ANGIOTENSIN II-SMAD SIGNALING IN CARDIAC FIBROSIS

说明(申请人提供)：Ang II在慢性心脏病中起着关键作用，然而，其导致心脏纤维化的信号通路仍很不清楚。已知Ang II通过刺激转化生长因子-b介导心肌纤维化，然而，我们的初步研究发现，Ang II能够通过两种机制直接激活转化生长因子-b信号通路：1)通过激活ERK/p38 MAP激酶的急性通路(5-30分钟)。这是不依赖于转化生长因子-b的，因为血管紧张素转换酶II能够激活缺乏转化生长因子-b受体的细胞中的Smad2和3；这一反应被ERK或p38抑制剂阻断；2)通过自分泌转化生长因子-b作用的晚期机制(24小时)，导致纤维化。此外，我们还发现，Smad2基因缺失的小鼠对心肌纤维化有保护作用，而Smad2基因有条件缺失的小鼠对Ang II的反应是促进纤维化的。因此，我们假设Smad信号是Ang II反应的心脏纤维化发展的关键。我们计划通过追求三个特定目标来验证这一假设。在特定的目标1中，我们建议确定Ang II介导心肌纤维化的新的信号通路。我们将证明Ang II通过AT1-R发出信号，并通过ERK/p38 MAPK依赖的机制(5-30分钟)激活急性Smad信号。我们还建议通过经典的转化生长因子-b依赖机制(24小时)激活晚期Smad信号，从而确定Ang II的长期效应。在特定的目标2中，我们将剖析Smad2或Smads在缺乏Smad2或Smads的小鼠胚胎成纤维细胞和不表达Smad2或具有Smad2条件性基因敲除的心脏成纤维细胞中所起的特定作用。在特定的目标3中，我们将进一步研究Smad2或Smads在小鼠心肌纤维化中的功能作用，而Smads KO小鼠为空白，或通过皮下注射大剂量Ang II获得Smad2的条件性KO。我们期望所获得的结果将支持中心假说，为AngⅡ介导的心肌纤维化的发病机制提供新的见解，并为开发新的治疗策略提供信息。

DESCRIPTION (provided by applicant): Ang II plays a pivotal role in chronic cardiac disease, however, its signaling pathways leading to cardiac fibrosis remain largely unclear. It is known that Ang II acts by stimulating TGF-b to mediate cardiac fibrosis, however, our preliminary studies found that Ang II is able to directly activate the TGF-b signaling pathway by two mechanisms: 1) an acute pathway (5-30 minutes) via activation of the ERK/p38 MAP kinases. This is TGF-b-independent since Ang II is able to activate Smad2 &3 in cells lacking TGF-b receptors; this response is blocked by ERK or p38 inhibitors; 2) a late mechanism (24 hours) that acts through autocrine TGF-b and leads to fibrosis. Furthermore, we also found that mice null for SmadS are protected against cardiac fibrosis, while mice that are conditionally deleted for Smad2 enhance fibrosis in response to Ang II. Thus, we hypothesize that Smad signaling is a key to the development of cardiac fibrosis in response to Ang II. We plan to test this hypothesis by pursuing three specific aims. In Specific Aim 1, we propose to identify new signaling pathways whereby Ang II mediates cardiac fibrosis. We will demonstrate that Ang II signals through the AT1-R and activates an acute Smad signaling via the ERK/p38 MAPK-dependent mechanism (5-30 mins). We also propose to identify that a long-term effect of Ang II by activating a late Smad signaling via the classic TGF-b-dependent mechanism (24hrs). In Specific Aim 2, we will dissect the specific role of Smad2 or SmadS in Ang ll-mediated fibrosis in mouse embryonic fibroblasts lacking Smad2 or SmadS and in cardiac fibroblasts that do not express SmadS or have conditional knockout for Smad2. In Specific Aim 3, we will further investigate the functional role of Smad2 or SmadS in cardiac fibrosis in mice null for SmadS KO mice or have conditional KO for Smad2 by subcutaneous infusion of large doses of Ang II. We expect that the outcomes obtained will support the central hypothesis, providing new insights into the pathogenesis of Ang ll-mediated cardiac fibrosis and information for the development of new therapeutic strategies.