Smad3 Mediates Angiotensin II– and TGF-β1–Induced Vascular Fibrosis
Smad3 Thickens the Plot
See related article, pages 1032–1039
Hypertension, diabetes, and atherosclerosis are three diseases responsible for the majority of cardiovascular morbidity in humans. Not only are they independent risk factors, but they clearly potentiate and complement each other in the pathogenesis of cardiovascular disease. The first manifestation of end-organ damage in these diseases is the vascular remodeling of small (resistance) and large (conductance), and this precedes the development of cardiac hypertrophy, renal insufficiency or stroke.1 This vascular remodeling is characterized by hyperplasia, hypertrophy, and apoptosis of smooth muscle cells (SMCs) and vascular fibrosis, caused by increased extracellular matrix deposition of total collagen, and changes in ratio of type I/III collagen, fibronectin, and proteoglycans.1 Angiotensin II (Ang II) has recently emerged as a key mediator of vascular fibrosis (sclerosis) both in humans and animal models of hypertension because of its pleiotropic effects of SMCs, fibroblasts, and inflammatory cells. Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor antagonists (ARB) diminish cardiac hypertrophy and vascular fibrosis in animal models of hypertension.1,2 In human clinical trials of hypertension, ARBs reverse vascular remodeling in resistance arteries and new incidence of strokes more potently than any other antihypertensive agents (beta-blockers or calcium channel blockers), despite similar reduction in blood pressure.3,4 This provides strong evidence that targeting the mechanisms involved in arterial remodeling process may provide the key in preventing the long-term consequences of hypertension or atherosclerosis in humans.
However, deciphering the intimate mechanisms of in vivo vascular remodeling has become very complex because of enormous reciprocal interactions between Ang II and other growth factors. Ang II exerts its effects not only by activating its specific receptors, but also by transactivating other growth factor receptors such as epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), platelet-derived growth factor receptor (PDGF-R),1 or type III transforming growth factor receptor (endoglin).5 Whereas these pathways mediate the acute effects of Ang II, long-term outcomes of Ang II are mediated by a second wave of paracrine release of other growth factors. Specifically, while acutely Ang II directly activates transcription of collagens and fibronectin, strong evidence supports the notion that long term in vitro and in vivo profibrotic effects of Ang II are mediated by paracrine release of transforming growth factor-β1 (TGF-β1).2 TGF-β1 is potently and rapidly upregulated after Ang II stimulation, and blockade of TGF-β1 diminishes Ang II–induced cardiac and vascular fibrosis and prevents hypertension-induced end-organ damage in hypertensive rat models and aging-induced cardiac fibrosis.2 This is not surprising because TGF-β1 is the most potent profibrotic cytokine identified to date. Exposure of fibroblasts to TGF-β1 results in upregulation of smooth muscle α-actin, which gives contractile properties to fibroblasts and irreversibly converts them into a more synthetic phenotype: the myofibroblasts.2 Furthermore, TGF-β1 causes potent upregulation and secretion of extracellular matrix proteins such as collagens, fibronectin, and connective tissue growth factor, which further amplifies the collagen production.
Although perivascular adventitial fibroblasts and the interstitial cardiac fibroblasts are the traditional sources of fibrosis in cardiovascular system, the smooth muscle cells (in particular the intimal synthetic SMCs) have also been demonstrated to be able to secrete extracellular matrix.6 As in fibroblasts, Ang II and TGF-β1 are able to activate extracellular matrix production and trigger a potent profibrotic response on direct stimulation of SMCs, and this may contribute to vascular sclerosis from hypertensive and atherosclerotic arteries.6 The intracellular mediators of TGF-β1 signaling are the Smads 2 and 3.7 On receptor activation, Smad2 and 3 are phosphorylated and form a heterotrimeric complex with Smad4. Together, they translocate to the nucleus and mediate most of the profibrotic transcriptional activation.7 Because only Smad3 has a DNA-binding and transactivation domain, it is the most likely Smad to be directly involved in transcription.7
Although inhibition of TGF-β1 receptors appears a logical step to prevent vascular fibrosis, the potential success of this approach in humans with hypertension or atherosclerosis has been recently called into question. First, reports have shown that the TGF-β1 receptors are essentially undetectable in fibrotic regions from atherosclerotic plaques, which makes it unlikely that they mediate active remodeling at those sites. However, these cells and macrophages express intracellularly the classic mediators of TGF-β1 signaling: Smads 2 and 3.8 Two articles may offer an answer to reconcile the apparent discrepancy. First, Rodriguez-Vita et al6 showed that Ang II directly phosphorylates and activates Smad 2 within 20 minutes. Together with Smad 4, Smad 2 translocates to the nucleus, binds to DNA, and mediates de novo transcription of connective tissue growth factor (CTGF) and collagen I. The activation of Smad 2 is mediated by type I angiotensin receptor and p38 MAPK, and also occurs in vivo in rat aortas after 3 days infusion with Ang II.
