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. 2015 Dec;185(12):3258-73.
doi: 10.1016/j.ajpath.2015.08.009.

Myofibroblastic Conversion and Regeneration of Mesothelial Cells in Peritoneal and Liver Fibrosis

Ingrid Lua  1 , Yuchang Li  1 , Lamioko S Pappoe  2 , Kinji Asahina  3
Affiliations

Myofibroblastic Conversion and Regeneration of Mesothelial Cells in Peritoneal and Liver Fibrosis

Ingrid Lua et al. Am J Pathol. 2015 Dec.

Abstract

Mesothelial cells (MCs) form a single epithelial layer and line the surface of body cavities and internal organs. Patients who undergo peritoneal dialysis often develop peritoneal fibrosis that is characterized by the accumulation of myofibroblasts in connective tissue. Although MCs are believed to be the source of myofibroblasts, their contribution has remained obscure. We determined the contribution of peritoneal MCs to myofibroblasts in chlorhexidine gluconate (CG)-induced fibrosis compared with that of phenotypic changes of liver MCs. CG injections resulted in disappearance of MCs from the body wall and the accumulation of myofibroblasts in the connective tissue. Conditional linage tracing with Wilms tumor 1 (Wt1)-CreERT2 and Rosa26 reporter mice found that 17% of myofibroblasts were derived from MCs in peritoneal fibrosis. Conditional deletion of transforming growth factor-β type II receptor in Wt1(+) MCs substantially reduced peritoneal fibrosis. The CG treatment also induced myofibroblastic conversion of MCs in the liver. Lineage tracing with Mesp1-Cre mice revealed that Mesp1(+) mesoderm gave rise to liver MCs but not peritoneal MCs. During recovery from peritoneal fibrosis, peritoneal MCs, but not liver MCs, contribute to the regeneration of the peritoneal mesothelium, indicating an inherent difference between parietal and visceral MCs. In conclusion, MCs partially contribute to myofibroblasts in peritoneal and liver fibrosis, and protection of the MC layer leads to reduced development of fibrous tissue.

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Figures

Supplemental Figure S1
Supplemental Figure S1
Immunohistochemistry of GFP (green) in the body wall with or without TAM injection into Wt1CreERT2/+;R26TGfl/fl mice. The endogenous fluorescence of Tomato was bleached with 3% H2O2 in methanol. A: Arrowheads indicate MCs. GFP expression is only observed in MCs after tamoxifen injection. Arrows indicate GFP− fibroblasts beneath MCs. B: Two weeks after TAM injection, the mice were treated with CG by 10 intraperitoneal injections. The body wall tissues were immunostained for GFP. After bleaching Tomato fluorescence, no red fluorescent is observed. Arrows indicate GFP+ myofibroblasts. Arrowheads indicate the body wall surface. Nuclei were counterstained with DAPI. Scale bar = 10 μm. CG, chlorhexidine gluconate; GFP, green fluorescent protein; MC, mesothelial cell; TAM, tamoxifen.
Supplemental Figure S2
Supplemental Figure S2
Deletion of Tgfbr2 gene in peritoneal MCs. A and B: Deletion of Tgfbr2 gene in cultured peritoneal MCs. GPM6A+ MCs were isolated from R26TGfl/fl;Tgfbr2fl/fl mouse body walls. Cultured MCs were infected with an Ad vector that carried LacZ or Cre (multiplicity of infection, 50) and were treated with TGF-β1 for 3 hours (A) or 12 hours (B). A: Immunocytochemistry of MCs with GFP (green) and P-SMAD3 (red). Arrowheads indicate P-SMAD3 in MCs treated with Ad-LacZ and TGF-β1. Nuclei were counterstained with DAPI. B: qPCR of MCs infected with Ad-LacZ or Cre, followed by treatment with TGF-β1. C–G: Lineage tracing of MCs during recovery from fibrosis. C: Experimental design of the conditional deletion of the Tgfbr2 gene. The tamoxifen-induced CreERT2 in peritoneal MCs activates the expression of GFP while deleting the Tgfbr2 gene. Two weeks after tamoxifen injection, peritoneal fibrosis was induced by 10 CG injections, and the mice were allowed to recover for 4 weeks by discontinuing the CG treatment. D: H&E staining of the body wall 4 weeks after the last CG injection in the Tgfbr2fl/+ (fl/+) or Tgfbr2fl/fl (fl/fl) mouse. E: Quantification of the area of connective tissue 4 weeks after the last CG injection in D. F and G: Immunohistochemistry of the body wall 4 weeks after the last CG injection in Tgfbr2fl/+ (fl/+) and Tgfbr2fl/fl (fl/fl) mice with the use of anti-GFP antibodies (green) and GPM6A or ACTA2 (red). Arrowheads and arrows indicate MCs and myofibroblasts, respectively. n = 3 (E). ∗∗P < 0.01. Scale bars: 10 μm (A, F, and G); 50 μm (D). ACTA2, α-smooth muscle actin; Ad, adenovirus; CG, chlorhexidine gluconate; GFP, green fluorescent protein; H&E, hematoxylin and eosin; MC, mesothelial cell; P-SMAD3, phosphorylated-SMAD3; qPCR, quantitative real-time PCR; TGF, transforming growth factor.
