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

血管平滑肌细胞（VSMC）浸润和胶原沉积共存于弥漫性增厚的内膜内，是随着年龄增长而出现的中央动脉壁炎症的显著特征。然而，所涉及的分子机制仍未明确。大鼠主动脉的免疫染色和免疫印迹显示有三组促炎分子；随着年龄的增长，主动脉壁内MCP-1、TGF-1和MMP-2升高。MCP-1效应通过cc趋化因子受体2 （CCR-2）信号通路暴露于8岁大鼠（年轻）分离的VSMC中，产生TGF-1活性增加，达到30岁（年老）FXBN大鼠未处理VSMC的水平，同时MMP-2激活增加。此外，年轻的VSMC暴露于TGF-1会使MCP-1水平和MMP-2激活水平增加到未治疗的老年大鼠VSMC水平。通过沉默-MCP-1基因、CCR2拮抗剂或抑制MMP-2有效抑制这种增强胶原生成和VSMC侵袭性的自催化信号环。MCP-1、MMP-2或TGF-1活性的阈值水平触发前馈信号机制，该机制涉及与年龄相关的不良动脉壁重塑的开始和进展。抑制这一信号循环的干预措施可能会潜在地延缓与年龄相关的不良动脉重塑。

The coexistence of vascular smooth muscle cell (VSMC) infiltration and collagen deposition within a diffusely thickened intima is a salient feature of central arterial wall inflammation that accompanies advancing age. However, the molecular mechanisms involved remain undefined. Immunostaining and immunoblotting of rat aortae demonstrate that a triad of proinflammatory molecules; MCP-1, TGF-1, and MMP-2 increase within the aortic wall with aging. Exposure of MCP-1 effects to VSMC isolated from 8-mo-old rats (young) , via CC-chemokine receptor 2 CCR-2 signaling, produce both an increase in TGF-1 activity, up to levels of untreated VSMC from 30-mo-old (old) FXBN rats, and a concurrent increase in MMP-2 activation. Furthermore, exposure of young VSMC to TGF-1 increases levels of MCP-1, and MMP-2 activation, to levels of untreated VSMC from old rats. This autocatalytic signaling loop that enhances collagen production and invasiveness of VSMC is effectively suppressed by silencing -MCP-1 gene, a CCR2 antagonist, or MMP-2 inhibition. Threshold levels of MCP-1, MMP-2, or TGF-1 activity trigger a feed-forward signaling mechanism that is implicated in the initiation and progression of adverse age-associated arterial wall remodeling. Interventions that suppress this signaling loop may potentially retard age-associated adverse arterial remodeling. Indeed, we proved that inhibition of MMP activation decelerates the age-associated arterial proinflammation and its attendant increase in arterial pressure. Chronic administration of a broad-spectrum MMP inhibitor, PD166793, 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 MCP-1 and TGF-1 activation; (5) a diminution in the activity of the profibrogenic signaling molecule Sma and Mad (Mothers against decapentaplegic)-2/3 (SMAD-2/3) phosphorylation; (6) inhibition of proendothelin 1 activation; and (7) downregulation of expression of V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1 ( 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, the vascular type Ehlers-Danlos syndrome (vEDS), a genetic disorder that is associated with increased MMP activity, reduced collagen content in the arterial walls, and spontaneous development of lesions of varying severity in the aorta. Chronic treatment with an MMP inhibitor increases the collagen content and prevents the development of spontaneous aortic lesions. Heterozygous collagen 3A1 gene (COL3A1)-deficient mice (HT) was treated after weaning with a broad spectrum MMP inhibitor, doxycycline, added to their food. At the age of 9 months, MMP-9 expression was twice as high in tunica media of aortae 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 aortae of HT were at the level of WT. In the aneurismal mouse model of collagen III haploinsufficiency, treatment with a 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 in the haploinsufficient variety of vEDS patients. A recent study has shown the angiotensin II (Ang II)/transforming growth factor beta1 (TGF-1)/vasorin signaling relationship within VSMC with aging. In vivo studies in old (30-month-old) versus young (8-month-old) 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 with treatment of 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-1 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, creating an imbalance of the Ang II/TGF-1/vasorin signaling cascade, a feature of the aged arterial wall, that enhances the collagen production by VSMC. In addition, a recent study indicates that vasorin protein expression is decreased in human grossly normal aortic walls. Thus, maintaining the balance of the vasorin/TGF-1 signaling is a novel approach to retard adverse age-associated extracellular matrix remodeling, a determinant of arterial stiffening. Interestingly, the cumulative effects of cellular senescence and cell loss over time in the vascular walls, similar to classical cellular replicative senescence, are considered major contributing factors to the aging proinflammation model such as activated MMP-2/9. Herein, an in vitro model of calorie restriction (CR) was used to study the effects of this dietary regime on replicative senescence, cellular lifespan, MMP-2/9 activation and modulation of the silent mating type information regulation 2 homolog (SIRT1) signaling pathway in normal human diploid fibroblasts. We found that serum from calorie-restricted animals was able to delay senescence, inactivate MMP-2/9 and significantly increased the replicative lifespan in these cells, when compared to serum from ad libitum fed animals. These effects correlated with CR-mediated increases in SIRT1 and decreases in p53 expression levels. Our results demonstrate that CR can delay senescence, MMP-2/9 inactvation, and increase replicative lifespan of normal human diploid fibroblasts in vitro and likely suggest that SIRT1 plays an important role in the activation of MMP-2/9 in the arterial all with 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. Blockade of this vicious cycle is a potential therapeutic approach to arterial health with advancing age