Effect of Metalloprotease Inhibition on Age-Associated Arterial Remodeling

National Institute On Aging

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Abstract

Age-associated arterial structural and functional remodeling includes intimal vascular smooth muscle cell (VSMCs) cellularity (invasion and proliferation), VSMCs senescence, collagen deposition, elastin fragmentation, amyloidosis, and calcification as well as an increase in arterial stiffening and blood pressure. This adverse arterial cellular, extracellular matrix and functional 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) This includes the activation of matrix metalloproteinases (MMPs) such as MMP2 via the transcriptional factors ETS-1 and nuclear factor kappa B (NF-B) as well as a decrease in anti-inflammatory molecules such as vasorin and transcription factor nuclear factor erythroid 2-related factor 2 (NrF-2). We have found that inhibiting MMP activation decelerates adverse age-associated arterial remodeling leading to a decrease in arterial blood pressure. Chronic administration (8 months) of the broad-spectrum MMP inhibitor, PD166793, via a daily gavage, to 16-month-old FXBN rats alleviates age-associated increases in arterial pressure. This is accompanied by an alleviation of the following age-associated effects: (1) increases in aortic gelatinase and interstitial collagenase activity; (2) increase of aortic wall thickening; (3) elastic fiber network destruction; (4) collagen deposition; (5) Increases in MCP-1 and TGF- 1 activity; (6) Increase in phosphorylation of the profibrogenic signaling molecule SMAD-2/3; (7) Increase in pro-ET1 activation; and (8) upregulation of transcriptional factor ETS-1. In addition, our in vitro study has shown that treating cultured VSMCs with pro-ET1 increases both the transcription and translation of ETS-1, and these effects are markedly reduced with the MMP inhibitor, PD166793. Furthermore, infecting VSMCs with an adenovirus containing full-length ETS-1 cDNA increases 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 the intact elastin fiber network, a reduction in collagen, and a blunting of age-associated increases in systolic blood pressure. The glycosylated protein vasorin physically interacts with TGF-1 and functionally attenuates its profibrogenic and proinflammatory signaling in VSMCs of the arterial wall. Angiotensin II (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 are significantly decreased both in the aortic wall and in primary cultured VSMCs from old vs. young FXBN rats. Treating young VSMCs with Ang II reduces vasorin protein expression down to the levels of old untreated cells. Conversely treating old VSMCs with the Ang II type AT1 receptor antagonist, Losartan, upregulates vasorin protein expression up to the levels of untreated young. The physical interactions between vasorin and TGF-1 are significantly decreased in old vs. young VSMCs. Further, treating young VSMCs with Ang II increases 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 are substantially inhibited by overexpressing vasorin. Treating young rats (8 mo) with Ang II for 28 days via an osmotic minipump markedly reduced the expression of vasorin and improved their arterial health such as inflammation, elasolysis, and collagen deposition. Importantly, the vasorin protein is 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 the levels of vasorin in the aortic wall. Thus, reduced vasorin amplifies Ang II profibrotic signaling very likely via the 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 such as hypertension, atherosclerosis, and stroke mainly due to arterial proinflammatory shifts at the molecular, cellular, and tissue levels within the arterial wall and systemic circulation. Calorie restriction (CR) in rats improves cardiovascular health and frailty; and extends both health span and lifespan. How CR effects the proinflammatory landscape of the molecular, cellular, and tissue proinflammatory phenotypic shifts within the arterial wall in rats are examined in the current study. 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 demonstrates that CR markedly reduces age-associated intimal medial thickening, collagen deposition, and increases the elastin fraction within the arterial walls. Aortic wall immunostaining/blotting shows that CR effectively prevents 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 alleviates the age-associated VSMC phenotypic shifts, pro-fibrogenic signaling, and proliferation in response to PDGF-BB. Collectively, CR reduces adverse matrix remodeling and cellular proinflammation associated with aging that occurs within the aortic wall and is attributable to PDGF signaling. Thus, CR reduces the PDGF-associated MMP activation signaling cascade and contributes to the postponement of biological aging, maintaining a more youthful aortic wall phenotype. Importantly, we have recently found that auto-antibody production is related to MMP2 activation during experimental atherosclerosis in ApoE-/- mice. B cells and antibodies function during atherosclerosis in two distinct ways based on antibody isotype, where IgM is protective, and IgG is inflammatory pathogenic. ApoE-/- Aid-/- are unable to produce IgG antibodies due to the absence of activation-induced deaminase (AID) but able to maintain high plasma cholesterol due to the absence of apolipoprotein E (ApoE). A dramatic decrease in plaque burdens and MMP2 expression has been observed in ApoE-/- Aid-/- mice compared to ApoE-/- mice. Rigorous evaluation of serum antibodies reveals both ApoE-/- and ApoE-/- Aid-/- mice have substantially elevated titers of IgM antibodies compared to C57BL/6J controls. Furthermore, ApoE-/- Aid-/- mice have elevated titers of antibodies specific to malondialdehyde-oxidized low-density lipoprotein (MDA-oxLDL), which has been shown to block macrophage infiltration into plaques. Conversely, ApoE-/- mice show low levels of MDA-oxLDL specificity but have antibodies specific to numerous self-proteins. These findings suggest that there is a hierarchical order of antibody specificity, where elevated levels of MDA-oxLDL specific IgM antibodies inhibit plaque formation and MMP2 expression. These findings suggest that both IgM and IgG are involved in age-associated atherogenesis.