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

本研究首次在体内和体外研究表明，MCP-1和转化生长因子-β1是一种强大的促纤维化细胞因子，在增厚的大鼠主动脉内膜中显著增加并共同表达。此外，我们还发现MCP-1与转化生长因子-β1相互作用，并位于中央，与炎症反应直接相连，而炎症反应与基质金属蛋白酶-2的激活密切相关。 众所周知，与年龄相关的动脉重塑包括动脉壁胶原沉积和弹性蛋白断裂，以及动脉压的增加。我们验证了这样一种假设，即抑制基质金属蛋白酶的激活可以减缓与年龄相关的动脉前炎症及其伴随的动脉压升高。事实上，通过每天灌胃给16个月大鼠长期服用广谱基质金属蛋白酶抑制剂PD166739 8个月，显著降低了预期的与年龄相关的动脉压升高。伴随而来的是：(1)抑制与年龄相关的主动脉明胶酶和间质胶原酶活性；(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-β1的激活；(5)促纤维化信号分子SMAD-2/3的磷酸化活性降低；(6)抑制前内皮素1的激活；以及(7)下调ETS-1的表达。总而言之，我们的结果表明，抑制基质金属蛋白酶延缓了与年龄相关的动脉促炎信号，同时伴随着完整的弹性蛋白纤维的保存，胶原的减少，以及与年龄相关的血压上升的钝化。 此外，对于血管型Ehler-Danlos综合征(VEDS)，还没有得到证实的治疗或预防方法，vEDS是一种遗传性疾病，与金属蛋白酶(MMP)活性增加、动脉壁胶原含量减少以及主动发展的不同严重程度的主动脉病变有关。我们推测，慢性应用基质金属蛋白酶抑制剂可以增加胶原含量，防止自发性主动脉病变的发展。事实上，COL3A1缺陷型杂合子小鼠(HT)在断奶后就在食物中添加了广谱的基质金属蛋白酶抑制剂多西环素。9个月龄时，未治疗组小鼠主动脉中膜基质金属蛋白酶-9的表达是野生型小鼠的两倍，而总胶原含量比野生型小鼠低30%，主动脉病变积分是野生型小鼠的8倍。经多西环素治疗9个月后，大鼠主动脉中的基质金属蛋白酶-9活性、胶原含量及病变程度均达到西药水平。在I型胶原单倍化不全的动脉瘤小鼠模型中，早期开始使用广谱基质金属蛋白酶抑制剂可使成年小鼠的主动脉中基质金属蛋白酶活性升高，胶原含量降低，从而防止自发性主动脉病变的发生。这些发现为临床评估多西环素的益处提供了实验依据，至少在单倍体缺陷的vEDS患者中是如此。 最近，体内研究表明，老年(30mo)FXBN大鼠与青年(8mo)FXBN大鼠相比，主动脉壁中糖基化蛋白Vasorin的转录和翻译显著减少。体外研究表明，原代培养的老年大动脉VSMC较年轻VSMC的VSMC中血管蛋白水平显著降低。此外，免疫共沉淀显示VSMC中血管生成素和转化生长因子-β1的相互作用随着年龄的增长而显著减少。重要的是，Ang II暴露于年轻的VSMC，模拟老年细胞或年轻的血管球蛋白基因沉默细胞，增加p-Smad2/3和I型胶原的产生。AT1拮抗剂暴露于陈旧的VSMC，就像Vasorin基因的过度表达一样，显著减少p-Smad2/3和胶原的产生。此外，Vasorin的过度表达显著抑制了衰老或Ang II处理的VSMC的迁移/侵袭能力，并伴随着MMP2的失活。综上所述，转化生长因子-β1/血管生成素与血管紧张素Ⅱ信号转导通路在年龄相关的VSMC促炎转变中起决定性作用。因此，血管紧张素是一种天然的延缓动脉衰老的分子。 综上所述，MCP-1/MMP-2/TGF-β1这个复杂的局部信号环在年龄相关动脉内膜细胞和纤维化以及包括动脉瘤在内的相关血管疾病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动脉健康治疗方法。

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.