The Senescent Heart-"Age Doth Wither Its Infinite Variety".

Cardiovascular diseases are a leading cause of morbidity and mortality world-wide. While many factors like smoking, hypertension, diabetes, dyslipidaemia, a sedentary lifestyle, and genetic factors can predispose to cardiovascular diseases, the natural process of aging is by itself a major determinant of the risk. Cardiac aging is marked by a conglomerate of cellular and molecular changes, exacerbated by age-driven decline in cardiac regeneration capacity. Although the phenotypes of cardiac aging are well characterised, the underlying molecular mechanisms are far less explored. Recent advances unequivocally link cardiovascular aging to the dysregulation of critical signalling pathways in cardiac fibroblasts, which compromises the critical role of these cells in maintaining the structural and functional integrity of the myocardium. Clearly, the identification of cardiac fibroblast-specific factors and mechanisms that regulate cardiac fibroblast function in the senescent myocardium is of immense importance. In this regard, recent studies show that Discoidin domain receptor 2 (DDR2), a collagen-activated receptor tyrosine kinase predominantly located in cardiac fibroblasts, has an obligate role in cardiac fibroblast function and cardiovascular fibrosis. Incisive studies on the molecular basis of cardiovascular aging and dysregulated fibroblast function in the senescent heart would pave the way for effective strategies to mitigate cardiovascular diseases in a rapidly growing elderly population.

Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2.

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a Collagen receptor tyrosine kinase, has an obligate regulatory role in Collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and overexpression approaches, the present study examined whether DDR2 is a target of metformin and whether, by targeting DDR2, it inhibits Fibronectin and Collagen type I expression in rat aortic adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-ß1/SMAD2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of Fibronectin and Collagen type I, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.

Discoidin Domain Receptor 2 Regulates AT1R Expression in Angiotensin II-Stimulated Cardiac Fibroblasts via Fibronectin-Dependent Integrin-ß1 Signaling.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-ß1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-ß1 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 a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-ß1.

Coordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts.

Relative resistance to apoptosis and the ability to proliferate and produce a collagen-rich scar determine the critical role of cardiac fibroblasts in wound healing and tissue remodeling following myocardial injury. Identification of cardiac fibroblast-specific factors and mechanisms underlying these aspects of cardiac fibroblast function is therefore of considerable scientific and clinical interest. In the present study, gene knockdown and overexpression approaches and promoter binding assays showed that discoidin domain receptor 2 (DDR2), a mesenchymal cell-specific collagen receptor tyrosine kinase localized predominantly in fibroblasts in the heart, acts via ERK1/2 MAPK-activated serum response factor (SRF) transcription factor to enhance the expression of antiapoptotic cIAP2 in cardiac fibroblasts, conferring resistance against oxidative injury. Furthermore, DDR2 was found to act via ERK1/2 MAPK-activated SRF to transcriptionally upregulate Skp2 that in turn facilitated post-translational degradation of p27, the cyclin-dependent kinase inhibitor that causes cell cycle arrest, to promote G1-S transition, as evidenced by Rb phosphorylation, increased proliferating cell nuclear antigen (PCNA) levels, and flow cytometry. DDR2-dependent ERK1/2 MAPK activation also suppressed forkhead box O 3a (FoxO3a)-mediated transcriptional induction of p27. Inhibition of the binding of collagen type I to DDR2 using WRG-28 indicated the obligate role of collagen type I in the activation of DDR2 and its regulatory role in cell survival and cell cycle protein expression. Notably, DDR2 levels positively correlated with SRF, cIAP2, and PCNA levels in cardiac fibroblasts from spontaneously hypertensive rats. To conclude, DDR2-mediated ERK1/2 MAPK activation facilitates coordinated regulation of cell survival and cell cycle progression in cardiac fibroblasts via SRF.NEW & NOTEWORTHY Relative resistance to apoptosis and the ability to proliferate and produce a collagen-rich scar enable cardiac fibroblasts to play a central role in myocardial response to injury. This study reports novel findings that mitogen-stimulated cardiac fibroblasts exploit a common regulatory mechanism involving collagen receptor (DDR2)-dependent activation of ERK1/2 MAPK and serum response factor to achieve coordinated regulation of apoptosis resistance and cell cycle progression, which could facilitate their survival and function in the injured myocardium.

Discoidin domain Receptor 2: A determinant of metabolic syndrome-associated arterial fibrosis in non-human primates.

Collagen accumulation and remodeling in the vascular wall is a cardinal feature of vascular fibrosis that exacerbates the complications of hypertension, aging, diabetes and atherosclerosis. With no specific therapy available to date, identification of mechanisms underlying vascular fibrogenesis is an important clinical goal. Here, we tested the hypothesis that Discoidin Domain Receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, is a determinant of arterial fibrosis. We report a significant increase in collagen type 1 levels along with collagen and ECM remodeling, degradation of elastic laminae, enhanced fat deposition and calcification in the abdominal aorta in a non-human primate model of high-fat, high-sucrose diet (HFS)-induced metabolic syndrome. These changes were associated with a marked increase in DDR2. Resveratrol attenuated collagen type I deposition and remodeling induced by the HFS diet, with a concomintant reduction in DDR2. Further, in isolated rat vascular adventitial fibroblasts and VSMCs, hyperglycemia increased DDR2 and collagen type I expression via TGF-ß1/SMAD2/3, which was attenuated by resveratrol. Notably, gene knockdown and overexpression approaches demonstrated an obligate role for DDR2 in hyperglycemia-induced increase in collagen type I expression in these cells. Together, our observations point to DDR2 as a hitherto unrecognized molecular link between metabolic syndrome and arterial fibrosis, and hence a therapeutic target.

Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II-stimulated cardiac fibroblasts.

Excessive collagen deposition by myofibroblasts during adverse cardiac remodeling leads to myocardial fibrosis that can compromise cardiac function. Unraveling the mechanisms underlying collagen gene expression in cardiac myofibroblasts is therefore an important clinical goal. The collagen receptors, discoidin domain receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, and integrin-ß1, are reported to mediate tissue fibrosis. Here, we probed the role of DDR2-integrin-ß1 cross-talk in the regulation of collagen α1(I) gene expression in angiotensin II (Ang II)-stimulated cardiac fibroblasts. Results from gene silencing/overexpression approaches, electrophoretic mobility shift assays, and ChIP revealed that DDR2 acts via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK)-dependent transforming growth factor-ß1 (TGF-ß1) signaling to activate activator protein-1 (AP-1) that in turn transcriptionally enhances the expression of collagen-binding integrin-ß1 in Ang II-stimulated cardiac fibroblasts. The DDR2-integrin-ß1 link was also evident in spontaneously hypertensive rats and DDR2-knockout mice. Further, DDR2 acted via integrin-ß1 to regulate α-smooth muscle actin (α-SMA) and collagen type I expression in Ang II-exposed cardiac fibroblasts. Downstream of the DDR2-integrin-ß1 axis, α-SMA was found to regulate collagen α1(I) gene expression via the Ca2+ channel, transient receptor potential cation channel subfamily C member 6 (TRPC6), and the profibrotic transcription factor, Yes-associated protein (YAP). This finding indicated that fibroblast-to-myofibroblast conversion is mechanistically coupled to collagen expression. The observation that collagen receptor cross-talk underlies α-SMA-dependent collagen type I expression in cardiac fibroblasts expands our understanding of the complex mechanisms involved in collagen gene expression in the heart and may be relevant to cardiac fibrogenesis.