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: 9147247
• 负责人: Edward Lakatta
• 金额: $ 38.59万
• 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/9147247
• 关键词: 17 year old; Actins; Adult; Age; Aging; Angiotensin II; Animals; Aorta; Apolipoprotein E; Area; Arterial Fatty Streak; Atherosclerosis; Behavior; Beryllium; Biological Assay; Blood Vessels; Bromodeoxyuridine; CAV1 gene; CDK4 gene; Cardiovascular Diseases; Cell Cycle; Cells; Chest; Cholesterol; Collagen; Diet; Elastin; Elderly; Elements; Fluorescence; Foam Cells; Gel; Gene Expression; Genes; Genetic Transcription; Growth; Human; Immunoprecipitation; In Vitro; Investigation; Label; Lesion; Lipids; Low-Density Lipoproteins; Maps; Messenger RNA; Methods; Molecular; Monkeys; Monocyte Chemoattractant Protein-1; Mus; Phosphorylation; Platelet-Derived Growth Factor Receptor; Play; Proliferating; Proteins; Proteome; Proteomics; RNA; Rat Protein; Rattus; Relative (related person); Role; Signal Transduction; Signaling Molecule; Signaling Protein; Smooth Muscle; Smooth Muscle Myocytes; Time; Translations; Western Blotting; age related; aged; arterial remodeling; caveolin 1; feeding; gel electrophoresis; glycosylation; in vivo; interest; macrophage; medin; monocyte chemoattractant protein 1 receptor; nonhuman primate; novel; novel strategies; overexpression; protein expression; receptor; uptake

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 Fluorescence Difference Gel Electrophoresis (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 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 one protein of interest, MFG-E8, significantly increases in abundance in old rat aortae. 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. 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 ERK-1/2, augments levels of PCNA and CDK4, increases BrdU incorporation and promotes growth. The knockdown of MFG-E8 reduces the rate of cell cycling, accelerating signaling molecules PCNA and CDK4 expression, facilitating cell entry into a growth-arrested state. Furthermore, we find that αvβ5 and PDGF receptor are upregulated with MFG-E8 and also are elements to relay proliferative signals to aging VSMC. In addition, MFG-E8 facilitates the profibrosis of VSMC with aging via an interaction with caveolin 1 (CAV1) signaling. Exposure of isolated aortic VSMC of old rats (30-mo) to MFG-E8 increases the profibrogenic signaling molecules TGF-β1, TGF-β receptor type II (TβRII), p-SMAD-2/3, and collagen I (Col I) while knockdown of MFG-E8 gene expression substantially reduces the expression of these molecules. Both co-immuno-labeling and co-immune-precipitation of MFG-E8, TβRII, and CAV1 in young VSMC (8-mo) indicates their physical interactions. In young VSMC, MFG-E8 up-regulates expression of CAV1. Knockdown of the CAV1 gene, similar to MFG-E8 exposure, significantly increases the expression of TGF-β1, TβRII, p-SMAD-2/3, and Col I. Over-expression of the CAV1 gene, similar to MFG-E8 gene knockdown, markedly decreases the expression of these fibrogenic molecules in both young and old VSMC. Furthermore, MFG-E8 treatment significantly increases the expression of TGF-β1, p-SMAD-2/3, and Col I in CAV1 silenced young VSMC. Interestingly, MFG-E8 exposure does not increase expression of these profibrogenic molecules in old VSMCs when CAV1 is overexpressed. These results, for the first time, demonstrate that MFG-E8 modulates TGF-β1 fibrogenic signaling in VSMC in a CAV1/age-dependent manner. Importantly, we find that the involvement of MFG-E8 in atherosclerotic lesions increases with age. In in vivo studies: 1) Immunostaining demonstrates that MFG-E8 protein in the aortic wall in ApoE-/- mice on a standard chow diet increases with age and is localized predominantly in advanced plaques and the elastin laminae . 2) In nonhuman primates (9 to 23- years old), iTRAQ assay indicates that levels of arterial MFG-E8 protein are highly correlated with age. Arterial MFG-E8 protein expression in older monkeys (>17-years-old) fed a high cholesterol diet for 2 years is further increased 1.6-fold compared to older animals on a standard diet. 3) In aortic thoracic intimae isolated from adult humans (age 22-to 69-years old), Western blot analysis indicates that MFG-E8 protein abundance is significantly increased in atherosclerotic areas compared to the adjoining grossly normal intimae. Notably, the glycosylated form of MFG-E8 is markedly increased in lesion areas and rarely detected in normal intimae. 4) Immunostaining indicates that MFG-E8 is mainly derived from smooth muscle cells (alpha-smooth actin positive) and macrophages (CD 68 positive). In in vitro studies: 1) Early passage thoracic aortic smooth muscle cells (SMC) isolated from young and old monkeys, and exposed to pathogenic oxidative low density lipoprotein (oxLDL) shows that the uptake capacity of lipid in old cells exceeds that of young cells, facilitating trans-differentiation into foam-like cells (CD68 positive). 2) Silencing of MFG-E8 RNA markedly reduced the uptake of oxLDL into SMC. In addition, an MFG-E8 fragment, medin, plays an important role in arterial aging and athersocelrosis. The effects of medin on the behavior of VSMCs with advancing age are under investigation. Taken together, MFG-E8 translation and glycosylation and its fragment medin are increased in atherosclerotic aortic walls with aging in various species, including humans, potentially playing a precipitating role in the formation of foam cells, the hallmark of atherosclerosis.

