The anti-fibrotic effects of mesenchymal stem cells on irradiated lungs via stimulating endogenous secretion of HGF and PGE2

Abstract

Radiation-induced pulmonary fibrosis is a common disease and has a poor prognosis owing to the progressive breakdown of gas exchange regions in the lung. Recently, a novel strategy of administering mesenchymal stem cells for pulmonary fibrosis has achieved high therapeutic efficacy. In the present study, we attempted to use human adipose tissue-derived mesenchymal stem cells to prevent disease in Sprague-Dawley rats that received semi-thoracic irradiation (15 Gy). To investigate the specific roles of mesenchymal stem cells in ameliorating radiation-induced pulmonary fibrosis, we treated control groups of irradiated rats with human skin fibroblasts or phosphate-buffered saline. After mesenchymal stem cells were infused, host secretions of hepatocyte growth factor (HGF) and prostaglandin E2 (PGE2) were elevated compared with those of the controls. In contrast, tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta1 (TGF-β1) levels were decreased after infusion of mesenchymal stem cells. Consequently, the architecture of the irradiated lungs was preserved without marked activation of fibroblasts or collagen deposition within the injured sites. Moreover, mesenchymal stem cells were able to prevent the irradiated type II alveolar epithelial cells from undergoing epithelial-mesenchymal transition. Collectively, these data confirmed that mesenchymal stem cells have the potential to limit pulmonary fibrosis after exposure to ionising irradiation.

Figure 1. Identification of Ad-MSCs.

(A)Spindle-like shape in vitro. (B) Phenotypic analysis by flow cytometry. (C) Adipogenic potential of Ad-MSCs: Oil Red O staining for lipid droplets in Ad-MSCs; (D): Osteogeneic potential of Ad-MSCs: Alizarin Red staining for calcium in Ad-MSCs.

Figure 2. Homing of Ad-MSCs to irradiated lung.

(A) Electrophesis in 1.5% agarose gel for verifying the specificity of hBeta2-MG primers to the human cells. (B) Expression of hBeta2-MG in lung tissue within 14 days post-irradiation. (C) Expression of rSDF-1α in lung tissue at 3 days post-irradiation. The expression levels of genes, including hBeta2-MG and rSDF-1α, were tested using quantitative real-time PCR. Rat Actin (rActin) was set as the internal control for determining ΔC_T values. Fold increases in expression were normalized to normal group by determining 2^−ΔΔCT values. Data shown are the mean ± S.D. of nine independent samples. This experiment was performed only once. One-way ANOVA analysis was used for comparing the differences of data among groups. *P ≤ 0.05 and **P ≤ 0.01 (significant high) versus the Normal + Ad-MSC group. ns: no statistical significance. (D) IHC-staining for Beta2-MG only in human cells and for SDF-1α in lung tissues at 3 days post-irradiation. Left rank: Magnification at 200×. Scale bar, 100 μm. Right rank: Magnification at 1000×. Scale bar, 20 μm. Black arrow: hBeta2-MG-positive cells. (E) IF-staining for CXCR4 in Ad-MSCs. Left: Propidium iodide for nuclear; Middle: Alexa Fluor 488 for detecting CXCR4 in Ad-MSCs; Right: Overlay. Magnification at 2000×. Scale bar, 10 μm. (F) FACS analysis for CD4⁺ or CD8⁺ T lymphocytes in peripheral blood samples at 14 days post-irradiation. (G) Percent of CD4⁺ or CD8⁺ T lymphocytes in peripheral blood monocytes (PBMCs). Upper: Percent of CD4⁺/CD8⁻ T lymphocytes; Lower: Percent of CD8⁺/CD4⁻ T lymphocytes. Data shown are the mean ± S.D. of nine independent samples. This experiment was performed only once. One-way ANOVA analysis was used for comparing the differences of data among groups. ^$P ≤ 0.05 and ^$$P ≤ 0.01 (significant low) versus the Normal group, 15 Gy group and Normal + Ad-MSC group.

Figure 3. Histological changes in irradiated lungs within 24 weeks following irradiation.

