Effect of Metalloprotease Inhibition on Age-Associated Arterial Remodeling

National Institute On Aging

Investigators

Linked publications, trials & patents

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 pressure and stiffening. This adverse arterial cellular and extracellular 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), and 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 of 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, our 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 containing 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. Treating young VSMCs with 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 their 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 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 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, in collaboration with Dr. Patricia Gearhart, an inter-lab collaboration at the NIA, we found that auto-antibody production related MMP2 activation during experimental atherosclerosis in ApoE-/- mic. B cells and antibodies function during atherosclerosis in two distinct ways based on antibody isotype, where IgM is protective, and IgG is inflammatory. ApoE-/- Aid-/- mice were generated, which are unable to produce IgG antibodies due to the absence of activation-induced deaminase (AID) but are able to maintain high plasma cholesterol due to the absence of apolipoprotein E (ApoE). A dramatic decrease in plaque formation and MMP2 production was seen in ApoE-/- Aid-/- mice compared to ApoE-/- mice. Rigorous analysis of serum antibodies revealed both ApoE-/- and ApoE-/- Aid-/- mice had substantially elevated titers of IgM antibodies compared to C57BL/6J controls. Furthermore, ApoE-/- Aid-/- mice had elevated titers of antibodies specific to malondialdehyde-oxidized low-density lipoprotein (MDA-oxLDL), which has been shown to block macrophage recruitment into plaques. Conversely, ApoE-/- mice showed low levels of MDA-oxLDL specificity but had 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. If the level of MDA-oxLDL specific IgM is insufficient, self-reactive IgM and IgG antibodies are generated against debris within the arterial plaque, resulting in increased inflammation, MMP2 activation, and further plaque expansion.