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

在这项研究中，第一个在体内和体外研究表明，MCP-1和TGF-1，一个强大的促纤维化细胞因子，显着增加和共表达的主动脉壁内增厚内膜的老龄大鼠。此外，我们证明MCP-1与TGF- 1相互作用，位于中心并直接与炎症级联反应相关，而炎症级联反应与MMP-2活化密切相关。 动脉粥样硬化相关的动脉重塑涉及动脉壁胶原沉积和弹性蛋白断裂，以及动脉压升高。我们测试了抑制MMP活化可以减缓年龄相关的动脉促炎症反应及其伴随的动脉压升高的假设。事实上，通过每日灌胃对16个月大的大鼠长期给予广谱MMP抑制剂PD 166739，持续8个月，显著减弱了预期的与年龄相关的动脉压升高。这伴随着以下情况：（1）抑制与年龄相关的主动脉明胶酶和间质胶原酶活性原位增加;（2）保持弹性纤维网络的完整性;（3）减少胶原沉积;（4）减少单核细胞趋化蛋白1和转化生长因子β 1活化;（5）减少促纤维化信号分子SMAD-2/3磷酸化的活性;（6）抑制内皮素原1活化;和（7）下调ets-1的表达。总的来说，我们的研究结果表明，MMP抑制延缓年龄相关的动脉促炎信号传导，这是伴随着保存完整的弹性蛋白纤维，减少胶原蛋白，并钝化与年龄相关的血压升高。 此外，对于血管型埃勒斯-当洛斯综合征（vEDS），没有经证实的治疗或预防，这是一种与金属蛋白酶（MMP）活性增加、动脉壁中胶原蛋白含量减少以及主动脉中不同严重程度病变的自发发展相关的遗传性疾病。我们假设MMP抑制剂的长期治疗会增加胶原含量，防止自发性主动脉病变的发展。事实上，杂合子COL 3A 1缺陷型小鼠（HT）从断奶时起就用添加到食物中的广谱MMP抑制剂强力霉素进行治疗。在9个月大时，MMP-9在未经治疗的HT的主动脉中膜图尼卡中的表达是野生型小鼠WT的两倍，而总胶原含量低30%，主动脉病变的累积评分高8倍。强力霉素治疗9个月后，HT组主动脉MMP-9活性、胶原含量及病变程度均与WT组相当。在III型胶原单倍不足的糖尿病小鼠模型中，在生命早期开始使用广谱MMP抑制剂治疗使MMP活性增加正常化，并降低成人的主动脉胶原含量，并防止自发性主动脉病变的发展。这些发现为临床评价多西环素至少在单倍不足的vEDS患者中的获益提供了实验依据。 近年来的研究表明血管平滑肌细胞内的Ang Ⅱ/TGF-1/vasorin信号通路与衰老密切相关。在老年（30月龄）与年轻（8月龄）Fisher 344杂交挪威棕（FXBN）大鼠中的体内研究表明，血管紧张素的主动脉转录和翻译随着年龄的增长而显著降低。在体外研究早期传代VSMC从老年人与年轻人相比，血管紧张素蛋白的丰度大大减少与老化。血管紧张素II受体AT 1拮抗剂Losartan（Los）可逆转年轻VSMC中血管紧张素II相关的vasorin蛋白丰度降低和血管紧张素蛋白水平的年龄相关变化，表明随着年龄的增长，动脉壁内AT 1信号传导的组成性激活。双重免疫标记和免疫共沉淀表明，血管紧张素和TGF- 1在血管平滑肌细胞内的共同发病率和物理相互作用显着下降，随着年龄的增长。 重要的是，年轻的VSMC暴露于Ang II增加p-SMAD 2/3和I型胶原蛋白的产生，模仿老年细胞，并且这种作用被Los治疗、异位血管紧张素的过表达或外源性重组人血管紧张素蛋白消除或基本上减轻。相比之下，旧VSMC暴露于Los会降低p-SMAD 2/3和I型胶原的产生。 因此，血管紧张素II/TGF-1/血管紧张素信号级联的失衡，一个老化的动脉壁的特征，增强了VSMC的胶原蛋白的生产。因此，维持血管紧张素/TGF- 1信号的平衡是一种新的措施，以延缓不利的年龄相关的细胞外基质重塑，动脉硬化的决定因素。 总之，MCP-1/MMP-2/TGF-β 1的这种复杂的局部信号传导回路在年龄相关的动脉内膜细胞结构和纤维化以及相关血管疾病（包括动脉瘤）的起始和进展中起着基础作用。中断这种恶性循环是动脉健康的潜在治疗方法。

In this study, the first in vivo and in vitro studies show that MCP-1 and TGF-β1, a powerful profibrogenic cytokine, markedly increase and co-expression within the aortic wall in the thickened intima of aging rats. Furthermore, we document that MCP-1 interacts with TGF-β1 and is centrally located and directly connected with the inflammation cascade, which is closely associated with MMP-2 activation. Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, as well as an increase in arterial pressure. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad-spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-month-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of monocyte chemoattractant protein 1 and transforming growth factor-beta1 activation; (5) a diminution in the activity of the profibrogenic signaling molecule SMAD-2/3 phosphorylation; (6) inhibition of proendothelin 1 activation; and (7) downregulation of expression of ets-1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by preservation of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure. In addition, there is no proven therapy or prevention for the vascular type Ehlers-Danlos syndrome (vEDS), a genetic disorder associated with increased metalloproteinase (MMP) activity, reduced collagen content