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Fibroblast-specific TGF-?-Smad2/3 signaling underlies cardiac fibrosis.

ABSTRACT: The master cytokine TGF-? mediates tissue fibrosis associated with inflammation and tissue injury. TGF-? induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-? signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-?-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-? receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-? mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-?-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

SUBMITTER: Khalil H

PROVIDER: S-EPMC5617658 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Fibroblast-specific TGF-β-Smad2/3 signaling underlies cardiac fibrosis.

Khalil Hadi H Kanisicak Onur O Prasad Vikram V Correll Robert N RN Fu Xing X Schips Tobias T Vagnozzi Ronald J RJ Liu Ruijie R Huynh Thanh T Lee Se-Jin SJ Karch Jason J Molkentin Jeffery D JD

The Journal of clinical investigation 20170911 10

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined press ...[more]

PMID: 28891814

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Project description:Rationale: Extracellular matrix (ECM) remodeling, a key pathological feature in diabetic cardiomyopathy (DCM), is triggered by oxidative stress, inflammation, and various metabolic disorders in the heart. Cardiac fibroblasts (CFs) are the primary source of ECM proteins and the ultimate effector cells in ECM remodeling. CFs are turned on and differentiated into myofibroblasts in response to profibrotic signaling. Rnd3 is a small Rho-GTPase involved in the regulation of cell-cycle distribution, cell migration, and cell morphogenesis. Emerging evidence suggests a link between Rnd3 expression and onset of cardiovascular diseases. However, the role of Rnd3 in DCM remains unknown. Methods: Flow cytometry was employed to separate different types of cardiac cells. Type 2 diabetes mellitus was established in Rnd3 fibroblast-specific knockout and transgenic mice. RNA sequencing and chromatin immunoprecipitation assay were used to discern signaling pathways involved. Results: Rnd3 expression was reduced in cardiac tissues of diabetic mice, with CFs being the primary cell type. Fibroblast-specific upregulation of Rnd3 in vivo was protective against DCM, whereas Rnd3 downregulation in fibroblasts accentuated cardiac oxidative stress, fibrosis, ventricular remodeling, and dysfunction. Moreover, in vitro Rnd3 overexpression significantly attenuated reactive oxygen species production, CF migration and proliferation under high levels of glucose (35 mmol/L) and palmitic acid (500 µmol/L) challenge. Furthermore, RNA sequencing indicated that Notch and TGF-β signaling were significantly suppressed upon Rnd3 overexpression. Mechanistically, Rnd3 regulated Notch and TGF-β signaling by interacting with NICD and ROCK1, respectively. Specifically, glucotoxicity and lipotoxicity control Rnd3 expression by regulating the binding of Nr1H2 and Rnd3 promoter. Conclusions: Our findings provide compelling evidence in that fibroblast-specific activation of Rnd3 protects against cardiac remodeling in DCM, indicating promises of targeting Rnd3 in the treatment of DCM.

Project description:Ethnopharmacological relevance: Curcumin is a bright yellow chemical produced by plants of the Curcuma longa species. Chemically, curcumin is a diarylheptanoid, belonging to the group of curcuminoids. The therapeutic potential of curcumin has been widely investigated, including its utilization in various of cardiovascular diseases. However, its effect in cardiac remodeling post myocardial infarction and underlying mechanism remains to be uncover. Aim: To evaluate the therapeutic effect and underlying mechanism of curcumin on cardiac fibrosis after myocardial infarction via macrophage-fibroblast crosstalk. Methods: Male C57BL/6 (C57) mice were subjected to left anterior descending coronary artery ligation to establish myocardial infarction and intragastrically fed vehicle or curcumin (50 mg/kg or 100 mg/kg) for 4 weeks. In parallel, neonatal rat cardiac fibroblasts were isolated and co-cultured with liposaccharide (LPS- or LPS+) curcumin-treated macrophages, followed by TGF-β stimulation for 24 h. Cardiac function was determined by 2-dimensional echocardiography, and cardiac fibrosis was measured by picrosirius red staining. Apoptosis of macrophages was investigated by flow cytometry; all pro-fibrotic protein expression (EDA-Fibronectin, Periostin, Vimentin, and α-SMA) as well as TGF-βR1 downstream signaling activation reflected by phosphorylated SMAD2/3 (p-SMAD2 and p-SMAD3) were demonstrated by western blotting. Results: Curcumin significantly ameliorated the inflammation process subsequent to myocardial infarction, reflected by decreased expression of CD68+ and CD3+ cells, accompanied by dramatically improved cardiac function compared with the placebo group. In addition, cardiac fibrosis is inhibited by curcumin administration. Interestingly, no significant reduction in fibrotic gene expression was observed when isolated cardiac fibroblasts were directly treated with curcumin in vitro; however, pro-fibrotic protein expression was significantly attenuated in CF, which was co-cultured with LPS-stimulated macrophages under curcumin treatment compared with the placebo group. Mechanistically, we discovered that curcumin significantly downregulated pro-inflammatory cytokines in macrophages, which in turn inhibited IL18 expression in co-cultured cardiac fibroblasts using bulk RNA sequencing, and the TGF-β1-p-SMAD2/3 signaling network was also discovered as the eventual target downstream of IL18 in curcumin-mediated anti-fibrosis signaling. Conclusion: Curcumin improves cardiac function and reduces cardiac fibrosis after myocardial infarction. This effect is mediated by the inhibition of macrophage-fibroblast crosstalk in the acute phase post-MI and retrained activation of IL18-TGFβ1-p-SMAD2/3 signaling in cardiac fibroblasts.

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Fibroblast-specific TGF-?-Smad2/3 signaling underlies cardiac fibrosis.

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Fibroblast-specific TGF-β-Smad2/3 signaling underlies cardiac fibrosis.

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