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

National Institute On Aging

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Abstract

The coexistence of vascular smooth muscle cell (VSMC) cellularity via invasion and proliferation and excessive collagen secretion within a diffusely thickened intima is a hallmark of central arterial wall inflammatory remodeling that accompanies advancing age. However, the molecular and cellular mechanisms involved remain undefined. Immunostaining and immunoblotting of rat aortic walls demonstrate that a triad of proinflammatory molecules monocyte chemoattractant protein-1 (MCP-1), transforming growth factor beta 1 (TGF-1), and matrix metalloprotein type II (MMP-2) increase within the aging aortic wall. Treating VSMCs, isolated from 8-mo-old Fisher 344 crossbreed Brown Norway (FBN) rats (young) with MCP-1, via the CC-chemokine receptor 2 (CCR-2), promotes both an increase in TGF-1 activity, up to levels of untreated VSMCs isolated 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 isolated from old rats. This autocatalytic signaling loop that enhances collagen production and invasiveness of VSMCs is effectively suppressed by silencing the MCP-1 gene, treating with a CCR2 antagonist, or through the inhibition of MMP-2. Certain levels of activated MCP-1, MMP-2, or TGF-1 trigger a feed-forward signaling mechanism that is implicated in the initiation and progression of age-associated adverse arterial senescent remodeling. Importantly, the active MMP-associated activation of TGF-1 plays an important role in the stiffening of aging VSMCs and arterial walls. Distinct mechanical properties of primary VSMCs isolated from thoracic aortae of young vs. aged F344XBN rats have been observed. Individual VSMCs derived from aged animals shows an internal network of the actin cytoskeleton exhibits increased stiffness and frictional (loss) moduli compared with those cells derived from the young animals. This discrete mechanical response is long-lived in culture and is persistent across a physiological range of matrix rigidity. Strikingly, TGF-1 has been reported to emerge as a specific modifier of age-associated VSMC stiffening. TGF-1 reinforces the mechanical phenotype of arterial aging in VSMCs on multiple time and length scales through clustering of mechanosensitive receptors 51 and v3 integrins. Taken together, our findings identify a novel signaling node for long-range regulation of VSMC stiffness and serve as further proof-of-concept that the broad-based inhibition of TGF-1 expression or TGF-1 signal transduction in VSMCs may be a useful therapeutic approach to mitigate the age associated arterial wall stiffening. Very Importantly, our findings indicate that inhibiting MMP-2 activation associated with TGF-1 activation decelerates age-associated arterial proinflammation and its attendant increase in arterial systolic blood pressure. Eight months of chronic administration 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 effects: (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) an increase of vasorin, an inhibitor of TGF-1 signaling; (6) a decrease in the activity of the profibrogenic signaling molecule SMAD 2/3 (Sma and Mad (Mothers against decapentaplegic)-2/3) phosphorylation; (7) an inhibition of proendothelin 1 activation; and (8)a downregulation of expression of V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1 (Ets-1). Collectively, our results indicate that MMP inhibition diminishes age-associated arterial proinflammation, which is accompanied by the preservation of an intact elastin network, a reduction in collagen, and a blockade of age-associated increases in blood pressure. More importantly, our recent study has demonstrated that a signaling interrelationship exists between angiotensin II (Ang II), TGF-1, MMP-2 and vasorin within aging VSMCs. In vivo studies in old (30-month-old) versus young FXBN rats have showed that the aortic transcription and translation levels of vasorin markedly decreases with aging. In vitro studies of early passage VSMCs from old versus young rat aortae indicate that the abundance of vasorin protein is substantially reduced. Ang II-associated reduction of vasorin protein abundance in young VSMCs and age-associated changes in vasorin protein levels are reversed when treated with Losartan, an Ang II receptor (AT1) antagonist, in both in vitro and in vivo conditions, suggesting constitutive activation of AT1 signaling within the aged arterial wall. Dual immunolabeling and co-immunoprecipitation demonstrate that the co-incidence and physical interaction of vasorin and TGF-1 within aging VSMCs are significantly decreased. Importantly, treating young VSMCs and young animals with Ang II increases p-SMAD2/3 and collagen type I production, mimicking old cells and this is i abolished or substantially mitigated by either treating with Losartan, or through the overexpression of vasorin or exogenous recombinant human-vasorin protein. In contrast, when old VSMCs are treated with Losartan, there is a decrease in the production of p-SMAD2/3 and collagen type I. In the aged arterial wall, an imbalance in the Ang II/TGF-1/vasorin signaling cascade is created that enhances collagen production in VSMCs. In addition, age-associated arterial vasorin is closely associated with an enhanced capacity of MMP activation. Activated MMP-2/9 cleaves the full-length of vasorin, and is blocked by the MMP Inhibitor, GM6001, in vitro and PD166793, in vivo. Thus, maintaining the balance of the full-length vasorin/TGF-1 signaling is a novel approach to hinder age-associated MMP-2 activation and arterial adverse extracellular matrix remodeling, a determinant of arterial stiffening. In addition, in collaboration with K Shivakumar, using gene knockdown and over-expression approaches, western blotting and promoter pull-down assay, we have showed that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Ang II-stimulated transcriptional up-regulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Further, siRNA-mediated fibronectin knockdown attenuates Ang II-stimulated expression of collagen type I and anti-apoptotic cIAP2 and enhances susceptibility to apoptosis. Importantly, an obligate role for fibronectin has been observed in Ang II-stimulated expression of AT1R, the Ang II receptor, which potentially link extracellular matrix with Ang II signaling in cardiac fibroblasts. Moreover, conditioned medium collected from DDR2- or fibronectin-silenced cardiac fibroblasts has been shown to reduce AT1R expression in H9c2 cardio-myoblasts. The regulatory role of fibronectin in Ang II-stimulated baculoviral IAP repeat-containing protein3 (cIAP2), collagen type I and AT1R expression is mediated by Integrin-1-integrin-linked kinase signaling. In vivo, we have observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, associated with the previously reported reduction in myocardial Integrin-1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest that there is a complex mechanism for regulating cardiac fibroblast function that involves two major extracellular matrix proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-1.