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，以分析年轻（8 个月）和老年（30 个月）大鼠的主动脉蛋白。使用 2-D DIGE，我们获得了 301 种来自大鼠主动脉的已鉴定非冗余蛋白的 2-D 凝胶图，并观察到 ​​18 种蛋白的丰度随着衰老而显着变化。利用 iTRAQ，对 921 种蛋白质进行了定量，并且在两种方法之间，50 种蛋白质显示出与年龄相关的丰度存在显着差异。 值得注意的是，蛋白质组分析表明，一种感兴趣的蛋白质 MFG-E8 在老年大鼠主动脉中的丰度显着增加。转录和翻译分析表明，在包括人类在内的多种哺乳动物中，主动脉 MFG-E8 mRNA 和蛋白质水平随着衰老而增加。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) 共定位于增厚的老化主动脉壁的血管平滑肌细胞 (VSMC) 内。将来自年轻主动脉的早期传代 VMSC 暴露于 Ang II 显着增加 MFG-E8，并将侵袭能力增强至老年大鼠 VSMC 中观察到的水平。用 MFG-E8 治疗 VSMC 会增加 MCP-1 和 VSMC 的侵袭，而 MCP-1 受体阻断剂 vCCI 可抑制这种侵袭。沉默 MFG-E8 RNA 显着降低 MFG-E8 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 侵袭信号级联中的关键中继元件。 重要的是，我们已经发现，富含 MFG-E8 的衰老动脉 VSMC 在体内和体外均被激活并增殖。 VSMC 中 MFG-E8 的增加会触发 ERK1/2 的磷酸化，增加 PCNA 和 CDK4 的水平，增加 BrdU 掺入并促进生长。 MFG-E8 的敲除降低了细胞周期的速率，加速信号分子 PCNA 和 CDK4 的表达，促进细胞进入生长停滞状态。此外，我们发现 MFG-E8 上调了 av5 和 PDGF，并且也是将增殖信号传递给老化 VSMC 的元件。 此外，促纤维化信号分子 TGF-β1、TβRII、p-SMAD 2/3 和胶原蛋白在年轻 VSMC 的 MFG-E8 治疗和 Vasorin 沉默中均上调，高达老年细胞中的水平。相反，这些促纤维化分子的表达在 MFG-E8 缺失和沉默的老 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 会增加旧 VSMC 中 TGF-β1、TβRII、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 促纤维化反应的抵抗通过 cav-1 信号传导的上调而消除。综上所述，目前的研究结果首次表明，随着年龄的增长，脂筏中 MFG-E8 和 Cav-1 信号传导的不平衡是 VSMC 胶原蛋白生成的关键分子事件。