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

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。利用二维荧光差异凝胶电泳（2-D DIGE）技术，我们获得了301个大鼠主动脉非冗余蛋白的二维凝胶图谱，并观察到18个随年龄增长而显著变化的蛋白。利用Isobaric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，有50种蛋白质显示出显著不同的年龄相关丰度。 蛋白质组学分析显示，一种感兴趣的蛋白质MFG-E8在老年大鼠睾丸中的丰度显著增加。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。 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MFG-E8上调v5和PDGF受体，它们也是向衰老VSMC传递增殖信号的元件。 此外，MFG-E8通过与caveolin 1（CAV 1）信号转导相互作用促进VSMC的促纤维化。老年大鼠（30个月）的分离的主动脉VSMC暴露于MFG-E8增加了促纤维化信号分子TGF-1、TGF-受体II型（TRII）、p-SMAD-2/3和胶原I（Col I），而MFG-E8基因表达的敲低基本上降低了这些分子的表达。MFG-E8、TRII和CAV 1在年轻VSMC（8-mo）中的共免疫标记和共免疫沉淀表明它们的物理相互作用。在年轻的VSMC中，MFG-E8上调CAV 1的表达。与MFG-E8暴露类似，CAV 1基因的敲低显著增加TGF-1、TRII、p-SMAD-2/3和Col I的表达。与MFG-E8基因敲低类似，CAV 1基因的过表达显著降低了这些纤维化分子在年轻和老年VSMC中的表达。此外，MFG-E8处理显著增加了CAV 1沉默的年轻VSMC中TGF- 1、p-SMAD-2/3和Col I的表达。 有趣的是，当CAV 1过表达时，MFG-E8暴露不会增加这些促纤维化分子在老年VSMC中的表达。这些结果首次证明MFG-E8以CAV 1/年龄依赖性方式调节VSMC中的TGF- 1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）免疫染色证明，在标准食物饮食的ApoE-/-小鼠中，主动脉壁中的MFG-E8蛋白随年龄增加，并且主要定位于晚期斑块和弹性蛋白板层中。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。在喂食高胆固醇饮食2年的老年猴（>17岁）中，动脉MFG-E8蛋白表达与喂食标准饮食的老年动物相比进一步增加1.6倍。3)在分离自成年人（年龄22- 69岁）的主动脉胸内膜中，蛋白质印迹分析表明，与相邻的大体正常内膜相比，MFG-E8蛋白质丰度在动脉粥样硬化区域中显著增加。值得注意的是，MFG-E8的糖基化形式在病变区域中显著增加，并且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-平滑肌肌动蛋白阳性）和巨噬细胞（CD 68阳性）。体外研究：1）分离自年轻和老年猴的早期传代胸主动脉平滑肌细胞（SMC）暴露于致病性氧化低密度脂蛋白（oxLDL）显示，老年细胞的脂质摄取能力超过年轻细胞，促进转分化为泡沫样细胞（CD 68阳性）。2)MFG-E8 RNA沉默显著降低SMC对oxLDL的摄取。此外，MFG-E8片段medin在动脉老化和动脉粥样硬化中起重要作用。 随着年龄的增长，medin对VSMCs行为的影响正在调查中。总之，MFG-E8翻译和糖基化及其片段medin在包括人类在内的各种物种的动脉粥样硬化主动脉壁中随着年龄的增长而增加，可能在泡沫细胞的形成中起沉淀作用，泡沫细胞是动脉粥样硬化的标志。

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.