(A) H&E staining. Upper panel: 4 weeks post-irradiation; Lower panel: 24 weeks post-irradiation. Magnification at 100×. Scale bar, 200 μm. (B) Masson's Trichrome staining. Magnification at 200×. Scale bar, 100 μm. (C) Expressions of ECM-related genes in whole right lung at 24 weeks post-irradiation. Left: Collagen I-α1 expression; Right: Collagen III-α1 expression. The primers of Collagen I-α1 and Collagen III-α1 are designed for rat species. The expression levels of genes, including Collagen I-α1 and Collagen III-α1, were tested using quantitative real-time PCR. rActin was set as the internal control for determining ΔC_T values. Fold increases in expression were normalized to normal group by determining 2^−ΔΔCT values. The data of three samples from each group were collected in each independent experiment. The experiment was repeated for two times. For statistical analysis, the data of six samples in all two independent experiments were calculated for the value of mean ± S.D. One-way ANOVA analysis was used for comparing the differences of data among groups. ^$P ≤ 0.05 and ^$$P ≤ 0.01 (significant low) versus the PBS and Fibroblast groups. ns: no statistical significance.

Figure 4. Anti-fibrotic effects of Ad-MSCs on irradiated lung.

(A) IHC-staining for α-SMA. Left rank: Magnification at 200×. Scale bar, 100 μm. Right rank: Magnification at 1000×. Scale bar, 20 μm (B) IHC-staining for TGF-β1. Left rank: Magnification at 200×. Scale bar, 100 μm. Right rank: Magnification at 1000×. Scale bar, 20 μm. (C) Concentrations of cytokines in serum. Data represent the mean ± S.D. of six independent samples. This experiment was performed only once. For statistical analysis, the data of six samples were calculated for the value of mean ± S.D.One-way ANOVA analysis was used for comparing the differences of data among groups. (D) Concentrations of cytokines in bronchoalveolar lavage fluid (BALF). The data of three samples from each group were collected in each independent experiment. The experiment was repeated for twice. For statistical analysis, the data of six samples in all two independent experiments were calculated for the value of mean ± S.D. One-way ANOVA analysis was used for comparing the differences of data among groups.*P ≤ 0.05 and **P ≤ 0.01 versus the PBS and Fibroblast groups. ^$P ≤ 0.05 and ^$$P ≤ 0.01 versus the PBS and Fibroblast groups. It should be noted that the assays used to test for HGF, PGE2, TGF-β1, and TNF-α were for rat tissues, not human tissues.(E) Expression of HGF in irradiated lung. (F) Expression of mPGES in irradiated lung. All primers of above genes are designed for rat species. The expression levels of HGF and mPGES were tested using quantitative real-time PCR. rActin was set as the internal control for determining ΔC_T values. Fold increases in expression were normalized to normal group by determining 2^−ΔΔCTvalues. The data of nine samples from each group were collected in only one independent experiment. For statistical analysis, the data of nine samples were calculated for the value of mean ± S.D. One-way ANOVA analysis was used for comparing the differences of data among groups. *P ≤ 0.05 and **P ≤ 0.01 (significant high) versus the PBS and Fibroblast groups. ^$P ≤ 0.05 and ^$$P ≤ 0.01 (significant low) versus PBS and Fibroblast groups. ns: no statistical significance.

Figure 5. EMT in type II AECs.

(A) Confocal imaging using Z-stacks technology at 24 weeks post-irradiation. The thickness of the section was set as 15 μm. The thickness between frames was set as 0.25 μm. The SP-C marker were used for identifying type II AECs, co-staining with α-SMA and E-cadherin in type II AECs. Magnification at 1500× in the first five ranks. Scale bar, 200 μm. Magnification at 630× in the last one rank. Scale bar, 200 μm. (B) Gene expression patterns in type II AECs. Type II AECs were isolated at 24 weeks post-irradiation using microbeads. The expression levels of genes, including E-cadherin, α-SMA, Smad2, Smad3, Smad7, JNK1 and JNK2, were tested using quantitative real-time PCR. All primers of these genes are designed for rat species. rActin was set as the internal control for determining ΔC_T values. Fold increases in expression were normalized to normal type II AECs by determining 2^−ΔΔCT values. The data of three samples from each group were collected in each independent experiment. The experiment was repeated for two times. For statistical analysis, the data of six samples in all two independent experiments were calculated for the value of mean ± S.D. One-way ANOVA analysis was used for comparing the differences of data among groups. *P ≤ 0.05 and **P ≤ 0.01(significant high) versus PBS and Fibroblast groups. ^$P ≤ 0.05 (significant low) versus PBS and Fibroblast groups.