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。使用二维荧光差异凝胶电泳 (2-D DIGE)，我们获得了来自大鼠主动脉的 301 个已识别非冗余蛋白的二维凝胶图，并观察到 ​​18 个蛋白的丰度随着衰老而显着变化。利用同量异位标签进行相对和绝对定量 (iTRAQ)，对 921 种蛋白质进行了定量，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。 蛋白质组学分析表明，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。 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 ERK-1/2 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 αvβ5 和 PDGF 受体，并且也是将增殖信号传递给老化 VSMC 的元件。 此外，MFG-E8 通过与小窝蛋白 1 (CAV1) 信号传导相互作用，促进 VSMC 随着衰老而促纤维化。将老年大鼠（30 个月）的离体主动脉 VSMC 暴露于 MFG-E8 会增加促纤维化信号分子 TGF-β1、TGF-β II 型受体 (TβRII)、p-SMAD-2/3 和胶原蛋白 I (Col I) ）而 MFG-E8 基因表达的敲低则大大降低了这些分子的表达。年轻 VSMC（8 个月）中 MFG-E8、TβRII 和 CAV1 的免疫共标记和免疫共沉淀均表明了它们的物理相互作用。在年轻的 VSMC 中，MFG-E8 上调 CAV1 的表达。与 MFG-E8 暴露类似，CAV1 基因的敲低显着增加 TGF-β1、TβRII、p-SMAD-2/3 和 Col I 的表达。 CAV1 基因的过度表达，与 MFG-E8 基因类似敲低，显着降低年轻和老年 VSMC 中这些纤维化分子的表达。此外，MFG-E8 处理显着增加了 CAV1 沉默的年轻 VSMC 中 TGF-β1、p-SMAD-2/3 和 Col I 的表达。 有趣的是，当 CAV1 过表达时，MFG-E8 暴露不会增加旧 VSMC 中这些促纤维化分子的表达。这些结果首次证明 MFG-E8 以 CAV1/年龄依赖性方式调节 VSMC 中的 TGF-β1 纤维化信号传导。 重要的是，我们发现 MFG-E8 在动脉粥样硬化病变中的参与随着年龄的增长而增加。 体内研究：1) 免疫染色表明，标准饲料饮食的 ApoE-/- 小鼠主动脉壁中的 MFG-E8 蛋白随着年龄的增长而增加，并且主要位于晚期斑块和弹性蛋白层中。 2) 在非人灵长类动物（9 至 23 岁）中，iTRAQ 测定表明动脉 MFG-E8 蛋白水平与年龄高度相关。与标准饮食的老年动物相比，饲喂高胆固醇饮食 2 年的老年猴子（>17 岁）的动脉 MFG-E8 蛋白表达进一步增加了 1.6 倍。 3) 在从成年人（22 岁至 69 岁）分离的主动脉胸内膜中，Western blot 分析表明，与邻近的总体正常内膜相比，动脉粥样硬化区域的 MFG-E8 蛋白丰度显着增加。值得注意的是，MFG-E8 的糖基化形式在病变区域显着增加，而在正常内膜中很少检测到。 4) 免疫染色表明MFG-E8主要来源于平滑肌细胞（α-smooth actin阳性）和巨噬细胞（CD 68阳性）。体外研究： 1）从幼猴和老年猴中分离的早期传代胸主动脉平滑肌细胞（SMC），并暴露于致病性氧化低密度脂蛋白（oxLDL），表明老年细胞对脂质的摄取能力超过年轻细胞，促进转分化为泡沫样细胞（CD68 阳性）。 2) MFG-E8 RNA 的沉默显着减少了 oxLDL 进入 SMC 的摄取。此外，MFG-E8片段medin在动脉老化和动脉粥样硬化中发挥着重要作用。 随着年龄的增长，medin 对 VSMC 行为的影响正在研究中。综上所述，在包括人类在内的不同物种中，随着年龄的增长，动脉粥样硬化主动脉壁中的 MFG-E8 翻译和糖基化及其片段 medin 都会增加，这可能在泡沫细胞（动脉粥样硬化的标志）的形成中发挥促进作用。