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

在本研究中，首次在体内和体外研究表明，MCP-1和转化生长因子-1，一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，我们还发现单核细胞趋化蛋白-1与转化生长因子-1相互作用，并且与炎症级联反应直接相连，而炎症反应与基质金属蛋白酶-2的激活密切相关。 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。 最近的研究表明血管紧张素Ⅱ/转化生长因子-1/血管紧张素转运蛋白信号转导关系与年龄有关。对老年(30个月大)和年轻(8个月大)的Fisher 344杂交挪威布朗(FXBN)大鼠的活体研究表明，随着年龄的增长，主动脉血管球蛋白的转录和翻译显著减少。体外研究表明，随着年龄的增长，VSMC的血管蛋白丰度显著降低。Ang II受体AT1拮抗剂氯沙坦(Losartan，LOS)逆转了年轻VSMC中Ang II相关的Vasorin蛋白丰度降低和年龄相关的Vasorin蛋白水平的变化，提示随着年龄的增长，动脉壁内AT1信号的结构性激活。双重免疫标记和免疫共沉淀显示血管球蛋白和转化生长因子-1在VSMC内的共存和物理相互作用随着年龄的增长而显著减少。重要的是，年轻的VSMC暴露于Ang II会增加p-Smad2/3和I型胶原的产生，模拟老年细胞，这种作用可以被LOS处理、异位血管球蛋白的过度表达或外源重组人血管球蛋白所消除或实质上减轻。相反，陈旧的VSMC暴露于LOS会减少p-Smad2/3和I型胶原的产生。因此，血管紧张素Ⅱ/转化生长因子-1/血管球蛋白信号级联的失衡，这是衰老动脉壁的一个特征，增强了VSMC产生胶原的能力。因此，维持血管蛋白/转化生长因子信号的平衡是延缓与年龄相关的不良细胞外基质重塑的新措施，细胞外基质重塑是动脉硬化的决定因素。 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-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.