Figure 1
Figure 1
Characterization of peritoneal MCs on the mouse body walls. The body walls from wild-type (A, B, and E–L), Upk1bRFP (C and D), and Col1a1GFP (M–P) mice were immunostained with GPM6A (A), PDPN and RFP (C and D), CD200 (E), VIM and PDPN (F and G), KRT8 (H), KRT19 (I), COL I (J), CDH1 (K), ACTA2 (L), GFP and PDPN (M and N), and GFP and COL IV (O and P). B: A bright field image of A is shown. Arrowheads indicate peritoneal MCs on the body wall. Fibroblasts beneath MCs are indicated by arrows. Peritoneal MCs express GPM6A, PDPN, CD200, VIM, KRT8, KRT19, and COL I but not CDH1 and ACTA2 in the normal mouse. The Upk1bRFP mouse shows specific expression of RFP in PDPN+ MCs. MCs and fibroblasts are separated by the basal lamina composed of COL IV, and both cell types express GFP in the Col1a1GFP mouse. Nuclei were counterstained with DAPI. n = 3 mice. Scale bars = 10 μm. ACTA2, α-smooth muscle actin; CDH1, E-cadherin; COL I, type I collagen; COL IV, type IV collagen; ct, connective tissue; GFP, green fluorescent protein; GPM6A, glycoprotein M6a; KRT8, cytokeratin 8; KRT19, cytokeratin 19; MC, mesothelial cell; PDPN, podoplanin; RFP, red fluorescent protein; sm, skeletal muscle; VIM, vimentin.
Figure 2
Figure 2
Peritoneal MCs give rise to myofibroblasts in peritoneal fibrosis. A: Experimental design. After two tamoxifen injections into Wt1CreERT2/+;R26TGfl/fl mice, MCs change from the expression of Tomato to membrane-tagged GFP. Two weeks after tamoxifen injection, the mice were treated with CG by 10 intraperitoneal injections. B: Immunohistochemistry of GFP (green) and GPM6A or PDPN (red). After tamoxifen injection into Wt1CreERT2/+;R26TGfl/fl mice, only GPM6A+ or PDPN+ MCs express GFP (arrowheads). Note that no GFP expression in fibroblasts (arrows) is evident. No induction of GFP was observed in the control Wt1+/+;R26TGfl/fl mice (Cre-) after tamoxifen treatment. C–E: Phenotypic changes of MCs in peritoneal fibrosis. The body wall tissues 1 day after CG injections (1, 3, or 10 times) were immunostained with GFP (green) and ACTA2, GPM6A, or COL IV (red). After a single CG injection, GFP+ MCs begin to express ACTA2 (arrowheads). After three CG injections, GFP+ cells in the connective tissue show a myofibroblastic structure with ACTA2 expression but not with GPM6A (double arrows). After 10 CG injections, a few MCs expressing GPM6A survive on the body wall (arrowheads), and no COL IV is observed on the surface of the body wall, indicating denudation of the MC layer. F: Immunohistochemistry of GFP (green) and cell markers (red) after 10 CG injections. Arrowheads indicate the surface of the body wall. MC-derived GFP+ myofibroblasts express PDPN, COL I, and DES but not KRT8, KRT19, F4/80, and CD31 (double arrows). Immunostaining without primary antibodies does not show signals (Negative). Nuclei were counterstained with DAPI. n = 3 (B); n = 2 (C–E, after a single injection and after 3 injections); n = 3 (C–E, after 10 CG injections); Scale bar = 10 μm. ACTA2, α-smooth muscle actin; CG, chlorhexidine gluconate; COL I, type I collagen; COL IV, type IV collagen; DES, desmin; GFP, green fluorescent protein; GPM6A, glycoprotein M6a; KRT8, cytokeratin 8; KRT19, cytokeratin 19; MC, mesothelial cells; PDPN, podoplanin.
