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

动脉蛋白质组和主动脉平滑肌信号与年龄相关的变化

• 批准号: 8931487
• 负责人: Edward Lakatta
• 金额: $ 39.09万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8931487
• 关键词: Age; Aging; Angiotensin II; Aorta; Beryllium; Blood Vessels; Bromodeoxyuridine; CDK4 gene; Cell Cycle; Cells; Collagen; Disease; Elderly; Elements; Event; Gel; Genetic Transcription; Growth; Human; In Vitro; MAPK3 gene; Maps; Membrane Microdomains; Messenger RNA; Methods; Molecular; Monocyte Chemoattractant Protein-1; Phosphorylation; Platelet-Derived Growth Factor; Production; Proliferating; Proteins; Proteome; Proteomics; RNA; Rat Protein; Rattus; Resistance; Role; Signal Transduction; Signaling Molecule; Signaling Protein; Smooth Muscle; Smooth Muscle Myocytes; Time; Translations; Up-Regulation; aged; arterial remodeling; in vivo; interest; monocyte chemoattractant protein 1 receptor; novel; novel strategies; response

In this study, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) rats. Using 2-D DIGE, we have obtained 2-D gel maps of 301 identified non-redundant proteins from rat aorta and observed 18 proteins that significantly change abundance with aging. Utilizing iTRAQ, 921 proteins were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Notably, proteomic analysis shows that one protein of interest, MFG-E8, significantly increases in abundance in old rat aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within vascular smooth muscle cells (VSMC) of the thickened aged aortic wall. Exposure of early passage VMSC from young aorta to Ang II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMC from old rats. Treatment of VSMC with MFG-E8 increases MCP-1 and VSMC invasion that are inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMC invasion capacity. Thus, arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the Ang II MCP-1/VSMC invasion signaling cascade. Importantly, we have identified that aging arterial MFG-E8-enriched VSMC are activated and proliferating both in vivo and in vitro. Increased MFG-E8 in VSMC triggers phosphorylation of ERK1/2, augments levels of PCNA and CDK4, increases BrdU incorporation and promotes growth. The knockdown of MFG-E8 reduces rate of cell cycling, accelerating signaling molecules PCNA and CDK4 expression, facilitating cell entry into a growth-arrested state. Furthermore, we find that av5 and PDGF are upregulated with MFG-E8 and also are elements to relay proliferative signals to aging VSMC. In addition, the profibrogenic signaling molecules TGF-β1, TβRII, p-SMAD 2/3, and collagen are up-regulated in both MFG-E8 treatment and Vasorin silencing of young VSMCs, up to the levels in old cells. Conversely, expression of these profibrogenic molecules is down-regulated in MFG-E8 null and silenced old VSMCs, reaching the levels of young cells. Impressively, exposure of VSMCs to exogenous MFG-E8 increases the profibrotic levels of TGF-β1, TβRII, p-SMAD 2/3, and collagen in the old VSMCs. Further, young VSMCs resistance to MFG-E8 profibrogic responses is abolished by up-regulation of cav-1 signaling. Taken together, the present findings, for the first time, suggest that the imbalance of MFG-E8 and Cav-1 signaling in the lipid raft is a key molecular event to collagen production of VSMCs with advancing age.

在这项研究中，我们进行了全面的定量蛋白质组学研究，分析主动脉蛋白质从年轻（8个月）和老年（30个月）大鼠。使用2-D DIGE，我们已经获得了301个从大鼠主动脉中鉴定的非冗余蛋白的2-D凝胶图，并观察到18个蛋白质的丰度随年龄的变化显着。利用iTRAQ，921种蛋白质被定量，并且在两种方法之间，50种蛋白质被证明具有显著不同的年龄相关丰度。 值得注意的是，蛋白质组学分析显示，一种感兴趣的蛋白质MFG-E8在老年大鼠主动脉中的丰度显著增加。转录和翻译分析表明，主动脉MFG-E8 mRNA和蛋白质水平随着包括人类在内的几种哺乳动物的衰老而增加。双重免疫标记显示MFG-E8与血管紧张素II（Ang II）和单核细胞趋化蛋白-1（MCP-1）在增厚的老年主动脉壁的血管平滑肌细胞（VSMC）内共定位。暴露早期通道VMSC从年轻的主动脉血管紧张素II显着增加MFG-E8和增强侵袭能力的水平观察到VSMC从老年大鼠。用MFG-E8处理VSMC增加了MCP-1和VSMC侵袭，其被MCP-1受体阻断剂vCCI抑制。沉默MFG-E8 RNA显著降低MFG-E8表达和VSMC侵袭能力。 因此，动脉MFG-E8随着年龄的增长而显著增加，并且是Ang II MCP-1/VSMC侵袭信号级联中的关键中继元件。 重要的是，我们已经确定，老化的动脉MFG-E8富集的VSMC在体内和体外都被激活和增殖。VSMC中MFG-E8的增加触发ERK 1/2的磷酸化，增加PCNA和CDK 4的水平，增加BrdU掺入并促进生长。MFG-E8的敲低降低了细胞周期的速率，加速了信号分子PCNA和CDK 4的表达，促进细胞进入生长停滞状态。此外，我们发现，av 5和PDGF上调MFG-E8，也是元件中继增殖信号老化VSMC。 此外，促纤维化信号分子TGF-1、T RII、p-SMAD 2/3和胶原蛋白在MGF-E8处理和年轻VSMC的Vasorin沉默中均上调，达到老年细胞中的水平。相反，这些促纤维化分子的表达在MFG-E8无效和沉默的老年VSMC中下调，达到年轻细胞的水平。 令人印象深刻的是，VSMC暴露于外源性MFG-E8增加了旧VSMC中TGF-1、TRII、p-SMAD 2/3和胶原蛋白的促纤维化水平。此外，年轻的VSMC对MFG-E8促纤维化反应的抗性通过上调cav-1信号传导而消除。综上所述，本研究结果首次表明，MFG-E8和Cav-1信号在脂筏中的不平衡是随着年龄增长VSMC胶原蛋白产生的关键分子事件。