Age-Associated Changes in Arterial Proteome and Aortic Smooth Muscle Signaling

National Institute On Aging

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Abstract

We have performed a comprehensive quantitative proteomic study of young (8 mo) and old (30 mo) FXBN rats. Using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), we obtained 2-D gel maps of 301 identified non-redundant proteins from rat aortae and observed 18 proteins whose abundance significantly changed with aging. Utilizing Isobaric tags for relative and absolute quantitation (iTRAQ), 921 proteins have been quantified, and 50 proteins are significantly different in old versus young rat aortic proteins. Of note proteomic analysis shows that milk fat globule protein EGF-8 (MFG-E8) abundance significantly increases in old versus young rat aortae, which is a inflammatory bridging molecule linked VSMCs to elastic fibers. Transcription and translational analysis demonstrate that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including mice, rats, nonhuman primate, and humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within rat vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Ang II treatment of early passage VSMCs, isolated from young FXBN rat aortae, markedly increases MFG-E8 expression and enhances the invasive capacity of VSMCs to levels observed in untreated old cells. MFG-E8 treatment of VSMCs increased the levels of MCP-1, matrix metalloproteinase type II (MMP2) and their invasion capacity but these effects were markedly inhibited by the MCP-1 receptor blocker, vCCI. Importantly, silencing MFG-E8 mRNA substantially reduces MFG-E8 expression, MMP2 activation, and eventually VSMCs invasiveness. Thus, arterial MFG-E8 significantly increases in aging VSMCs, which is a key component within the Ang II-MCP-1/MMP2/VSMCs invasion signaling cascade within the arterial wall. We have identified that aging arteries and VSMCs are enriched with MFG-E8 and display increased levels of MMP2 activation, invasiveness, and proliferation. Increased MFG-E8 in VSMCs triggers phosphorylation of extracellular signal regulated kinases-1/2(ERK-1/2), augments levels of proliferation cellular nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4), increases bromodeoxyuridine /5-bromo-2'-deoxyuridine (BrdU) incorporation and promotes growth. When MFG-E8 mRNA is effectively silenced, the rate of cell cycling is reduced, the expression of the signaling molecules PCNA and CDK4 is lowered , thus, facilitating the cells entry into a growth-arrested state known as senescence. Furthermore, we find that integrin v5 and platelet derived growth factor (PDGF) beta receptor are upregulated with MFG-E8 treatment, which is the key elements of extracellular proliferative signals in aging VSMC. We have also identified that MFG-E8 facilitates profibrosis of aging VSMCs. Treating aortic VSMCs isolated from both young and old rats with MFG-E8 increases the profibrogenic signaling molecules transforming growth factor-beta1 (TGF-1), TGF- receptor type II (TRII), p-SMAD (Sma and Mad: Mothers against decapentaplegic)-2/3, and collagen I (Col I) and collagen III (Col III) while knockdown of MFG-E8 gene expression substantially reduces the expression of these molecules. These results demonstrate that MFG-E8 modulates the TGF-1 fibrogenic signaling in VSMCs with advancing age. We have found that both proinflammatory molecules Ang II and MFG-E8 increase with age or hypertension during arterial remodeling. MFG-E8 is a downstream molecule of Ang II signaling and activates MMP2 in VSMCs in vitro and in vivo. MFG-E8 plays an inflammatory role in Ang II-induced blood pressure and structural remodeling. A list of our specific findings include: MFG-E8 knockout (KO) and age-matched wild type (WT) mice chronically infused with Ang II or saline control via an osmotic mini-pump for four weeks show that systolic blood pressure (SBP) is significantly increased in both mice infused with Ang II vs saline, after day 7 post-implantation onward. Histochemical staining and immunostaining demonstrate that the intimal-medial thickness, VSMC proliferation, elastin lamina breaks, and collagen type I deposits are significantly increased within aortae harvested from WT vs. age-matched MFG-E8 KO mice despite the change in SBP seen similarly in both genotypes. Furthermore, chronic Ang II infusion significantly enhances the expression of the inflammatory molecule MFG-E8, MMP2 protein and up-regulated activated TGF-1 levels and its downstream signaling molecule p-Smad2 in the aortic walls of WT vs MFG-E8 KO mice. Notably, Ang II treated WT mice exhibits 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-1) in aortic wall. However, these effects are substantially diminished in MFG-E8 KO mice. Taken together, 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 curb the adverse effects of arterial aging. Importantly, MFG-E8 serves as a structural bridging molecule that connects smooth muscle cells to elastic fibers, forming elastin-smooth muscle units, an elementary component found in arterial walls. The bridging and signaling effects in the presence or absence of MFG-E8 between cells and matrix on arterial remodeling with advancing age have been observed. Western blotting analysis indicates that MFG-E8 protein is deficient in the aortic walls of homogeneous MFG-E8 knockout (KO) mice but is enriched in the aortic walls of WT mice. In WT, aortic MFG-E8 protein is markedly increased in 96-week-old (old) versus 40-week-old (young). Aortic elastin proportion, an index of elastin degradation, significantly decreases in old versus young while collagen proportion and calcium deposits proportion are increased in both WT and MFG-E8 KO; however, MFG-E8 deficiency significantly lessens these age-associated increases in elastin degradation and collagen and calcium deposition. The pro-calcification molecules alkaline phosphatase and runt-related transcription factor 2 are markedly increased in old WT versus old MFG-E8 KO. Notably, MFG-E8 deficiency slightly effected changes in age-associated systolic blood pressure compared to WT and significantly extended the lifespan of MFG-E8 deficient mice compared to WT. Taken together, these findings suggest that MFG-E8 is a novel signaling protein in age-associated adverse aortic remodeling such as elastin degradation, collagen deposition, and calcification. Targeting MFG-E8 is a novel molecular approach to maintaining arterial health with advancing age and potentially prolonging life expectancy.