Figure 3
Figure 3
Lineage tracing of MCs during recovery from fibrosis. Regeneration of the MC layer on the body wall during recovery from peritoneal fibrosis. A: By tamoxifen injection into Wt1CreERT2/+;R26TGfl/fl mice, MCs begin to express GFP. Two weeks after tamoxifen injection, peritoneal fibrosis was induced by 10 CG injections, and the mice were allowed to recover for 4 weeks by discontinuing the CG treatment. B: H&E staining of the body wall before (N), after 10 CG injections (CG), and 4 weeks after the last CG injection (CG+4W). An arrowhead and arrow indicate MCs and fibroblasts, respectively. C: Quantification of the areas of connective tissue in the body wall in B. D: qPCR of mRNA expression in body wall tissues from mice that were N, fibrotic (CG), and recovered 4 weeks after the CG injection (CG+4W). E–J: Immunohistochemistry of the body wall with GFP (green) and GPM6A (E), PDPN (F), KRT8 (G), KRT19 (H), COL IV (I), or ACTA2 (J, red) at 4 weeks after the last CG injection. Arrowheads indicate regenerated peritoneal MCs expressing GFP and markers. Arrows indicate GFP+ fibroblasts in the connective tissue. K: BrdU incorporation in the body wall. One week after the last CG injection, the peritoneum was analyzed 4 hours after BrdU treatment. Immunohistochemistry for cytokeratin (Keratin, green) and BrdU (red) shows the incorporation of BrdU in 2.2% MCs (arrowheads, 910 MCs were counted). An arrow indicates rare myofibroblasts showing nuclear BrdU staining. Nuclei were counterstained with DAPI. n = 3 (B and K); ∗∗P < 0.01. Scale bars: 50 μm (B); 10 μm (E–K). ACTA2, α-smooth muscle actin; BrdU, bromodeoxyuridine; CG, chlorhexidine gluconate; COL IV, type IV collagen; GFP, green fluorescent protein; GPM6A, glycoprotein M6a; H&E, hematoxylin and eosin; KRT8, cytokeratin 8; KRT19, cytokeratin 19; MC, mesothelial cell; N, normal; PDPN, podoplanin; qPCR, quantitative real-time PCR; 4W, 4 weeks.
Figure 4
Figure 4
Induction of peritoneal MMT by TGF-β in vitro. Peritoneal MCs were isolated from the mouse body wall with the use of anti-GPM6A antibodies and MACS. A: qPCR of GPM6A− (−) and GPM6A+ cells (+) separated from body wall cells by MACS. The GPM6A+ fraction highly expresses mRNAs for Gpm6a, Cd200, Msln, Pdpn, Upk1b, Wt1, Krt8, and Krt19. B: Culture of GPM6A+ peritoneal MCs. MCs form epithelial colonies. Some MCs lose epithelial cell structure in culture. C: qPCR of peritoneal MCs in culture. MCs increase the expression of Acta2 mRNA in accordance with the structural changes shown in B. D: Peritoneal MCs were cultured in the presence or absence of TGF-β and SB431542. TGF-β induces MMT, whereas SB431542 blocks TGF-β–induced structural changes. E: Immunocytochemistry of cultured peritoneal MCs with antibodies against ACTA2 (red) and GPM6A (green). TGF-β strongly induces the expression of ACTA2, whereas SB431542 blocks the induction of ACTA2. F: qPCR of cultured peritoneal MCs in the presence of DMSO (−), TG, SB, and both TG and SB. TGF-β induces the expression of Acta2, Ctgf, Gpm6a, and Pdpn mRNAs. G: Immunocytochemistry of cultured peritoneal MCs. TGF-β induces the nuclear localization of P-SMAD3 (green). H and I: Lineage tracing of Wt1+ peritoneal MCs in culture. After tamoxifen injection to Wt1CreERT2/+;R26TGfl/fl mice, GPM6A+ MCs were isolated by MACS. H: MCs express either Tomato (red) or GFP (green). Arrowheads indicate MCs co-expressing GFP (green) and GPM6A (red). I: With TGF-β treatment (+TGF-β), both GFP+ (arrowheads) and GFP− MC-derived myofibroblasts (arrow) express ACTA2. No signals were detected in a negative control without primary antibodies (negative). Nuclei were counterstained with DAPI. ∗P < 0.05, ∗∗P < 0.01. Scale bars: 20 μm (B and D); 10 μm (E, and G–I). ACTA2, α-smooth muscle actin; DMSO, dimethyl sulfoxide; GFP, Green fluorescent protein; GPM6A, glycoprotein M6a; MACS, magnetically activated cell sorting; MC, mesothelial cell; MMT, mesothelial–mesenchymal transition; P-SMAD3, phosphorylated-SMAD3; qPCR, quantitative real-time PCR; SB, presence of SB431542; SB431542, inhibitor for TGFBR1; TG, presence of TGF-β; TGF, transforming growth factor.
