Differential effects of Smad2 and Smad3 in regulation of macrophage phenotype and function in the infarcted myocardium. Differential effects of Smad2 and Smad3 in regulation of macrophage phenotype and function in the infarcted myocardium

TGF-βs regulate macrophage responses, by activating Smad2/3. We have previously demonstrated that macrophage-specific Smad3 stimulates phagocytosis and mediates anti-inflammatory macrophage transition in the infarcted heart. However, the role of macrophage Smad2 signaling in myocardial infarction remains unknown. We studied the role of macrophage-specific Smad2 signaling in the healing infarct, and we explored the basis for the distinct effects of Smad2 and Smad3. Infarct macrophages exhibited both Smad2 and Smad3 activation. In contrast to the effects of Smad3 loss, myeloid cell-specific Smad2 disruption had no effects on mortality, ventricular dysfunction and adverse remodeling, after myocardial infarction. Phagocytic removal of dead cells, macrophage and myofibroblast infiltration, collagen deposition, angiogenesis and scar remodeling were not affected by macrophage Smad2 loss. In isolated macrophages, TGF-β1, -β2 and -β3, activated both Smad2 and Smad3, whereas BMP6 triggered only Smad3 activation. Smad2 and Smad3 had similar patterns of nuclear translocation in response to TGF-β1. Smad3, and not Smad2, was the main mediator of transcriptional effects of TGF-β on macrophages and Smad3 loss resulted in enrichment of genes associated with RAR/RXR signaling, cholesterol biosynthesis and lipid metabolism. In conclusion, the in vivo and in vitro effects of TGF-β on macrophage function involve Smad3, and not Smad2. Overall design: This study is designed to compare gene expression profiles between Smad2fl/fl and MyS2KO (Smad2fl/fl + LyzMCre), and Smad3fl/fl and MyS3KO (Smad3fl/fl + LyzMCre) bone marrow derived macrophages in the presence or absence of 10 ng/ml TGF-β1. The loss of Smad2 and Smad3 in MyS2KO, MyS3KO macrophages has been documented by qPCR and western blot.

转化生长因子βs（Transforming Growth Factor-βs, TGF-βs）可通过激活Smad2/3调控巨噬细胞应答。我们既往研究证实，巨噬细胞特异性Smad3可促进梗死心脏内的吞噬作用，并介导抗炎巨噬细胞表型转化。然而，巨噬细胞Smad2信号通路在心肌梗死中的作用仍未明确。
本研究探讨了巨噬细胞特异性Smad2信号通路在梗死灶修复中的作用，并解析了Smad2与Smad3发挥不同生物学效应的分子基础。梗死灶内巨噬细胞可同时激活Smad2与Smad3通路。与Smad3缺失的效应不同，心肌梗死发生后，髓系细胞特异性Smad2敲除对小鼠死亡率、心室功能障碍及不良重构均无显著影响。巨噬细胞Smad2缺失不会改变死细胞的吞噬清除、巨噬细胞与肌成纤维细胞浸润、胶原沉积、血管生成及瘢痕重构过程。
在分离培养的巨噬细胞中，TGF-β1、TGF-β2与TGF-β3均可同时激活Smad2与Smad3，而骨形态发生蛋白6（Bone Morphogenetic Protein 6, BMP6）仅能激活Smad3。TGF-β1刺激下，Smad2与Smad3的核转位模式相似。Smad3而非Smad2是TGF-β对巨噬细胞产生转录调控效应的核心介导因子；Smad3缺失会导致与维甲酸受体/类视黄醇X受体（RAR/RXR）信号通路、胆固醇生物合成及脂质代谢相关的基因表达富集。
综上，TGF-β对巨噬细胞功能的体内外调控作用均依赖于Smad3，而非Smad2。
整体实验设计：本研究旨在比较Smad2fl/fl与MyS2KO（Smad2fl/fl + LyzMCre）、Smad3fl/fl与MyS3KO（Smad3fl/fl + LyzMCre）来源的骨髓来源巨噬细胞（Bone Marrow Derived Macrophages, BMDM）在添加或不添加10 ng/ml TGF-β1时的基因表达谱差异。已通过定量聚合酶链反应（Quantitative Polymerase Chain Reaction, qPCR）与蛋白质印迹法（Western Blot）验证了MyS2KO与MyS3KO巨噬细胞中Smad2、Smad3的敲除效果。