Mesenchymal stem cells ameliorate experimental peritoneal fibrosis by suppressing inflammation and inhibiting TGF-β1 signaling

Abstract

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have regenerative capability and exert paracrine actions on damaged tissues. Since peritoneal fibrosis is a serious complication of peritoneal dialysis, we tested whether MSCs suppress this using a chlorhexidine gluconate model in rats. Although MSCs isolated from green fluorescent protein-positive rats were detected for only 3 days following their injection, immunohistochemical staining showed that MSCs suppressed the expression of mesenchymal cells, their effects on the deposition of extracellular matrix proteins, and the infiltration of macrophages for 14 days. Moreover, MSCs reduced the functional impairment of the peritoneal membrane. Cocultures of MSCs and human peritoneal mesothelial cells using a Transwell system indicated that the beneficial effects of MSCs on the glucose-induced upregulation of transforming growth factor-β1(TGF-β1) and fibronectin mRNA expression in the human cells were likely due to paracrine actions. Preincubation in MSC-conditioned medium suppressed TGF-β1-induced epithelial-to-mesenchymal transition, α-smooth muscle actin, and the decrease in zonula occludens-1 in cultured human peritoneal mesothelial cells. Although bone morphogenic protein 7 was not detected, MSCs secreted hepatocyte growth factor and a neutralizing antibody to this inhibited TGF-β1 signaling. Thus, our findings imply that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-β1 signaling in a paracrine manner.

Figure 1

Mesenchymal stem cells (MSCs) suppressed peritoneal cell density and thickening in chlorhexidine gluconate (CG)-injected rats. (a, b) Representative light microscopic features of peritoneal tissues on days 7 and 14 (a: hematoxylin–eosin stain; b: Masson's trichrome stain, original magnification × 200) in control rats, CG-injected rats treated with the vehicle, and CG-injected rats treated with MSCs. (c, d) The thickness of the submesothelial compact zone increased along with its cellularity until day 14 in rats treated with the vehicle, whereas cell density and thickening of the zone were suppressed in rats treated with MSCs. Bars show the compact zone area. *P<0.01 versus control group, ^#P<0.05 versus CG+vehicle group.

Figure 2

Mesenchymal stem cells (MSCs) suppressed α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) expressions in rats with peritoneal fibrosis. Immunohistochemical analyses of (a) α-SMA and (b) FSP-1 expression in peritoneal tissues on days 7 and 14 (original magnification × 200) in control rats, chlorhexidine gluconate (CG)-injected rats treated with the vehicle alone, and CG-injected rats treated with MSCs. (c, d) Accumulation of α-SMA+ and FSP-1+ cells was observed on days 7 and 14 in CG-injected rats treated with the vehicle, whereas the areas (percentage) of α-SMA expression and the number of FSP-1+ cells were significantly smaller on both days 7 and 14 in CG-injected rats treated with MSCs. *P<0.01 versus control group, ^#P<0.05 versus CG+vehicle group.

Figure 3

Mesenchymal stem cells (MSCs) suppressed collagen I and III expressions in rats with peritoneal fibrosis. Immunohistochemical analyses of (a) collagen I and (b) collagen III expression in peritoneal tissues on days 7 and 14 (original magnification × 200) in control rats, chlorhexidine gluconate (CG)-injected rats treated with the vehicle alone, and CG-injected rats treated with MSCs. (c, d) The numbers of collagen I+ and III+ pixels were increased on days 7 and 14 in CG-injected rats treated with the vehicle, whereas the numbers of collagen I+ and III+ pixels were significantly smaller on days 7 and 14 in CG-injected rats treated with MSCs. *P<0.01 versus control group, ^#P<0.05 versus CG+vehicle group.

Figure 4

Mesenchymal stem cells (MSCs) suppressed monocyte/macrophage infiltration and transforming growth factor-β1 (TGF-β1) expression in rats with peritoneal fibrosis. (a) Immunohistochemical analysis of CD68 expression in peritoneal tissues on days 7 and 14 in control rats, chlorhexidine gluconate (CG)-injected rats treated with vehicle alone, and CG-injected rats treated with MSCs. (b) Two-color immunohistochemical analysis of CD68 (brown) and TGF-β1 (blue–gray) expression in peritoneal tissues on day 14 in CG-injected rats treated with the vehicle alone and CG-injected rats treated with MSCs. (c) The majority of CD68+ cells showed immunoreactivity for TGF-β1 (arrows). (d) The number of CD68+ cells increased until day 14 in CG-injected rats treated with the vehicle alone, whereas it was significantly smaller on days 7 and 14 in CG-injected rats treated with MSCs. (e) The number of cells double positive for CD68 and TGF-β1 increased until day 14 in CG-injected rats treated with vehicle alone, whereas it was significantly smaller on days 7 and 14 in CG-injected rats treated with MSCs. *P<0.01 versus control group, ^#P<0.05 versus CG+vehicle group (original magnification: a and c × 400; b × 200).

