TGF-β-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness

Purpose This study aimed to investigate the effects of substratum stiffness on the sensitivity of human conjunctival fibroblasts to transforming growth factor (TGF)-β, and to explore the molecular mechanism of action. Methods Human conjunctival fibroblasts were cultured on collagen-coated plastic or silicone plates. The stiffness of the silicone plates was 0.2 or 64 kPa. Cells were treated by 2.5 ng/mL TGF-β2 with or without fibroblast growth factor (FGF)-2 (0–100 ng/mL) for 24 h or 48 h. The protein expression levels were determined by Western blot analysis. Cell proliferation was assessed using the WST-8 assay. Results FGF-2 suppressed the TGF-β-induced expression of α-smooth muscle actin (SMA) and collagen type I (Col I), but not fibronectin (FN). Both FGF-2 and TGF-β2 increased cell proliferation without an additive effect. The induction of α-SMA by TGF-β2 was decreased on the soft substratum, without any change in the expression level or subcellular location of Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). FGF-2 suppressed TGF-β-induced α-SMA expression even on the soft substratum. Conclusions FGF-2 treatment and a soft substratum suppressed TGF-β-induced transdifferentiation of conjunctival fibroblasts into myofibroblasts. FGF-2 attenuated the TGF-β-induced expression of α-SMA, even on a soft substratum.

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Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00095.2018 ◽

2019 ◽

Vol 316 (3) ◽

pp. H596-H608 ◽

Cited By ~ 4

Author(s):

Rachel C. Childers ◽

Ian Sunyecz ◽

T. Aaron West ◽

Mary J. Cismowski ◽

Pamela A. Lucchesi ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Pressure Overload ◽

Volume Overload ◽

Transforming Growth Factor Β ◽

Collagen Type I ◽

Tissue Growth ◽

Type I

Hemodynamic load regulates cardiac remodeling. In contrast to pressure overload (increased afterload), hearts subjected to volume overload (VO; preload) undergo a distinct pattern of eccentric remodeling, chamber dilation, and decreased extracellular matrix content. Critical profibrotic roles of cardiac fibroblasts (CFs) in postinfarct remodeling and in response to pressure overload have been well established. Little is known about the CF phenotype in response to VO. The present study characterized the phenotype of primary cultures of CFs isolated from hearts subjected to 4 wk of VO induced by an aortocaval fistula. Compared with CFs isolated from sham hearts, VO CFs displayed a “hypofibrotic” phenotype, characterized by a ~50% decrease in the profibrotic phenotypic markers α-smooth muscle actin, connective tissue growth factor, and collagen type I, despite increased levels of profibrotic transforming growth factor-β1 and an intact canonical transforming growth factor-β signaling pathway. Actin filament dynamics were characterized, which regulate the CF phenotype in response to biomechanical signals. Actin polymerization was determined by the relative amounts of G-actin monomers versus F-actin. Compared with sham CFs, VO CFs displayed ~78% less F-actin and an increased G-actin-to-F-actin ratio (G/F ratio). In sham CFs, treatment with the Rho kinase inhibitor Y-27632 to increase the G/F ratio resulted in recapitulation of the hypofibrotic CF phenotype observed in VO CFs. Conversely, treatment of VO CFs with jasplakinolide to decrease the G/F ratio restored a more profibrotic response (>2.5-fold increase in α-smooth muscle actin, connective tissue growth factor, and collagen type I). NEW & NOTEWORTHY The present study is the first to describe a “hypofibrotic” phenotype of cardiac fibroblasts isolated from a volume overload model. Our results suggest that biomechanical regulation of actin microfilament stability and assembly is a critical mediator of cardiac fibroblast phenotypic modulation.

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Hierarchical Coculture of Hepatocyte and Hepatic Non-Parenchymal Cells for Liver Fibrosis Studies in vitro

10.20944/preprints202010.0409.v1 ◽

2020 ◽

Author(s):

Qiao You Lau ◽

Fuad Gandhi Torizal ◽

Marie Shinohara ◽

Yasuyuki Sakai

Keyword(s):

Growth Factor ◽

Liver Fibrosis ◽

Oxygen Tension ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Liver Sinusoidal Endothelial Cell ◽

Type I ◽

Parenchymal Cells

During chronic liver injury, inflammation leads to the development of liver fibrosis— particularly due to the activation of hepatic stellate cells (HSCs). However, the involvement of inflammatory cytokines in HSC activation is unclear. Many existing in vitro liver models do not include these non-parenchymal cells (NPCs), and hence, do not represent the physiological relevance found in vivo. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with NPCs such as the human-derived HSC line (LX-2) and the human-derived liver sinusoidal endothelial cell line (TMNK-1). The coculture tissue had higher albumin production and hepatic cytochrome P450 3A4 activity compared to the monoculture. We then further studied the effects of stimulation by both oxygen tension and key pro-fibrogenic cytokines, such as the transforming growth factor beta (TGF-β), on HSC activation. Gene expression analysis revealed that lower oxygen tension and TGF-β1 stimulation enhanced collagen type I, III, and IV, alpha-smooth muscle actin, platelet-derived growth factor, and matrix metallopeptidase expression from LX-2 cells in the hierarchical coculture after fibrogenesis induction. This hierarchical in vitro cocultured liver tissue could, therefore, provide an improved platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in liver fibrosis studies.

