Friday, April 26, 2024
4/26/2024
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.
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