Figure 5

Mesenchymal stem cells (MSCs) suppressed phosphorylated Smad2 (pSmad2) expression in rats with peritoneal fibrosis. (a) Immunohistochemical analysis of pSmad2 expression in peritoneal tissues on days 7 and 14 (original magnification × 200) in control rats, chlorhexidine gluconate (CG)-injected rats treated with vehicle alone, and CG-injected rats treated with MSCs. (b) The number of pSmad2+ cells increased until day 14 in CG-injected rats treated with vehicle alone, whereas it was significantly smaller on days 7 and 14 in CG-injected rats treated with MSCs. *P<0.01 versus control group, ^#P<0.05 versus CG+vehicle group.

Figure 6

Mesenchymal stem cells (MSCs) reduced the functional impairments of the peritoneal membrane in rats with peritoneal fibrosis. (a) The peritoneal absorption of glucose from the dialysate (D/D0) and the (b) dialysate-to-plasma (D/P) ratio of blood urea nitrogen (BUN) were assessed in control rats, chlorhexidine gluconate (CG)-injected rats treated with vehicle alone, and CG-injected rats treated with MSCs during 30-min dwell of dialysate (4.25% Dianeal) at 100 ml/kg body weight. The peritoneal permeabilities of glucose and BUN were significantly higher in CG-injected rats treated with the vehicle alone than in control rats, whereas they were significantly improved in CG-injected rats treated with MSCs. *P<0.01, ^#P<0.05.

Figure 7

Green fluorescent protein (GFP)-positive mesenchymal stem cells (MSCs) were briefly retained in the peritoneum after stimulation by chlorhexidine gluconate (CG). At 3 days after the injection of cells, GFP+ MSCs were present in the (a) parietal peritoneum, (b) omentum, and (c) diaphragm of CG-injected rats. (d) GFP-positive MSCs were not found in the parietal peritoneum of control rats treated with vehicle alone (original magnification × 400, all sections).

Figure 8

Mesenchymal stem cells (MSCs) suppressed glucose-induced upregulation of transforming growth factor-β1 (TGF-β1) and fibronectin expression in human peritoneal mesothelial cells. Incubation with glucose significantly induced expressions of (a) TGF-β1 mRNA and (b) fibronectin mRNA compared with control cells, whereas coculture with human MSCs led to a significant reduction of TGF-β1 and fibronectin mRNA expression (n=6). *P<0.01, ^#P<0.05.

Figure 9

Mesenchymal stem cell (MSC)-conditioned media (CM) blocked transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT). Representative photomicrograph shows characteristic cobblestone-like appearances of (a) control human peritoneal mesothelial cells (HPMCs) and (b) HPMCs incubated with MSC-CM without TGF-β1 stimulation. Fibroblast-like change was observed following TGF-β1 stimulation for 48 h, but the change was reduced in (d) HPMCs incubated with MSC-CM than in (c) HPMCs incubated with normal medium. HPMCs were observed by an independent investigator who was blinded to the experimental conditions. (e) Western blot analysis shows that MSC-CM inhibited phosphorylation of Smad2 in HPMCs after 30 min of TGF-β1 stimulation. (f, g) TGF-β1 treatment for 48 h caused increased α-smooth muscle actin (α-SMA) protein expression and decreased zonula occludens-1 (ZO-1) protein expression. MSC-CM attenuated these TGF-β1-induced EMT responses (n=6). *P<0.01, ^#P<0.05.

Figure 10

Mesenchymal stem cells (MSCs) secreted hepatocyte growth factor (HGF) and inhibited transforming growth factor-β1 (TGF-β1) signaling. (a) Human peritoneal mesothelial cells (HPMCs) and human MSCs were cultured in 0.1% fetal bovine serum (FBS) containing Dulbecco's modified Eagle's medium (DMEM) for 48 h and HGF concentration in the conditioned media (CM) was measured in triplicate by enzyme-linked immunosorbent assay (ELISA) and the mean data were used. HGF concentration in the MSC-CM was increased in a time-dependent manner, whereas it was below the detection level in the HPMC-CM. (b) MSC-CM and normal medium were preincubated with or without HGF-neutralizing antibody (HGF-Ab; 10 μg/ml) for 1 h before its addition to HPMC cultures. Western blot analysis shows that MSC-CM inhibited phosphorylation of Smad2 (pSmad2) in HPMCs after 30 min of TGF-β1 stimulation and HGF-neutralizing antibody blocked the inhibitory effect of MSC-CM (n=6). *P<0.05.