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

National Institute On Aging

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Abstract

A comprehensive quantitative proteomic study was conducted on young (8 months) and old (30 months) Fischer 344 crossbreed Brown Norway (FXBN) rats. Utilizing 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), 2-D gel maps of 301 identified non-redundant proteins from rat aortae were obtained, and 18 proteins showed significant changes in abundance with aging. Using Isobaric tags for relative and absolute quantitation (iTRAQ), 921 proteins were quantified, and 50 proteins were significantly different in old versus young rat aortic proteins. Signaling network analysis revealed that angiotensin II (Ang II) associated proinflammation is key to arterial aging. Proteomic analysis indicated that the abundance of Ang II signaling downstream molecule, milk fat globule protein EGF-8 (MFG-E8), significantly increases in old versus young rat aortae, serving as an inflammatory bridging molecule linking VSMCs to elastic fibers. Transcriptional and translational analyses demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including mice, rats, nonhuman primates, and humans. Dual immunolabeling showed that MFG-E8 colocalizes with both Ang II and monocyte chemoattractant protein-1 (MCP-1) within rat or human VSMCs of the thickened aged aortic wall. Ang II treatment of early passage VSMCs isolated from young FXBN rat aortae markedly increased MFG-E8 expression and enhanced 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 C-C motif chemokine receptor 2 (CCR2) blocker, vCCI. Silencing MFG-E8 mRNA substantially reduced MFG-E8 protein expression, MMP2 activation, and VSMC invasiveness. Thus, arterial MFG-E8 significantly increases in aging VSMCs, playing a key role in the Ang II-MCP-1/MMP2/VSMCs proinflammatory and invasion signaling cascade within the arterial wall. In aging arteries, MFG-E8 was found to enrich in VSMCs and display increased levels of proliferation. Increased MFG-E8 in VSMCs triggered phosphorylation of extracellular signal-regulated kinases-1/2 (ERK-1/2), augmented levels of proliferation cellular nuclear antigen (PCNA) and cyclin-dependent kinase 4 (CDK4), increased bromodeoxyuridine (BrdU) incorporation, and promoted growth. Silencing MFG-E8 mRNA reduced the rate of cell cycling, lowered the expression of PCNA and CDK4, and enhanced the expression of p21 and p16, facilitating the cell's entry into a growth-arrested state known as senescence. Integrin αv5 and platelet-derived growth factor (PDGF) beta receptor, key elements of extracellular proliferative signals in aging VSMCs, were upregulated with MFG-E8 treatment. MFG-E8 also facilitates profibrosis in aging VSMCs. Treating aortic VSMCs isolated from both young and old rats with MFG-E8 increased the profibrogenic signaling molecules transforming growth factor-beta1 (TGF-β1), TGF-β receptor type II (TRII), p-SMAD2/3, collagen I (Col I), and collagen III (Col III). Knockdown of MFG-E8 gene expression substantially reduced the expression of these molecules, demonstrating that MFG-E8 modulates TGF-β1 fibrogenic signaling in VSMCs with advancing age. Both proinflammatory molecules Ang II and MFG-E8 increase with age or hypertension during arterial remodeling. MFG-E8, a downstream molecule of Ang II signaling, activates MMP2 in VSMCs in vitro and in vivo. MFG-E8 plays an inflammatory role in Ang II-induced blood pressure and structural remodeling. MFG-E8 knockout (KO) and age-matched wild-type (WT) mice chronically infused with Ang II or saline control showed that systolic blood pressure (SBP) significantly increased in both genotypes infused with Ang II. Histochemical staining and immunostaining demonstrated that the intimal-medial thickness, VSMC proliferation, elastic lamina breaks, and collagen type I deposits were significantly increased in aortae from WT versus age-matched MFG-E8 KO mice, despite similar SBP changes. Chronic Ang II infusion significantly enhanced the expression of inflammatory molecule MFG-E8, MMP2 protein/activation, and upregulated activated TGF-β1 levels and its downstream signaling molecule p-Smad2 in the aortic walls of WT versus MFG-E8 KO mice. Notably, Ang II treated WT mice exhibited increased expression of proinflammatory molecules such as nuclear factor-kappa beta p65 (Nf-kB p65), MCP-1, and tumor necrosis factor-alpha 1 (TNF-α1) in the aortic wall, which were substantially diminished in MFG-E8 KO mice. Taken together, MFG-E8 modulates Ang II-induced arterial cellular and matrix remodeling mainly via reducing proinflammation. Targeting MFG-E8 is a novel approach to curb the adverse effects of arterial aging. Importantly, MFG-E8 serves as a structural bridging molecule that connects VSMCs to elastic fibers, forming elastic-smooth muscle units, an elementary component found in elastic arterial walls. The bridging and signaling effects of MFG-E8 on arterial remodeling with advancing age were observed. Western blotting analysis indicated that MFG-E8 protein is deficient in the aortic walls of MFG-E8 KO mice but enriched in WT mice, with marked increases in 96-week-old (old) versus 40-week-old (young) WT mice. Aortic elastin proportion, an index of elastin degradation, significantly decreased in old versus young, while collagen proportion and calcium deposits proportion increased in both WT and MFG-E8 KO. However, MFG-E8 KO significantly lessened these age-associated increases in elastin degradation and collagen and calcium deposition. Pro-calcification molecules alkaline phosphatase and runt-related transcription factor 2 were markedly increased in old WT versus old MFG-E8 KO. MFG-E8 KO slightly affected age-associated systolic blood pressure changes compared to WT and significantly extended the health span of MFG-E8 KO compared to WT animals. These findings suggest that MFG-E8 is not only a signaling protein but also a bridging molecule linking VSMCs and elastic lamina in age-associated adverse aortic remodeling, such as elastin degradation, collagen deposition, and calcification. Targeting MFG-E8 is a novel molecular approach to preventing proinflammation, maintaining arterial health with advancing age, and potentially prolonging healthy life expectancy.