BACKGROUND Cardiac fibroblast activation contributes to adverse remodeling, fibrosis, and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β (transforming growth factor-β)/Smad (small mother against decapentaplegic)-3 activation protects the pressure-overloaded heart by preserving the matrix, sustained TGF-β activation is deleterious, accentuating fibrosis and dysfunction. Thus, endogenous mechanisms that negatively regulate the TGF-β response in fibroblasts may be required to protect from progressive fibrosis and adverse remodeling. We hypothesized that Smad7, an inhibitory Smad that restrains TGF-β signaling, may be induced in the pressure-overloaded myocardium and may regulate fibrosis, remodeling, and dysfunction. METHODS The effects of myofibroblast-specific Smad7 loss were studied in a mouse model of transverse aortic constriction, using echocardiography, histological analysis, and molecular analysis. Proteomic studies in S7KO (Smad7 knockout) and overexpressing cells were used to identify fibroblast-derived mediators modulated by Smad7. In vitro experiments using cultured cardiac fibroblasts, fibroblasts populating collagen lattices, and isolated macrophages were used to dissect the molecular signals responsible for the effects of Smad7. RESULTS Following pressure overload, Smad7 was upregulated in cardiac myofibroblasts. TGF-β and angiotensin II stimulated fibroblast Smad7 upregulation via Smad3, whereas GDF15 (growth differentiation factor 15) induced Smad7 through GFRAL (glial cell line-derived neurotrophic factor family receptor α-like). MFS7KO (myofibroblast-specific S7KO) mice had increased mortality, accentuated systolic dysfunction and dilative remodeling, and accelerated diastolic dysfunction in response to transverse aortic constriction. Increased dysfunction in MFS7KO hearts was associated with accentuated fibrosis and increased MMP (matrix metalloproteinase)-2 activity and collagen denaturation. Secretomic analysis showed that Smad7 loss accentuates secretion of structural collagens and matricellular proteins and markedly increases MMP2 secretion. In contrast, Smad7 overexpression reduced MMP2 levels. In fibroblasts populating collagen lattices, the effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Surprisingly, MFS7KO mice also exhibited significant macrophage expansion caused by paracrine actions of Smad7 null fibroblasts that stimulate macrophage proliferation and fibrogenic activation. Macrophage activation involved the combined effects of the fibroblast-derived matricellular proteins CD5L (CD5 antigen-like), SPARC (secreted protein acidic and rich in cysteine), CTGF (connective tissue growth factor), ECM1 (extracellular matrix protein 1), and TGFBI (TGFB induced). CONCLUSIONS The antifibrotic effects of Smad7 in the pressure-overloaded heart protect from dysfunction and involve not only reduction in collagen deposition but also suppression of MMP2-mediated matrix denaturation and paracrine effects that suppress macrophage activation through inhibition of matricellular proteins.

中文翻译：

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成纤维细胞 Smad7 诱导可保护重塑压力超载的心脏。


背景技术心脏成纤维细胞活化导致压力超负荷的心脏中的不良重塑、纤维化和功能障碍。尽管早期成纤维细胞 TGF-β（转化生长因子-β）/Smad（小母体对抗十肢瘫痪）-3 激活可以通过保留基质来保护压力超载的心脏，但持续的 TGF-β 激活是有害的，会加剧纤维化和功能障碍。因此，可能需要负向调节成纤维细胞中 TGF-β 反应的内源机制来防止进行性纤维化和不良重塑。我们假设 Smad7（一种抑制 TGF-β 信号传导的抑制性 Smad）可能在压力超载的心肌中被诱导，并可能调节纤维化、重塑和功能障碍。方法 使用超声心动图、组织学分析和分子分析，在横主动脉缩窄的小鼠模型中研究肌成纤维细胞特异性 Smad7 缺失的影响。 S7KO（Smad7 敲除）和过表达细胞的蛋白质组学研究用于鉴定受 Smad7 调节的成纤维细胞衍生介质。使用培养的心脏成纤维细胞、胶原蛋白格中的成纤维细胞和分离的巨噬细胞进行体外实验，以剖析负责 Smad7 作用的分子信号。结果 压力超负荷后，心肌成纤维细胞中的 Smad7 表达上调。 TGF-β 和血管紧张素 II 通过 Smad3 刺激成纤维细胞 Smad7 上调，而 GDF15（生长分化因子 15）通过 GFRAL（胶质细胞系衍生的神经营养因子家族受体 α 样）诱导 Smad7。 MFS7KO（肌成纤维细胞特异性 S7KO）小鼠死亡率增加，收缩功能障碍和扩张重塑加剧，并加速舒张功能障碍以响应主动脉横缩。 MFS7KO 心脏功能障碍的增加与纤维化加剧、MMP（基质金属蛋白酶）-2 活性增加和胶原变性有关。分泌组学分析表明，Smad7 缺失会加剧结构胶原和基质细胞蛋白的分泌，并显着增加 MMP2 的分泌。相反，Smad7 过度表达会降低 MMP2 水平。在胶原蛋白晶格中的成纤维细胞中，Smad7 对成纤维细胞诱导的胶原变性和垫收缩的影响部分是通过 MMP2 下调介导的。令人惊讶的是，MFS7KO 小鼠还表现出显着的巨噬细胞扩张，这是由 Smad7 缺失成纤维细胞的旁分泌作用引起的，刺激巨噬细胞增殖和纤维化激活。巨噬细胞活化涉及成纤维细胞衍生的基质细胞蛋白 CD5L（CD5 抗原样）、SPARC（富含半胱氨酸的酸性分泌蛋白）、CTGF（结缔组织生长因子）、ECM1（细胞外基质蛋白 1）和 TGFBI 的综合作用（TGFB 诱导）。结论 Smad7 在压力超负荷的心脏中的抗纤维化作用可以防止功能障碍，不仅涉及减少胶原蛋白沉积，还涉及抑制 MMP2 介导的基质变性和旁分泌作用，通过抑制基质细胞蛋白来抑制巨噬细胞活化。