Int J Stem Cells.  2021 Aug;14(3):320-330. 10.15283/ijsc20186.

Galectin-3 Derived from HucMSC Exosomes Promoted Myocardial Fibroblast-to-Myofibroblast Differentiation Associated with β-catenin Upregulation

Affiliations
  • 1School of Medicine, Jiangsu University, Zhenjiang, China
  • 2Department of Obstetrics, Affiliated Hospital of Jiangsu University, Zhenjiang, China
  • 3Department of Clinical Laboratory, Zhenjiang Provincial Blood Center, Zhenjiang, China

Abstract

Background and Objectives
Galectin-3 promotes fibroblast-to-myofibroblast differentiation and facilitates injury repair. Previous studies have shown that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-ex) promote the differentiation of myocardial fibroblasts into myofibroblasts under inflammatory environment. Whether hucMSC-ex derived Galectin-3 (hucMSC-ex-Galectin-3) plays an important role in fibroblast-to-myofibroblast differentiation is the focus of this study.
Methods and Results
Galectin-3 was knocked-down by siRNA in hucMSCs, and then exosomes were extracted. Fibroblasts were treated with LPS, LPS+hucMSC-ex, LPS+negative control-siRNA-ex (NC-ex), or LPS+ Galectin-3-siRNA-ex (si-ex) in vitro. The coronary artery of the left anterior descending (LAD) branch was permanently ligated, followed by intramyocardial injection with phosphate buffered saline(PBS), hucMSC-ex, hucMSC-NC-ex, or hucMSC-si-ex in vivo. Western blot, RT-PCR, and immunohistochemistry were used to detect the expression of markers related to fibroblast-to-myofibroblast differentiation and inflammatory factors. Migration and contraction functions of fibroblasts were evaluated using Transwell migration and collagen contraction assays, respectively. β-catenin expression was detected by western blot and immunofluorescence. The results showed that hucMSC-ex increased the protein expression of myofibroblast markers, anti-inflammatory factors, and β-catenin. HucMSC-ex also reduced the migration and promoted the contractility of fibroblasts. However, hucMSC-si-ex did not show these activities.
Conclusions
HucMSC-ex-Galectin-3 promoted the differentiation of cardiac fibroblasts into myofibroblasts in an inflammatory environment, which was associated with increased β-catenin levels.

Keyword

Mesenchymal stem cell; Galectin-3; Cardiac fibroblasts; Myofibroblasts

Figure

  • Fig. 1 Characterization of hucMSC-ex. (A) The particle sizes of hucMSC-ex. (B) Image of hucMSC-ex. (C) Exoso-mal marker proteins CD63, CD81, and TSG-101 and the negative control calnexin were detected by western blot. (D) The internalization of exosomes by fibroblasts. Exosomes were stained with CM-Dil (red) and nuclei were stained with Hoechst (blue). Scale bar=50 μm.

  • Fig. 2 HucMSC-ex promoted fibroblast-to-myofibroblast differentiation in inflammatory environments. Cardiac fibroblasts were treated with LPS (100 ng/ml) or LPS+hucMSC-ex (200 μg/ml) for 24 h; the control group did not receive any treatment. (A, B) Colla-gen I, Periostin, and α-SMA expre-ssion were detected by western blot. (C) Collagen contraction analysis was used to determine the collagen contractility of cardiac fibroblasts treated with LPS (100 ng/ml) or LPS+hucMSC-ex (200 μg/ml) for 24 h. (D) Cardiac fibroblasts were treated with LPS (100 ng/ml) or LPS+hucMSC-ex (200 μg/ml) for 24 h, Transwell migration assays were used to detect the migration ability of cardiac fibro-blasts. Scale bar=100 μm; *p< 0.05, **p<0.01, and ***p<0.001.

  • Fig. 3 SiRNA-mediated knockdown of hucMSC-Galectin-3. (A) Galectin-3 protein expression in hucMSC and hucMSC-ex were detected by western blot. (B, C) HucMSCs were transfected with Galectin-3 siRNA, and then Galectin-3 protein and mRNA expression were detected by western blot and qRT-PCR, respecti-vely. (D) Levels of Galectin-3 protein in hucMSC-ex following Galectin-3 knockdown were detected by western blot. *p<0.05, **p<0.01, and ***p<0.001.

  • Fig. 4 HucMSC-ex-Galectin-3 promoted the differentiation of cardiac fibroblasts into myofibroblasts. Car-diac fibroblasts were treated with LPS (100 ng/ml), LPS+hucMSC-ex (200 μg/ml), LPS+hucMSC-NC-ex (200 μg/ml), or LPS+hucMSC-si-ex (200 μg/ml) for 24 h. (A) Collagen I, Periostin, and α-SMA levels were detected by western blot. (B) Two days after myocardial infarction was induced in SD rats, Collagen I, Periostin, and α-SMA expression in the infarcted area were detected by immunohistochemistry. Scale bar= 20 μm. *p<0.05, **p<0.01, and ***p<0.001.

  • Fig. 5 HucMSC-ex-Galectin-3 alte-red the function of cardiac fibroblasts. Cardiac fibroblasts were treated with LPS (100 ng/ml), LPS+hucMSC-ex (200 μg/ml), LPS+hucMSC-NC-ex (200 μg/ml), or LPS+hucMSC-si-ex (200 μg/ml) for 24 h. (A) Transwell migration assays were used to detect the migration ability of cardiac fibro-blasts. Scale bar=100 μm. (B) Co-llagen contraction analysis detected the collagen contractility of cardiac fibroblasts.*p<0.05 and ***p<0.001.

  • Fig. 6 HucMSC-ex-Galectin-3 promoted the transformation of fibroblasts to an anti-inflammatory phe-notype. Cardiac fibroblasts were treated with LPS (100 ng/ml), LPS+hucMSC-ex (200 μg/ml), LPS+hucMSC-NC-ex (200 μg/ml), or LPS+hucMSC-si-ex (200 μg/ml) for 24 h. (A, B) IL-1β, TNF-α, and TGF-β protein levels were detected by western blot. (C∼E) The mRNA expression of IL-1β, TNF-α, and TGF-β were detected by qRT-PCR. (F) Two days after myocardial infarction was induced, IL-1β, TNF-α, and TGF-β expression in the infarcted area were detected by immunohistochemistry. Scale bar= 20 μm. *p<0.05, **p<0.01, and ***p<0.001.

  • Fig. 7 HucMSC-ex-Galectin-3-induced fibroblast-to-myofibroblast transfor-mation was associated with increased β-catenin expression. Cardiac fibroblasts were treated with LPS (100 ng/ml), LPS+hucMSC-ex (200 μg/ml), LPS+hucMSC-NC-ex (200 μg/ml), or LPS+hucMSC-si-ex (200 μg/ml) for 24 h. (A) β-catenin expression was detected by western blot. (B) Nuclear β-catenin levels were also measured by western blot. (C) β-catenin expression as detected by immuno-fluorescence. Scale bar=100 μm. (D) Cardiac fibroblasts were treated with LPS (100 ng/ml), LPS+hucMSC-ex (200 μg/ml), LPS+hucMSC-ex (200 μg/ml)+ICG-001 (0, 5, 10, 15 μM) for 24 h, and then β-catenin levels were detected by western blot. (E) Cardiac fibroblasts were treated with LPS (100 ng/ml), LPS+hucMSC-ex (200 μg/ml), LPS+hucMSC-ex (200 μg/ml)+ICG-001 (5 μM) for 24 h, and then Collagen I, Periostin, and α-SMA expression were detected by western blot.


Reference

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