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 secreted collagen deposition within a diffusely thickened intima is a salient feature of central arterial wall inflammatory remodeling that accompanies advancing age. However, the molecular and cellular 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 aging aortic wall. Treating VSMCs, isolated from 8-mo-old rats (young) with MCP-1, via the CC-chemokine receptor 2 (CCR-2), produces 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 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 MCP-1, MMP-2, or TGF-1 activity trigger a feed-forward signaling mechanism that is implicated in the initiation and progression of age-associated adverse arterial wall senescent remodeling. Interventions that suppress this signaling loop may potentially lessen age-associated adverse arterial remodeling. The active MMP-associated activation of TGF-1 plays an important role in the stiffening of aging VSMCs. Distinct material properties of primary VSMCs isolated from thoracic aortae of young vs. aged F344XBN rats were observed. Individual VSMCs derived from aged animals shows an internal network of the actin cytoskeleton, exhibiting increased stiffness and frictional (loss) moduli than those are 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 emerged as a specific modifier of age-associated VSMC stiffening in vitro. TGF-1 reinforced the mechanical phenotype of arterial aging in VSMCs on multiple time and length scales through clustering of mechanosensitive 51 and v3 integrins. Taken together, these findings identify a novel nodal point 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 pathologic progression of age associated central arterial wall stiffening. Indeed, we proved that inhibiting MMP-2 activation associated with TGF-1 activation decelerates the 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: (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 proinflammatory signaling and is accompanied by the preservation of intact elastin fibers, a reduction in collagen, and a blunting of age-associated increases in blood pressure. Our recent study showed that a signaling relationship exists between angiotensin II (Ang II), TGF-1 and vasorin within aging VSMCs. 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 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 (Los), 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 are abolished or substantially mitigated by either treating with Los, or through the overexpression of vasorin or exogenous recombinant human-vasorin protein. In contrast, when old VSMCs are treated with Los, 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 adverse extracellular matrix remodeling, a determinant of arterial stiffening. In addition, in collaboration with K Shivakumar (Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, India), using gene knockdown and over-expression approaches, western blotting and promoter pull-down assay, we 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 attenuated Ang II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Ang II-stimulated expression of AT1R, the Ang II receptor, which would link extracellular matrix and Ang II signaling in cardiac fibroblasts. Moreover, conditioned medium collected from DDR2- or fibronectin-silenced cardiac fibroblasts reduced 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 was mediated by Integrin-1-integrin-linked kinase signaling. In vivo, we 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.