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 VSMC isolated from 8-mo-old rats (young) to MCP-1 , 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, treating young VSMCs with TGF-1 increases the 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 either silencing the MCP-1 gene, treating with a CCR2 antagonist, or MMP-2 inhibition. Certain 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 (8 months) of a broad-spectrum MMP inhibitor, PD166793, via a daily gavage, to 16-month-old rats 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 SMAD 2/3 (Sma and Mad (Mothers against decapentaplegic)-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 increased the collagen content in arterial walls and prevented the development of spontaneous aortic lesions in mice. Heterozygous collagen 3A1 gene (COL3A1)-deficient mice (HT) had doxycycline, a broad spectrum MMP inhibitor added to their food after weaning. At nine months of age, 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 eight times higher than in wild type mice (WT). After nine months of doxycycline treatment, MMP-9 activity, collagen content and lesions in aortae of HT were at the same level asWT. In the aneurismal COL3A1, treatment with a broad spectrum MMP inhibitor started early in life, normalized MMP activity and reduced aortic collagen content in adult animals and prevented the development of spontaneous aortic lesions. These findings provide experimental justification for clinical evaluation in the use of doxycycline in the haploinsufficient variety of vEDS patients. Our recent study has shown that a signaling relationship exists between angiotensin II (Ang II), TGF-1 and vasorin within VSMCs with aging. In vivo studies in old (30-month-old) versus young (8-month-old) FXBN rats show that the aortic transcription and translation levels of vasorin markedly decreases with aging. In vitro studies of early passage VSMCs from old versus young aortae indicate that abundance of vasorin protein 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 when treated with Losartan (Los), an Ang II receptor (AT1) antagonist, 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 these effects are abolished or substantially mitigated by either Los treatment, overexpression of vasorin, or exogenous recombinant human-vasorin protein. In contrast, when old VSMCs are exposed to Los, decreases in the production of p-SMAD2/3 and collagen type I are seen and create an imbalance in the Ang II/TGF-1/vasorin signaling cascade, a feature of the aged arterial wall, that enhances the collagen production by VSMCs. 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, senescence associated secretory phenotypes (SASP), senescence associated protein degradation (SASD) and cell loss over time in the vascular walls, like classical cellular replicative senescence, are considered major contributing factors in the aging proinflammation model such as activated MMP-2/9. 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 fibroblast cell line. We found that serum from CR rats could delay senescence, inactivate MMP-2/9 and significantly increased the replicative lifespan determined cellular population doubling 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 inactivation, and increase replicative lifespan of normal human diploid fibroblasts in vitro and suggest that SIRT1 plays a key role in the activation of MMP-2/9 in the arterial wall with aging. Taken together, this complex local signaling loop of MCP-1/MMP-2/TGF-1 plays a vital role in the initiation and progression of age-associated arterial intimal cellularity, fibrosis and relevant vascular diseases. Blocking this vicious cycle is a potential therapeutic approach to preserving arterial health with advancing age
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