Effect of Metalloprotease Inhibition on Age-Associated Arterial Remodeling

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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, calcification as well as an increase in arterial pressure and stiffening. This adverse arterial cellular and matrix 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), the activation of extracellular MMPs via the transcriptional factor, ETS-1, as well as a decreases in the anti-inflammatory molecule vasorin. We hypothesized that inhibiting MMP activation can decelerate adverse age-associated arterial remodeling leading to a decrease 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) Age-associated decreases in aortic gelatinase and interstitial collagenase activity in situ; (2) The age-associated decrease aortic vasorin in situ was diminished; (3) the elastic fiber network integrity was preserved; (4) Collagen deposition was reduced; (5) MCP-1 and TGF- 1 activity levels were reduced; (6) Phosphorylation of the profibrogenic signaling molecule SMAD-2/3 was reduced; (7) Blockade of pro-ET1 activation; and (8) The expression of ETS-1 was downregulated. In addition, in vitro study shows that treating cultured VSMCs with pro-ET1 increased both the transcription and translation levels of ETS-1, and these effects were markedly reduced with the MMP inhibitor, PD166793. Furthermore, infecting VSMCs with an adenovirus harboring the 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 the 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 are 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 reduces vasorin protein expression 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 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 arterial health. 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 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. Calorie restriction (CR) in rats improves cardiovascular function and extends both healthspan and lifespan. How CR effects the proinflammatory landscape of 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 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. Recently, we found that both proinflammatory molecules Ang II and MFG-E8 increase with age or hypertension associated arterial remodeling. MFG-E8 is a downstream molecule of Ang II signaling and effectively activates MMP2 in cells VSMC in vitro and in vivo. MFG-E8 plays a crucial role in Ang II-induced blood pressure, arterial proinflammation and structural remodeling. We identified that MFG-E8 knock out (MFG-E8-/-) and wide-type (WT) mice chronically infused with Ang II or a saline control via an osmotic mini-pump for four weeks showed an significant increase in systolic blood pressure in both WT and MFG-E8-/- mice infused with Ang II vs saline after day 7 post-implant onward. Histochemical staining and immunostaining demonstrated that the intimal-medial thickness, VSMC proliferation, elastin lamina breaks, and collagen type I deposit were significantly increased within the aortae harvested from WT vs. age-matched MFG-E8-/- mice despite the change in systolic blood pressure seen similarly in both genotypes . Furthermore, chronic Ang II infusion significantly enhanced the expression of inflammatory molecule MFG-E8, MMP2 protein and up-regulated the activation of TGF-1 and its downstream signaling molecule p-Smad2 in the aortic walls of WT vs MFG-E8-/- mice. Notably, Ang II treated WT mice exhibited increased expression of proinflammatory leading molecules such as nuclear factor-kappa beta p65 (Nf-kB p65), MCP-1 and tumor necrosis factor-alpha 1 (TNF-a1) in aortic walls. However, these effects were substantially diminished in MFG-E8-/- mice. Taken together, the current findings demonstrate that MFG-E8 modulates Ang II-induced arterial cellular and matrix remodeling via the reduction of proinflammation. Thus, targeting the MFG-E8 molecule is a novel approach to retard arterial aging.