In cardiac fibroblasts, interferon-beta attenuates differentiation, collagen synthesis, and TGF-β1-induced collagen gel contraction

Cardiac fibrosis plays an essential role in cardiac pathogenic processes that occur as a result of myocardial infarction or hypertrophic cardiomyopathy. The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGFβ is one of the essential molecules that promote transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Phorbol 12-Myristate 13-Acetate (PMA) regulates metabolism and functions of multiple cells via PKC activation mostly. To study effects of PMA on differentiation of de novo formed cardiac myofibroblasts, human cardiac fibroblasts were utilized. Human cardiac fibroblasts (HCF) cultured in fibroblast medium (FM)-2 were converted into myofibroblasts in the presence of 2 ng/mL of TGF-β1 for 48 hours. Expression of α-SMA, the biomarker of myofibroblasts, and FSP1, the biomarker of fibroblasts, was detected using Western blot and immunofluorescence. Collagen gel contraction induced by fibroblasts was determined as well. TGF-β1 increased the expression of α-SMA and reduced the expression of FSP1. Distinct cellular morphology changes in the shape and size of HCF were observed after incubation with TGF-β1 for 48 hours. To investigate effect of PMA on dedifferentiation of formed myofibroblasts, these TGF-β1-pretreated cells were divided into four groups for additional 48 hours incubation: PMA groups (10, 50, and 100 ng/mL) or DMSO (vehicle control). Both 50 and 100 ng/mL of PMA reduced the expression of α-SMA but only 100 ng/mL of PMA increased the expression of FSP1. The shape and size of cells changed after treatment with PMA. PMA also reduced TGF-β1-induced collagen gel contraction (P<0.05, compared to DMSO group). These data indicated that PMA can reverse the differentiation of de novo formed human cardiac myofibroblasts induced by TGF-β1 to fibroblasts and other unidentified type of cells. Although the mechanism of dedifferentiation remains to be identified, the novel finding of this study shed light on future development of agents to treat fibrotic diseases.

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Role of the ClC-2 Chloride Channel in TGF-β1-induced Proliferation, Collagen Synthesis, and Collagen Gel Contraction Mediated by Human Conjunctival Fibroblasts

10.21203/rs.2.20695/v1 ◽

2020 ◽

Author(s):

Lixia Sun ◽

Renzhe chui ◽

Meng Huan ◽

Xiwen Liu ◽

Xin Liu ◽

...

Keyword(s):

Cell Proliferation ◽

Chloride Channel ◽

Chloride Channels ◽

Cell Cycle Progression ◽

Collagen Synthesis ◽

Collagen Gel ◽

Sirna Transfection ◽

Collagen Gel Contraction ◽

Tgf Β1

Abstract Background: Excessive scar tissue can reduce postoperative survival of filtering blebs in patients with glaucoma. Previous studies have highlighted the role of chloride channels in wound healing. whereas The role of chloride channels in the formation of follicular scar has not been studied. Objectives: To investigate the effects of the ClC-2 chloride channel on scar formation of filtering blebs after glaucoma filtering surgery. Methods: We Inhibited ClC-2 chloride channels of Human Conjunctival Fibroblasts (HConFs) by transfecting HConFs with ClC-2 siRNA, Then cell proliferation, cycle and collagen synthesis of HConFs were measured. ClC-2 siRNA-transfected HConFs were cultured in type I collagen gels in the presence of transforming growth factor (TGF)-β1. Collagen gel contraction was evaluated based on the gel area. The expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in HConFs were assessed by western blotting and q-PCR. Results: TGF-β1induced cell proliferation, cell cycle progression, collagen synthesis, and collagen gel contraction in HConFs. TGF-β1 increased MMP-2 and MMP-9 levels but inhibited the expression of TIMPs. ClC-2 siRNA transfection inhibited TGF-β1-induced cell proliferation, cell cycle progression, collagen synthesis, and collagen gel contraction, mediated by HConFs. TGF-β1-induced increases in MMP-2 and MMP-9 were also inhibited by NPPB and ClC-2 siRNA transfection, but TIMP expression was increased by ClC-2 siRNA transfection. Conclusions: These findings demonstrate that ClC-2 gene knockout inhibited TGF-β1-induced cell proliferation, collagen synthesis, and collagen gel contraction of HConFs by attenuating MMP-2 and MMP-9 production and by stimulating TIMP-1 production. Keywords: ClC-2 chloride channel; conjunctival fibroblasts; TGF-β1; wound heal

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