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-β 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的内膜老化大鼠。此外，我们证明MCP-1与TGF-β 1相互作用，位于中心并与炎症级联反应直接相关，而炎症级联反应与MMP-2活化密切相关。 众所周知，年龄相关的动脉重塑涉及动脉壁胶原沉积和弹性蛋白断裂，以及动脉压的增加。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）从断奶开始就接受了添加到食物中的广谱MMP抑制剂强力霉素的治疗。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。 最近，体内研究表明，血管紧张素（一种糖基化蛋白）的转录和翻译在老年（30个月）与年轻（8个月）FXBN大鼠的主动脉壁内显着降低。体外研究表明，与年轻人相比，来自老年人动脉瘤的原代培养的早期传代VSMC中的血管紧张素蛋白水平显著降低。此外，免疫共沉淀显示，血管紧张素和TGF-β 1的相互作用是显着减少VSMC与老化。 重要的是，将Ang II暴露于年轻的VSMC模仿老年细胞或年轻的血管紧张素基因沉默细胞， 增加p-SMAD 2/3和I型胶原蛋白的产生。这些作用被AT 1拮抗剂氯沙坦或血管紧张素过度表达所消除。AT 1拮抗剂暴露于旧VSMC，如vasorin基因的过表达，显著降低p-SMAD 2/3和胶原蛋白的产生。 此外，过度表达血管紧张素实质上抑制迁移/侵袭能力的血管平滑肌细胞与老化或血管紧张素II治疗，伴随着失活的MMP-2。 总之，TGF-β 1/vasorin与Ang II信号级联在年龄相关的VSMC促炎性转变中起决定性作用。因此，血管紧张素是延缓动脉老化的天然分子。 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-beta1, 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-beta1 and is centrally located and directly connected with the inflammation cascade, which is closely associated with MMP-2 activation. It is well known that 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. Recently, in vivo studies show that transcription and translation of vasorin, a glycosylated protein, are markedly decreased within the aortic walls in old (30 mo) vs. young (8 mo) FXBN rats. In vitro studies indicate that levels of vasorin protein in primary cultured early passage VSMC from old aortas are substantially reduced compared to those of young. Furthermore, co-immunoprecipitation reveals that interaction of vasorin and TGF-beta1 is significantly decreased within VSMC with aging. Importantly, exposure of Ang II to young VSMC mimicking old cells or young vasorin gene silenced cells, increases p-SMAD2/3 and collagen type I production. These effects are abolished by an AT1 antagonist, Losartan, or overexpression of vasorin. Exposure of an AT1 antagonist to old VSMC, like overexpression of the vasorin gene, markedly reduces p-SMAD2/3 and collagen production. In addition, overexpression of vasorin substantially inhibited the migration/invasion capacity of VSMC with aging or Ang II treatment, accompanied by an inactivation of MMP-2. Taken together, the TGF-beta1/vasorin-linked with Ang II signaling cascade plays a determinant role in age-associated VSMC pro-inflammatory shift. Thus, vasorin is a naive molecule to retard arterial aging. 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.