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 deposition 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, immunoblotting, and RT-PCR 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) mRNA and protein 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 in culture, 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 potentially drives the initiation and progression of age-associated adverse arterial senescent proinflammatory remodeling. 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 exhibiting 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. Notably, similar observations have been found in the perivascular adventitial fibroblasts with advancing age, The broad-based inhibition of TGF-1 expression or signal transduction in VSMCs and adventitial fibroblasts may be a useful therapeutic approach to mitigate the age associated arterial wall stiffening, hypertension, atherosclerosis, and age-associated vascular-related dementia. 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). Importantly, MMP inhibition significantly reduce the degradation of tropoelastin in VSMCs, a core element of elastin fibrils. Collectively, MMP inhibition diminishes age-associated arterial proinflammation and age-associated increases in systolic blood pressure. 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. In addition, age-associated arterial vasorin is closely associated with an enhanced capacity of MMP activation. Thus, maintaining the critical 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. In collaboration with K Shivakumar, we have demonstrated 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. In vivo, modestly reduced basal levels of AT1R in DDR2-null mouse myocardium is associated with a reduction of 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. Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt cardiovascular function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. ARA290 ameliorates age-associated cardiac inflammation and is linked to structural and functional changes in the heart. We found that chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-B, and p-NF-B. Thus, administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system with aging leading to amelioration of frailty and preserved health span. We also recently found that an increased microRNA 34 (miR34) in the arterial wall and VSMCs is known to accompany aging in rats and nonhuman primates, which strongly activate MMP2. Importantly, the age-associated increase in miR-34a was causally linked to Ang II signaling within arterial all or VSMCs. Conversely the age-associated increase in Ang II signaling was potentially associated with the miR-34a associated downregulation in Ang II Receptor-Associated Protein (AGTRAP) and situin1 (SIRT1) within the arterial walls or cells. In vitro studies show that MFG-E8 increases the proliferation of VSMCs via an acceleration of cell cycle while miR-34a increases the senescence and invasion of VSMCs via a deceleration of cell cycle and activation of MMP-2. Thus, controlling the age-associated arterial miR-34a is a potential approach to curbing Ang II-linked proinflammation and VSMC heterogeneity and slowing arterial aging