Methylglyoxal-Stimulated Mesothelial Cells Prompted Fibroblast-to-Proto-Myofibroblast Transition

Abstract

During long-term peritoneal dialysis, peritoneal fibrosis (PF) often happens and results in ultrafiltration failure, which directly leads to the termination of dialysis. The accumulation of extracellular matrix produced from an increasing number of myofibroblasts was a hallmark characteristic of PF. To date, glucose degradation products (GDPs, i.e., methylglyoxal (MGO)) that appeared during the heating and storage of the dialysate are considered to be key components to initiating PF, but how GDPs lead to the activation of myofibroblast in fibrotic peritoneum has not yet been fully elucidated. In this study, mesothelial cell line (MeT-5A) and fibroblast cell line (MRC-5) were used to investigate the transcriptomic and proteomic changes to unveil the underlying mechanism of MGO-induced PF. Our transcriptomic data from the MGO-stimulated mesothelial cells showed upregulation of genes involved in pro-inflammatory, apoptotic, and fibrotic pathways. While no phenotypic changes were noted on fibroblasts after direct MGO, supernatant from MGO-stimulated mesothelial cells promoted fibroblasts to change into proto-myofibroblasts, activated fibroblasts in the first stage toward myofibroblasts. In conclusion, this study showed that MGO-stimulated mesothelial cells promoted fibroblast-to-proto-myofibroblast transition; however, additional involvement of other factors or cells (e.g., macrophages) may be needed to complete the transformation into myofibroblasts.

Keywords: fibroblast-to-myofibroblast; mesothelium; methylglyoxal; peritoneal fibrosis; proto-myofibroblast.

Figure 1

Examination of myofibroblast activation on MGO-treated fibroblasts. (A) Immunofluorescence results showed a significantly increased expression of αSMA (labeled in green) as well as fibronectin (labeled in red) in cells from the TGFβ1 group but not in that from the MGO groups. (B) Quantification analyses of immunofluorescence staining showed a 2-more fold increase in fibronectin expression in cells from the TGFβ1 group compared with the VC group. No differences were observed between the MGO-treated groups and the VC group. (C) Results of the Western blot showed that only cells treated with TGFβ1 induced a significant increase in protein expression of αSMA, fibronectin, and collagen I. (D) Collagen assay showed a significant shrinkage of fibroblast-laden collagen gels in the TGFβ1 group when compared with the VC group at all time points. However, treatment of 0.5 mM MGO causes no differences regarding the area of collagen gels, indicating that MGO-treated fibroblasts shared similar contraction ability with normal fibroblasts. Asterisks indicated significant differences between groups (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). Images from 10 different fields in each group were evaluated, and representative images were presented.

Figure 2

NGS results of MGO-stimulated mesothelial cells revealed distinct transcriptomic signatures between different treating times. (A) Experimental design of the samples that were sent to perform NGS analyses. (B) Distinct characteristics of transcriptome can be seen in cells among different MGO-treated times by PCA. Samples collected at time points of 0 h, 6 h, and 24 h were labeled in blue, green, and red colors, respectively. (C) 1182 upregulated DEGs and 828 downregulated DEGs were detected when comparing transcriptomic profiles at the time point of 6 h with 0 h. (D) 1725 upregulated DEGs and 791 downregulated DEGs were detected when comparing transcriptomic profiles at the time point of 24 h with 0 h. (E) Venn diagram showed that more than half of the DEGs from “6 h vs. 0 h” and “24 h vs. 0 h” were overlapped. DEGs from areas I, II, and III were sent to analyze the corresponding pathways, and the significant pathways (p < 0.05) were listed respectively.

Figure 3

Pathways associated with inflammation and fibrosis were significantly altered upon MGO stimulation on mesothelial cells. (A,B) Pathway enrichment analysis of “6 h vs. 0 h” and “24 h vs. 0 h” using the KEGG database showed that MGO initiated pathways that related with inflammation, apoptosis, cytokine–receptor response, and ECM–receptor response on MeT-5A. (C) DEGs involved in those significantly changed pathways were shown in the heatmap. Some DEGs were upregulated gradually along the treating time, while others were downregulated after being treated MGO for 6 h. (D) DEGs of cytokines and growth factors that commonly participated in inflammatory or fibrotic processes were listed.

Figure 4

Characterization of the cytoskeleton on fibroblasts treated with supernatant generated by MGO-stimulated mesothelial cells. (A) Experimental setting to investigate the effects of MGO-stimulated mesothelial cells on fibroblasts. (B) Immunofluorescence staining showed significantly increased expression of αSMA (labeled in green) and F-actin (labeled in yellow) on cells from the TGFβ1 group. Although no signals of αSMA were observed on supernatant-treated cells, increased signals of F-actin were noted compared with cells in the VC group. (C) Quantification analyses showed significantly elevated signals of F-actin on cells treated with TGFβ1 and cells treated with supernatant for 24 and 48 h. (D) Western blot showed that fibroblasts treated with TGFβ1 expressed significantly higher αSMA, and fibroblasts treated with supernatant expressed similar αSMA with normal fibroblasts. (E) In the contraction assay, the area of MRC-5-laden collagen gels was significantly decreased in the TGFβ1 group, but no differences were found between the supernatant group and the VC group. Asterisks indicated significant differences between groups (**** p < 0.0001, * p < 0.05). For each experimental condition, 10 images were evaluated, and representative images were presented.

Figure 5

Evaluation of inter- and extracellular expression of ECM proteins on supernatant-treated fibroblasts. (A) Immunofluorescence staining showed increased signals of fibronectin and EDA-FN on both TGFβ1- and supernatant-treated fibroblasts. (B) Quantification analyses showed that both fibronectin and EDA-FN were significantly increased on fibroblasts from the TGFβ1 group and supernatant groups. (C) Protein expressions of fibronectin, EDA-FN, and collagen I were examined using a Western blot. For fibronectin and collagen I, elevated expressions were only observed on fibroblasts treated with TGFβ1. As for EDA-FN, fibroblasts treated with TGFβ1, as well as those treated with supernatant for 24 h, expressed significantly higher amounts than normal fibroblasts. (D) Illustration for samples that were used to probe fibronectin and EDA-FN in the following Western blot experiment. (E) The same volume of the cultured medium collected from each condition was loaded into each well of the SDS page and ran the Western blot. Significantly increased expressions of fibronectin and EDA-FN in the cultured medium were noted in the TGFβ1 group, supernatant 48 h, and supernatant 72 h group. Asterisks indicated significant differences between groups (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). Images from 10 different fields in each group were evaluated, and representative images were presented.

Figure 6

Summarized illustration of the findings in this study. Upon the administration of MGO, genes involved in pro-inflammatory and fibrotic pathways were significantly upregulated in mesothelial cells. Then, the secretions from these stimulated mesothelial cells further activated the fibroblasts into proto-myofibroblasts, which were distinct from αSMA+ myofibroblasts but also demonstrated an increase in stress fibers and elevated ECM production ability. Upregulated genes being responsible for recruiting macrophages and promoting M1 polarization were also noted and indicated a possible role for macrophages to complete the activation of myofibroblasts.