Figure 5
Figure 5
Essential role of TGF-β signaling in peritoneal MMT in fibrosis. TGF-β signaling was inhibited with the use of STR (A–D) or conditional deletion of the Tgfbr2 gene in peritoneal MCs (E–K). A: Experimental design. After labeling peritoneal MCs as GFP+ cells by tamoxifen injection in Wt1CreERT2/+;R26TGfl/fl mice, peritoneal fibrosis was induced by CG injections with either STR or IgG injection five times. B: H&E staining of the body wall treated with an IgG control or STR in CG-induced fibrosis. C: Quantification of the area of the connective tissue treated with an IgG control or STR in B. D: Immunohistochemistry of the body wall with the use of anti-GFP antibodies (green) and ACTA2 or GPM6A antibodies (red). Arrowheads and arrows indicate MCs and myofibroblasts, respectively. The STR treatment inhibits MMT on the body wall. E: Experimental design of the conditional deletion of the Tgfbr2 gene. The tamoxifen-induced CreERT2 in peritoneal MCs activates the expression of GFP while deleting the Tgfbr2 gene. F: FACS of MCs isolated from the fl/+ or fl/fl mouse. After isolation of MCs with the use of the anti-GPM6A antibody and MACS, the cells (MACS+) were further incubated with anti-rat IgG Alexa Fluor 647 to efficiently detect GPM6A antibody+ MCs. GFP+ GPM6A+ and GFP− GPM6A+ MCs underwent FACS from the fl/+ or fl/fl Tgfbr2 knockout mouse. MCs (Cre− MACS+) isolated from Wt1+/+;R26TGfl/fl;Tgfbr2fl/+ or Tgfbr2fl/fl mice and Cre+ MACS− fractions were used as negative controls. G: qPCR of the GFP+ GPM6A+ (+) and GFP− GPM6A+ (−) MCs. GFP+ GPM6A+ MCs decrease Tgfbr2 mRNA expression, while increasing Gfp mRNA in the homozygous (fl/fl) MCs. H: H&E staining of the fibrotic body wall induced by CG from the Wt1CreERT2/+;R26TGfl/fl;Tgfbr2fl/+ (fl/+) or Wt1CreERT2/+;R26TGfl/fl;Tgfbr2fl/fl (fl/fl) mouse. I: Quantification of the area of connective tissue in Tgfbr2fl/+ and Tgfbr2fl/fl mice treated with 10 CG injections in H. J and K: Immunohistochemistry of the body wall from Tgfbr2fl/+ (fl/+) or Tgfbr2fl/fl (fl/fl) mice treated with 10 CG injections with the use of anti-GFP antibodies (green) and ACTA2, GPM6A (J), or P-SMAD2/3 antibodies (red) (K). Arrowheads and arrows indicate MCs and myofibroblasts, respectively. J: The MC layer remains on the body wall after the conditional deletion of Tgfbr2 in MCs (an arrowhead). K: Double arrowheads indicate GFP+ MCs without P-SMAD2/3 expression. Nuclei were counterstained with DAPI. n = 3 (C and I). ∗P < 0.05, ∗∗P < 0.01. Scale bars: 100 μm (B and H); 10 μm (D and K). ACTA2, α-smooth muscle actin; CG, chlorhexidine gluconate; FACS, fluorescence-activated cell sorter; GFP, green fluorescent protein; GPM6A, glycoprotein M6a; H&E, hematoxylin and eosin; KO, knockout; MACS, magnetically activated cell sorting; MC, mesothelial cell; MMT, mesothelial–mesenchymal transition; P-SMAD3, phosphorylated-SMAD3; qPCR, quantitative real-time PCR; STR, soluble form of TGFBR2; TGF, transforming growth factor.
