Aging

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Abstract

Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation as well as an increase in arterial pressure. This 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 proinflammation and its increase in arterial pressure. Indeed, chronic administration of the broad-spectrum MMP inhibitor, PD166793, via a daily gavage, to 16-month-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) inhibition of the 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. Acute exposure of cultured vascular smooth muscle cells (VSMC) in vitro to pro-ET1 increased both the transcription and translation of ETS-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of VSMC with an adenovirus harboring a full-length ETS-1 cDNA increased levels of activated proteins of both TGF-1 and MCP-1. 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 MMP inhibition effects, significantly improves arterial health. Immunostaining shows that the number of intimal VSMC 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 old AL rats. Impressively, aortic collagen and elastin fibers did not significantly change in the CR rat during aging. Notably, 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, were 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 increased in old AL rats when compared to young AL rats, but this was attenuated in 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 this was attenuated in CR rats. In addition, CR decreased early passage VSMC invasive capability in vitro in response to PDGF-BB, both in young (26%) and old (15%) compared with AL cells . Interestingly, CR substantially decreased MCP-1 expression in early passage VSMC compared to cells from AL rats; and medin treatment up-regulated PDGFR- in both young and old AL rat VSMC. 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) VSMC invasive capability. Notably, we recently found that the vasorin gene encoding a 170-190 kDa secreted vasorin protein markedly decreases in the arterial wall and VSMC with aging. Vasorin has a high affinity for TGF-1 and also has a strong inhibitory ability on the 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 VSMC. 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 in or secreted by primary cultured early passage VSMC from old aortae are substantially reduced compared to those from young aortae. Furthermore, co-immunoprecipitation reveals that the interaction of vasorin and TGF-1 in VSMC v is significantly decreased with aging. Exposure of young VSMC to Angiotensin (Ang II) mimics increased p-SMAD-2/3 and collagen type I production in old cells or in young VSMC with siRNA of vasorin . These effects are abolished by an Ang II type I receptor (AT1) antagonist, Losartan, or by overexpression of the vasorin gene in old VSMCs. Old VSMC exposed to Losartan show a markedly reduction of p-SMAD2/3 and collagen production. Furthermore, overexpression of vasorin of young VSMCs inactivates and substantially inhibits the invasive proliferation capacity with aging, accompanied by 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. 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.