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, calcification as well as an increase in arterial pressure. This adverse arterial remodeling is linked to an increase in proinflammatory signaling molecules, including angiotensin II (Ang II), 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), activation of extracellular MMPs via the transcriptional factor, ETS-1, as well as a decreases in the anti-inflammatory molecule vasorin. We tested whether an inhibition of MMP activation can decelerate adverse age-associated arterial remodeling leading to an 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) inhibition of age-associated decrease aortic vasorin in situ; (3) preservation of the elastic fiber network integrity; (4) a reduction in collagen deposition; (5) a reduction of MCP-1 and TGF- 1 activity; (6) a decrease in the phosphorylation activity of the profibrogenic signaling molecule SMAD-2/3; (7) inhibition of pro-ET1 activation; and (8) a downregulation of the expression of ETS-1. In addition, our in vitro study shows that treating cultured vascular smooth muscle cells (VSMCs) with pro-ET1 increased both the transcription and translation levels of ETS-1, and these effects were markedly reduced with MMP inhibition. Furthermore, infecting VSMCs with an adenovirus harboring full-length ETS-1 cDNA, increased the levels of activated forms of both TGF-1 and MCP-1 proteins. Collectively, our results indicate that MMP inhibition impedes age-associated arterial proinflammatory signaling and is accompanied by the preservation of intact elastin fiber network, a reduction in collagen, and a blunting of age-associated increases in blood pressure. The glycosylated protein vasorin physically interacts with TGF-1 and functionally attenuates its fibrogenic signaling in VSMCs of the arterial wall. Ang II amplifies TGF-1 activation in aging VSMCs of the arterial wall via a decrease in vasorin signaling mediated by MMP2 cleavage. Vasorin mRNA and protein expression were significantly decreased both in the aortic wall and in VSMCs from old (30 mo) vs. young (8 mo) FXBN rats. Exposing young VSMCs to Ang II reduced vasorin protein expression to the levels of old untreated cells while treating old VSMCs with the Ang II type AT1 receptor antagonist Losartan upregulated vasorin protein expression up to the levels of young. The physical interaction between vasorin and TGF-1 was significantly decreased in old vs. young VSMCs. Further, treating young VSMCs with Ang II increased the levels of MMP-2 activation and TGF-1 downstream molecules p-SMAD-2/3 and collagen type I production up to the levels of old untreated VSMCs, and these effects were substantially inhibited by overexpressing vasorin. Administration of Ang II to young rats (8 mo) for 28 days via an osmotic minipump markedly reduced the expression of vasorin. Importantly, the vasorin protein was effectively cleaved by activated MMP-2 both in vitro and in vivo. Administration of the MMP inhibitor, PD 166793, for 6 months, to young adult rats (18 mo) via a daily gavage markedly increased levels of vasorin in the aortic wall. Thus, reduced vasorin amplifies Ang II profibrotic signaling via an activation of MMP-2 in VSMCs within the aging arterial wall. Aging exponentially increases the incidence of morbidity and mortality of quintessential inflammatory cardiovascular disease mainly due to arterial proinflammatory shifts at the molecular, cellular, and tissue levels within the arterial wall. Calorie restriction (CR) in rats improves arterial function and extends both healthspan and lifespan. How CR effects the proinflammatory landscape of molecular, cellular, and tissue phenotypic shifts within the arterial wall in rats, however, remains to be elucidated. Aortae were harvested from young (6-month-old) and old (24-month-old) Fisher 344 rats, fed ad libitum (AL) and a second group maintained on a 40% CR beginning at one month of age. Histopathologic and morphometric analysis of the arterial wall demonstrated that CR markedly reduced age-associated intimal medial thickening, collagen deposition, and increased elastin fraction within the arterial walls. Aortic wall immunostaining/blotting showed that CR effectively prevented an age-associated increase in the density of platelet derived growth factor (PDGF-BB), MMP2 activity, TGF-1 and its downstream signaling molecule, p-SMAD-2/3. In early passage cultured VSMCs isolated from both AL and CR rat aortae, CR alleviated the age-associated VSMC phenotypic shifts, pro-fibrogenic signaling, and proliferation in response to PDGF-BB. Collectively, CR reduces matrix and cellular proinflammation associated with aging that occurs within the aortic wall and is attributable to PDGF signaling. Thus, CR reduces PDGF-associated MMP activation signaling cascade and contributes to the postponement of biological aging, preserving a more youthful aortic wall phenotype.