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 to analyze aortic proteins from 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 were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Proteomic analysis shows that milk fat globule protein EGF-8 (MFG-E8) abundance significantly increases in old rat aortae. Transcription and translational analysis demonstrated 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 old. MFG-E8 treatment of VSMCs increased the levels of MCP-1, matrix metalloproteinase type II (MMP2) and their invasion capacity but these effects were inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 mRNA substantially reduced MFG-E8 expression, MMP2 activation and VSMCs invasiveness. Thus, arterial MFG-E8 significantly increases in aging VSMCs and is a key component within the Ang II-MCP-1/MMP2/VSMCs invasion signaling cascade the arterial wall. We have identified that in aging arteries, VSMCs are enriched with MFG-E8 and display increased levels of MMP2 activation, invasiveness, and proliferation, both in vivo and in vitro. 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 cell growth. When MFG-E8 mRNA is effectively silenced, the speed of cell cycling is reduced, the expression of the signaling molecules PCNA and CDK4 is declined, thus, facilitating the cells entry into a growth-arrested state. Furthermore, we find that integrin v5 and platelet derived growth factor (PDGF) receptor are upregulated with MFG-E8 treatment and are also 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. 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 activated MMP2 in VSMCs in vitro and in vivo. MFG-E8 plays a crucial role in Ang II-induced blood pressure, arterial proinflammation and structural remodeling. Here is our findings: MFG-E8 KO and age-matched WT mice chronically infused with Ang II or saline control via an osmotic mini-pump for four weeks showed that SBP is significantly increased in both mice infused with Ang II vs saline, after day 7 post-implant 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 enhanced 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 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-1) in aortic walls. However, these effects were substantially diminished in MFG-E8 KO 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 diminish the effects arterial aging. Interestingly, MFG-E8 serves as a structural bridging molecule that connects smooth muscle cells to elastic fibers, forming elastin-smooth muscle units, an essential 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 were recently documented. Western blot analysis confirmed that MFG-E8 protein was deficient in the aortic walls of homogeneous MFG-E8 knockout (KO) mice and was enriched in the aortic walls of WT mice. In WT, aortic MFG-E8 protein was markedly increased (18-fold, p<0.0001) in 96-week-old (old) versus 40-week-old (young). Aortic elastin proportion (an index of elastin degradation) significantly decreased in old versus young while collagen proportion and calcium deposits proportion were 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 proteins alkaline phosphatase and runt-related transcription factor 2 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 (p=0.032). Taken together, these findings suggest that MFG-E8 is a novel signaling protein between vascular cells and matrix in age-associated adverse aortic remodeling such as elastin degradation, collagen deposition, and calcification. MFG-E8 deficiency had beneficial effects on arterial health with advancing age and potentially prolongs life expectancy.