Progression of Arterial Aging: the Local MCP-1/MMP-2/TGF-beta 1 Signaling Loop

在本研究中，双重染色显示，随着年龄的增长，MCP-1和转化生长因子-β1(一种强大的促纤维化细胞因子)在大鼠、非人灵长类动物和人类增厚的动脉内膜中显著增加并共存于主动脉壁内。心血管信号网络分析表明，MCP-1与转化生长因子-β1相互作用，处于中心位置，与炎症级联反应直接相连，炎症反应与基质金属蛋白酶-2的激活密切相关。体外研究表明，MCP-1通过CCR-2信号途径，以剂量或时间依赖的方式上调已知的潜在转化生长因子-β1激活剂MMP2，使幼年(8mo)大鼠主动脉VSMC的MMP2水平达到老年细胞(30mo)水平。单核细胞趋化蛋白-1处理增加激活的细胞内外转化生长因子-β1及其下游分子I型和III型胶原，这依赖于年轻VSMC中MMP2的激活，达到未处理的老年细胞的水平。此外，随着年龄的增长，细胞激活的转化生长因子-β1在VSMC亚组分中分布和增加，包括胞浆、细胞器和细胞核。有趣的是，通过siRNA敲除MCP-1或通过腺病毒转染过表达异位TIMP-2会以一种依赖于MMP-2激活的方式减少年轻细胞的激活和转化生长因子-β1的激活以及年轻细胞侵袭能力的产生，类似于未经处理的老年细胞。CCR-2和一种基质金属蛋白酶抑制剂GM 6001都能显著降低这些影响。值得注意的是，转化生长因子-β1处理以剂量依赖的方式增加年轻VSMC的MCP-1、MMP-2和VSMC侵袭力，达到老年未处理细胞的水平。 此外，我们还证明了一种新的蛋白质Vasorin在老年动脉壁中显著下调，这与转化生长因子-β1活性增加和动脉纤维化密切相关。进一步的体外研究表明，增龄还上调了VSMC中转化生长因子-β1、Smad2和I型胶原的表达；血管紧张素转换酶抑制剂对年轻大鼠VSMC中的转化生长因子-β1、Smad2和I型胶原的表达上调至与老年细胞相同的水平；与年轻细胞相比，增龄大鼠VSMC中Vasorin的表达下调；增龄大鼠VSMC中的转化生长因子-β1与血管生成素相互作用，增龄影响转化生长因子-β1信号转导通路和I型胶原的表达；血管生成素的过度表达拮抗血管紧张素诱导的转化生长因子-β1信号通路和I型胶原的表达。 综上所述，MCP-1/MMP-2/TGF-β1这个复杂的局部信号环在AGE相关动脉内膜细胞化和纤维化的发生和发展中起着基础性的作用。阻断这种恶性循环是一种潜在的老年人动脉健康的治疗方法。

In this study, dual staining shows that MCP-1 and TGF-beta1, a powerful profibrogenic cytokine, markedly increase and co-localize within the aortic wall in the thickened intima of rats, nonhuman primates, and humans with aging. Cardiovascular signaling network analysis indicates that MCP-1 interacts with TGF-beta1 and is centrally located and directly connected with the inflammation cascade, which is closely associated with MMP-2 activation. In vitro study shows that MCP-1 elevates MMP2, a known activator of latent TGF-beta1 in a dose- or time-dependent manner through CCR-2 signaling in cultured VSMC from young (8 mo) rat aortae, reaching the levels of old cells (30mo). MCP-1 treatment increases activated intracellular and extracellular TGF-beta1 and its downstream molecules collagen types I and III, which is dependent upon MMP-2 activation in young VSMC, reaching levels of untreated old cells. Furthermore, cellular activated TGF-beta1 is distributed and increased in VSMC sub-fractions, including cytosol, the organelles and the nuclei, with aging. Interestingly, knockdown of MCP-1 via siRNA or overexpression of ectopic TIMP-2 by adenovirus transfection reduces activation of MMP-2 and TGF-beta1 and production of invasive capacity of young cells in an MMP-2 activation-dependent manner, resembling that of untreated old cells. These effects are substantially reduced by both CCR-2 and an MMP inhibitor, GM 6001. Of note, TGF-beta1 treatment increases MCP-1, MMP-2, and VSMC invasiveness in a dose-dependent manner in young VSMC, up to levels of old untreated cells. Furthermore, we demonstrate that a novel protein, Vasorin, is markedly downregulated in aged arterial wall, which is closely associated with an increase of TGF-beta1 activity and arterial fibrosis. Further in vitro studies indicate that aging also upregulated TGF-beta1, SMAD 2 and collagen I expression VSMC; AngII treatment of young VSMC upregulated the levels of TGF-beta1, SMAD 2 and collagen I expression up to the same levels as from old cells; Aging downregulated Vasorin expression in old rat VSMC as compared with young; TGF-beta1 interacts with Vasorin in rat VSMC, Aging affects the interaction of TGF-beta1 with Vasorin; Overexpression of vasorin counteract TGF-beta1 signaling pathway and collagen I expression in VSMC from old rat; Overexpression of Vasorin counteracts TGF-beta1 signaling pathway and collagen I expression induced by AngII treatment in VSMC from in young rat. Taken together, this complex local signaling loop of MCP-1/MMP-2/TGF-beta1 plays a bedrock role in the initiation and progression of age-associated arterial intimal cellularity and fibrosis. Interruption of this vicious cycle is a potential therapeutic approach to arterial health in the elderly.