Figure 6
Figure 6
Lineage tracing of liver and peritoneal MCs in CG-induced injury. A–H: Lineage tracing of MCs in the liver with the use of Wt1CreERT2/+;R26TGfl/fl;Tgfbr2fl/fl mice. A: Experimental design. After two tamoxifen injections into Wt1CreERT2/+;R26TGfl/fl;Tgfbr2fl/fl mice, MCs begin to express GFP and lose Tgfbr2 mRNA expression. Two weeks after tamoxifen injection, the mice were treated with CG by 10 intraperitoneal injections. Four weeks after the last CG injection, the liver tissues were analyzed to examine the recovery. B: Immunohistochemistry of GFP (green) and GPM6A or ACTA2 (red) in the normal liver. GFP expression is observed in GPM6A+ MCs (arrowhead) but not fibroblasts (arrow) beneath MCs. Liver MCs do not express ACTA2 (arrowheads). C: Sirius red staining of the liver after 10 CG injections (CG) or 4 weeks after the last CG injection (CG+4W). Sirius red staining shows thin collagen lamella beneath the liver surface. D: Quantification of the length of the collagen lamella in C. E–G: After labeling MCs as GFP+ cells in Tgfbr2fl/+ or Tgfbr2fl/fl mice, liver injury was induced by 10 CG injections. E: GFP+ ACTA2+ myofibroblasts (arrows) are observed beneath MCs (arrowheads) in the Tgfbr2fl/+ (fl/) mice. MCs do not express ACTA2 (arrowheads). Tgfbr2fl/fl (fl/fl) liver shows less accumulation of ACTA2+ myofibroblasts. F: After CG injections, MCs remain on the liver surface and express GPM6A (arrowheads). Arrows indicate GFP+ MC-derived myofibroblasts beneath MCs. G: P-SMAD2/3 (red) is localized in the nuclei of MCs (arrowheads), myofibroblasts (arrows), and hepatocytes (large round nuclei) by CG treatment in the Tgfbr2fl/+ liver. Tgfbr2fl/fl liver shows down-regulation of P-SMAD2/3 in GFP+ cells (double arrowheads). H: Four weeks after the last CG injection, the liver tissues were analyzed by immunohistochemistry for GFP (green) and ACTA2 or GPM6A (red) in Tgfbr2fl/+ or Tgfbr2fl/fl mice. GFP+ MCs (arrowheads) are negative for ACTA2 and positive for GPM6A. An arrow indicates GFP+ cells beneath MCs in regenerating liver. I–K: Mesoderm lineage tracing in the body wall and liver. I: Mesodermal lineage was traced with the use of Mesp1Cre/+;R26TGfl/fl mice. J: Normal liver and body wall tissues from the Mesp1Cre/+;R26TGfl/fl mouse were immunostained with GFP (green) and PDPN (red). Liver MCs, but not peritoneal MCs, express GFP (arrowheads). Arrows indicate GFP+ hepatic stellate cells in the liver and fibroblasts in the body wall. K: Peritoneal fibrosis was induced by 10 CG injections into Mesp1Cre/+;R26TGfl/fl mice, and the mice were allowed to recover for 4 weeks by discontinuing the CG treatment. The body wall tissues were immunostained with GFP (green) and PDPN or ACTA2 (red) 4 weeks after 10 CG injections. Note that no GFP expression is evident in regenerated peritoneal MCs (arrowheads). Arrows indicate fibroblasts expressing GFP in the connective tissue. Nuclei were counterstained with DAPI. n = 3 (D); n = 4 mice (I). ∗P < 0.05, ∗∗P < 0.01. Scale bars: 10 μm (B, E–H, J, and K); 20 μm (C). ACTA2, α-smooth muscle actin; CG, chlorhexidine gluconate; GFP, green fluorescent protein; GPM6A, glycoprotein M6a; KO, knockout; MC, mesothelial cell; P-SMAD3, phosphorylated-SMAD3; PDPN, podoplanin; 4W, 4 weeks.
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