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，我们进行了一项全面的定量蛋白质组学研究，以分析来自年轻（8个月）和年龄（30 mo）大鼠的主动脉蛋白。使用2-D DIGE，我们获得了来自大鼠主动脉的301个鉴定出非冗余蛋白的2-D凝胶图，并观察到18种随着衰老的丰富而显着改变丰度的蛋白质。利用ITRAQ，量化了921种蛋白质，两种方法之间，50种蛋白质均显示出明显不同的年龄相关丰度。 值得注意的是，蛋白质组学分析表明，一种感兴趣的蛋白质MFG-E8显着增加了旧大鼠主动脉的丰度。转录和翻译分析表明，包括人类在内的几种哺乳动物物种中主动脉MFG-E8 mRNA和蛋白水平随着衰老而增加。双重免疫标记表明，MFG-E8与血管紧张素II（ANG II）和单核细胞化学吸收剂蛋白-1（MCP-1）均共定位在增厚的老年主动脉壁的血管平滑肌细胞（VSMC）中。早期通过VMSC暴露于年轻主动脉到ANG II显着增加了MFG-E8，并提高了侵入性能力，从旧大鼠的VSMC中观察到的水平。 MFG-E8对VSMC的处理增加了MCP-1受体阻滞剂VCCI抑制的MCP-1和VSMC侵袭。沉默的MFG-E8 RNA显着降低了MFG-E8的表达和VSMC侵袭能力。 因此，动脉MFG-E8随着衰老而显着增加，并且是ANG II MCP-1/VSMC入侵信号级联的关键继电器元件。 重要的是，我们已经确定富含动脉MFG-E8富集的VSMC被激活并在体内和体外增殖。 VSMC中MFG-E8的增加触发了ERK1/2的磷酸化，PCNA和CDK4的水平增加，可增加BRDU掺入并促进生长。 MFG-E8的敲低降低了细胞循环，加速信号分子PCNA和CDK4表达的速率，从而促进细胞进入生长降落的状态。此外，我们发现AV5和PDGF被MFG-E8上调，并且也是将增殖信号传递到衰老VSMC的元素。 此外，在MFG-E8处理和Young VSMC的血管素沉默中，TGF-β1，TβRII，P-SMAD 2/3和胶原蛋白的纤维化信号分子TGF-β1，TβRII，P-SMAD 2/3和胶原蛋白上调，最高到旧细胞的水平。相反，在MFG-E8 null中，这些纤维化分子的表达下调并沉默的旧VSMC，达到了幼小细胞的水平。 令人印象深刻的是，VSMC暴露于外源MFG-E8会增加旧VSMC中TGF-β1，TβRII，P-SMAD 2/3和胶原蛋白的纤维化水平。此外，通过上调CAV-1信号传导，消除了对MFG-E8的年轻VSMC对MFG-E8的抗性。综上所述，目前的发现首次表明，脂质筏中MFG-E8和CAV-1信号的失衡是胶原蛋白产生vSMC的关键分子事件，其增长率不断。