In an article in the current issue of Circulation Research, Wang et al9 extend these observations and complete the story by demonstrating that activation of Smads 2 and 3 precedes in vivo accumulation of collagen and fibrosis in the thickened intima of human renal arteries isolated from patients with hypertension and atherosclerosis. They show that Ang II induces a bimodal activation of Smad 2/3 in primary isolates of human artery, at 15 minutes and 24 hours. Whereas the initial activation is mediated by type 1 AT1 receptor and via p42-44 MAPK, the delayed response involves paracrine production of TGF-β1. They further demonstrate that induction of collagen and CTGF requires Smad 3 but not Smad 2 by using vascular SMCs isolated from Smad 2 and Smad 3 knockout mice (Figure).
Is Smad3 a Therapeutic Target for Vascular and Cardiac Fibrosis?
These studies suggest a potential answer for the question: what is the best way to prevent vascular remodeling associated with long standing hypertension and atherosclerosis? Angiotensin-converting enzyme inhibitors and type 1 angiotensin receptor antagonists are already in clinical use and clearly provide some benefit, although the magnitude of the effect is likely small. Most patients are treated late in disease when the remodeling has already occurred. According to the present study, this approach is unlikely to lead to reversal of remodeling when the arteries have already developed extensive vascular sclerosis, particularly after Smads are activated on long term. Inhibiting the activity of TGF-β1 may reduce both vascular and cardiac fibrosis; however, it can be risky because of the potent and sometimes contradictory effects of TGF-β1 on almost every cell type involved in remodeling. For example, TGF-β1 inhibits proliferation of SMCs, causes extracellular matrix deposition, chemotaxis, and activation of macrophages, and, importantly, inhibits activation of T lymphocytes.7 This later outcome is critical for maintenance of T cell homeostasis as it was proven by the fact that depletion of TGF-β1 in mice causes a lethal autoimmune cardiomyopathy attributable to overactivation of T cells.7 In human atherosclerosis, overactivation of T cells at the shoulder region of the plaques has been implicated in initiation of destabilization of the plaques and rupture by activating the macrophages abundant in the shoulder region (exposed to higher oscillatory shear stress); therefore, overall inactivation of TGF-β1 in the plaques may lead to unstable plaques and myocardial infarction in humans. However, inhibition of Smad 3 in vascular cells may provide a more specific tool to reverse vascular fibrosis. Whereas Smad 2 knockout mice are lethal, Smad 3 knockout are viable but have impaired immune functions.7 Paradoxically, deletion of Smad 3 accelerates skin wound healing by stimulating keratinocyte proliferation and reducing monocyte infiltration and inflammation while the matrix production is not affected.7,10 Thus the healing process in Smad 3 KO mice appears faster and the scar formation and fibrosis are minimal.7,10 Because Smad 3 appears to be a key player in mediating the fibrosis independent of the agonist (Ang II or TGF-β1) or cell type (vascular SMCs or fibroblasts), it may offer a cleaner way to reverse fibrosis in a safe predictable manner (Figure). Further studies with tissue-specific knockout of Smad 3 in smooth muscle cells, fibroblasts, macrophages, or T lymphocytes should help document the overall benefit of Smad 3 inhibition in atherosclerosis or hypertension.
Work in the author’s laboratory is supported by National Scientist Development grant no. 0335244N from the American Heart Association and an American Federation for Aging Research grant to D.S.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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