in the arterial walls, and spontaneous development of lesions of varying severity in the aorta. We hypothesized that chronic treatment with MMP inhibitor would increase the collagen content and prevent the development of spontaneous aortic lesions. Indeed, heterozygous COL3A1-deficient mice (HT) were treated since weaning with broad spectrum MMP inhibitor, doxycycline, added to food. At the age of 9 months MMP-9 expression was twice as high in tunica media of aorta in untreated HT, while total collagen content was 30% lower and the cumulative score of aortic lesions was 8 times higher than in wild type mice WT. After 9 months of doxycycline treatment, MMP-9 activity, collagen content and lesions in aorta of HT were at the level of WT. In the aneurismal mouse model of collagen III haploinsufficiency, treatment with broad spectrum MMP inhibitor started early in life normalized increased MMP activity and reduced aortic collagen content in adult and prevented development of spontaneous aortic lesions. These findings provide experimental justification for clinical evaluation of the benefit of doxycycline at least in the haploinsufficient variety of vEDS patients. Recent study has shown the Ang II/TGF-β1/vasorin signaling relationship within VSMC with aging. In vivo studies in old (30-month-old) versus young (8-month-old) Fisher 344 Crossbred Norway Brown (FXBN) rat show that aortic transcription and translation of vasorin markedly decreases with aging. In vitro studies in early passage VSMC from old versus young aortae indicate that vasorin protein abundance is substantially reduced with aging. Ang II-associated reduction of vasorin protein abundance in young VSMC and age-associated changes in vasorin protein levels are reversed by the Ang II receptor AT1 antagonist, Losartan (Los), suggesting constitutive activation of AT1 signaling within the arterial wall with aging. Dual immunolabeling and co-immunoprecipitation demonstrate that the co-incidence and physical interaction of vasorin and TGF-β1 within VSMC are significantly decreased with aging. Importantly, exposure of young VSMC to Ang II increases p-SMAD2/3 and collagen type I production, mimicking old cells, and this effect is abolished or substantially mitigated by Los treatment, overexpression of ectopic vasorin, or exogenous recombinant human-vasorin protein. In contrast, exposure of old VSMC to Los decreases p-SMAD2/3 and collagen type I production. Thus, an imbalance of the Ang II/TGF-β1/vasorin signaling cascade, a feature of the aged arterial wall, enhances the collagen production by VSMC. Thus, maintaining the balance of vasorin/TGF-β1 signaling is a novel measure to retard adverse age-associated extracellular matrix remodeling, a determinant of arterial stiffening. 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 and relevant vascular diseases, including aneurysm. Interruption of this vicious cycle is a potential therapeutic approach to arterial health.