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CXXC5 Attenuates Pulmonary Fibrosis in a Bleomycin-Induced Mouse Model and MLFs by Suppression of the CD40/CD40L Pathway

BioMed Research International ◽

10.1155/2020/7840652 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15 ◽

Cited By ~ 1

Author(s):

Wei Cheng ◽

Fangfang Wang ◽

Airan Feng ◽

Xiaodan Li ◽

Wencheng Yu

Keyword(s):

Pulmonary Fibrosis ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Feedback Regulation ◽

Collagen Type I ◽

Mouse Lung ◽

Type I ◽

Negative Feedback Regulation ◽

Protein Levels ◽

Proliferation And Apoptosis

Objective. To investigate the role of CXXC5 and the CD40/CD40L pathway in lung fibrosis. Methods. (1) We constructed mouse models of bleomycin-induced pulmonary fibrosis and transfected them with a CXXC5 overexpression vector to evaluate the severity of pulmonary fibrosis. (2) Mouse lung fibroblast (MLF) models stably overexpressed or knockout of CXXC5 vector were constructed. After transforming growth factor-β1 (TGF-β1) stimulation, we examined the proliferation and apoptosis of the MLF model and evaluated the expression of mesenchymal markers and the CXXC5/CD40/CD40L pathway. Results. (1) Compared with other groups, the overexpressed CXXC5 group had less alveolar structure destruction, thinner alveolar septum, and lower Ashcroft score. (2) In bleomycin-induced mice, the expression of CD40 and CD40L increased at both transcriptional and protein levels, and the same changes were observed in α-smooth muscle actin (α-SMA) and collagen type I (Colla I). After upregulation of CXXC5, the increase in CD40, CD40L, α-SMA, and Colla I was attenuated. (3) Stimulated with TGF-β1, MLF proliferation was activated, apoptosis was suppressed, and the expression of CD40, CD40L, α-SMA, and Colla I was increased at both transcriptional and protein levels. After upregulation of CXXC5, these changes were attenuated. Conclusion. CXXC5 inhibits pulmonary fibrosis and transformation to myofibroblasts by negative feedback regulation of the CD40/CD40L pathway.

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Collagen Type I Containing Hybrid Hydrogel Enhances Cardiomyocyte Maturation in a 3D Cardiac Model

Polymers ◽

10.3390/polym11040687 ◽

2019 ◽

Vol 11 (4) ◽

pp. 687 ◽

Cited By ~ 4

Author(s):

Sam G. Edalat ◽

Yongjun Jang ◽

Jongseong Kim ◽

Yongdoo Park

Keyword(s):

Growth Factor ◽

Collagen Type ◽

Transforming Growth Factor ◽

Embryonic Stem ◽

Collagen Type I ◽

Transforming Growth Factor Β1 ◽

Type I ◽

Hybrid Hydrogel ◽

Cardiac Model

In vitro maturation of cardiomyocytes in 3D is essential for the development of viable cardiac models for therapeutic and developmental studies. The method by which cardiomyocytes undergoes maturation has significant implications for understanding cardiomyocytes biology. The regulation of the extracellular matrix (ECM) by changing the composition and stiffness is quintessential for engineering a suitable environment for cardiomyocytes maturation. In this paper, we demonstrate that collagen type I, a component of the ECM, plays a crucial role in the maturation of cardiomyocytes. To this end, embryonic stem-cell derived cardiomyocytes were incorporated into Matrigel-based hydrogels with varying collagen type I concentrations of 0 mg, 3 mg, and 6 mg. Each hydrogel was analyzed by measuring the degree of stiffness, the expression levels of MLC2v, TBX18, and pre-miR-21, and the size of the hydrogels. It was shown that among the hydrogel variants, the Matrigel-based hydrogel with 3 mg of collagen type I facilitates cardiomyocyte maturation by increasing MLC2v expression. The treatment of transforming growth factor β1 (TGF-β1) or fibroblast growth factor 4 (FGF-4) on the hydrogels further enhanced the MLC2v expression and thereby cardiomyocyte maturation.

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