Resolution of inflammation in healing myocardial infarcts

摘要： 转化生长因子（TGF）-β超家族的成员（TGF-β，Bone 形态发生蛋白质/BMP、生长分化因子/GDF、激活素等）在肿瘤的发生和发展中起核心作用。 修复、重塑和纤维化。TGF-β信号通过异四聚体 由两种II型和两种I型TGF-β受体（分别为TBetaRII和TBetaRI）组成的复合物。TBetaRI 激活通过一个细胞内效应子家族，受体激活的Smads， （R-Smads：Smad 1/2/3/5/8），或通过刺激非Smad级联。1型受体的概况 由特定的TGF-β超家族成员激活决定了下游信号级联 将被激活。传统观念认为，3种TGF-β亚型（TGF-β 1、-β 2和-β 3）信号 通过Smad 2或Smad 3，而BMP激活Smad 1和Smad 5。内皮细胞研究 我挑战了这一概念，认为TGF-β可以刺激Smad 1和Smad 2/3 信号传导，其中2个途径发挥拮抗作用。我们之前已经证明了细胞- TGF-β/Smad 3信号在心肌梗死中特异性作用促进肌成纤维细胞 激活，调节心肌细胞存活和功能，并调节巨噬细胞吞噬活性， 和抗炎的过渡。然而，Smad 1级联反应在修复、重塑和 梗死心脏的纤维化仍不清楚，Smad 1对心肌细胞表型和功能的影响， 免疫和修复细胞尚未被研究。 我们的提案探讨了Smad 1信号在修复和重塑中的作用， 梗塞的心脏我们的初步数据显示Smad 1主要在梗死灶中被激活， 肌成纤维细胞和巨噬细胞，而且在边界区心肌细胞和周细胞中。在体外， TGF-β亚型有效激活心脏修复细胞（巨噬细胞和成纤维细胞）中的Smad 1。在 在体内，我们的细胞特异性功能丧失实验揭示了Smad 1意想不到的抗纤维化作用 在骨髓细胞中，Smad 1调节肌成纤维细胞的血管生成特性， 巨噬细胞因此，我们将探索Smad 1在心脏修复中的细胞特异性作用， 从4个具体目标剖析Smad 1作用的分子机制： 具体目的1：研究Smad 1在成纤维细胞表型调节中的作用 并探讨Smad 1介导的抗纤维化作用的分子基础。核糖核酸 序列分析表明Smad 1的抗纤维化作用可能与Smad 3竞争 信号传导或与STAT 6和p53纤维化途径的相互作用。这些机制的作用将 在体外和体内进行研究。 具体目标2：研究巨噬细胞Smad 1在修复和重塑中的作用。 梗塞的心脏我们的初步数据表明，巨噬细胞特异性Smad 1干扰了 心肌梗死通过抑制巨噬细胞的血管生成特性，通过下调 血管生成趋化因子CXCL 12。我们将探讨血管生成调控的机制， 巨噬细胞Smad 1在体内外的作用。 具体目标3：研究Smad 1与Smad 2/3激活的分子基础， 为了验证TBetaRI谱可能是一个关键因素的假设， 修复细胞的转录组和功能异质性的决定因素。我们将使用 流式细胞术和单核RNA-seq在体内，和细胞生物学测定在体外，研究的作用 特异性TBetaRI在调节成纤维细胞的表型和功能特性中的作用， 巨噬细胞 具体目标4：研究Smad 1通路在心肌细胞和心肌细胞中的作用。 梗死和重塑心肌中的周细胞。 这些研究将首次深入了解Smad 1在调控中的作用。 心肌炎症和纤维化，将剖析Smad 1介导的行动机制， 将探索免疫和免疫调节功能和转录组异质性的分子基础， 在梗塞心肌中的修复细胞。

ABSTRACT: The members of the Transforming Growth Factor (TGF)-Beta superfamily (TGF-Betas, Bone Morphogenetic Proteins/BMPs, Growth differentiation factors/GDFs, activins, etc), play a central role in repair, remodeling and fibrosis of the infarcted heart. TGF-Betas signal through heterotetrameric complexes composed of two type II and two type I TGF-Beta receptors (TBetaRII and TBetaRI respectively). TBetaRI activation transduces signals through a family of intracellular effectors, the receptor-activated Smads (R-Smads: Smad1/2/3/5/8), or by stimulating non-Smad cascades. The profile of type 1 receptors activated by a specific TGF-Beta superfamily member determines which downstream signaling cascade will be activated. Traditional concepts suggest that the 3 TGF-Beta isoforms (TGF-Beta1, -Beta2 and -Beta3) signal through Smad2 or Smad3, whereas BMPs activate Smad1 and Smad5. Studies in endothelial cells have challenged this concept, suggesting that TGF-Betas may stimulate both Smad1 and Smad2/3 signaling, with the 2 pathways exerting antagonistic effects. We have previously demonstrated cell- specific actions of TGF-Beta/Smad3 signaling in myocardial infarction that contribute to myofibroblast activation, modulate cardiomyocyte survival and function, and regulate macrophage phagocytic activity, and anti-inflammatory transition. However, the role of the Smad1 cascade in repair, remodeling and fibrosis of the infarcted heart remains unknown, and the effects of Smad1 on phenotype and function of immune and reparative cells has not been investigated. Our proposal explores the role of Smad1 signaling in repair and remodeling of the infarcted heart. Our preliminary data show that Smad1 is activated predominantly in infarct myofibroblasts and macrophages, but also in border zone cardiomyocytes and in pericytes. In vitro, TGF-Beta isoforms potently activate Smad1 in cardiac reparative cells (macrophages and fibroblasts). In vivo, our cell-specific loss-of-function experiments, revealed unanticipated anti-fibrotic effects of Smad1 in myofibroblasts and showed that in myeloid cells, Smad1 regulates the angiogenic properties of macrophages. Accordingly, we will explore cell-specific actions of Smad1 in cardiac repair and we will dissect the molecular mechanisms of Smad1 effects in 4 specific aims: Specific aim 1: to investigate the role of Smad1 in regulation of fibroblast phenotype following MI and to explore the molecular basis for Smad1-mediated anti-fibrotic effects. RNA- seq analysis suggests that the antifibrotic effects of Smad1 may involve competition with Smad3 signaling, or interactions with STAT6 and p53 fibrogenic pathways. The role of these mechanisms will be investigated in vitro and in vivo. Specific aim 2: to study the role of macrophage Smad1 in repair and remodeling of the infarcted heart. Our preliminary data show that macrophage-specific Smad1 perturbs repair of the infarcted heart by restraining the angiogenic properties of macrophages through downregulation of the angiogenic chemokine CXCL12. We will explore the mechanisms for the angiogenesis-regulating actions of macrophage Smad1 in vitro and in vivo. Specific aim 3: to investigate the molecular basis for Smad1 vs Smad2/3 activation in fibroblasts and macrophages and to test the hypothesis that the TBetaRI profile may be a key determinant of the transcriptomic and functional heterogeneity of reparative cells. We will use flow cytometry and single nucleus RNA-seq in vivo, and cell biological assays in vitro, to study the role of specific TBetaRIs in regulation of the phenotype and functional properties of fibroblasts and macrophages. Specific aim 4: to study the role of the Smad1 pathway in cardiomyocytes and in pericytes in the infarcted and remodeling myocardium. The proposed studies will provide for the first time insights into the role of Smad1 in regulation of myocardial inflammation and fibrosis, will dissect the mechanisms for Smad1-mediated actions, and will explore the molecular basis for the functional and transcriptomic heterogeneity of immune and reparative cells in the infarcted myocardium.