siRNA-Targeting Transforming Growth Factor-β Type I Receptor Reduces Wound Scarring and Extracellular Matrix Deposition of Scar Tissue  Yi-Wen Wang, Nien-Hsien Liou, Juin-Hong Cherng, Shu-Jen Chang, Kuo-Hsing Ma, Earl Fu, Jiang-Chuan Liu, Niann-Tzyy Dai  Journal of Investigative Dermatology  Volume 134, Issue 7, Pages 2016-2025 (July 2014) DOI: 10.1038/jid.2014.84 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Transforming growth factor-β type I receptor (TGFBRI) small interfering RNA (siRNA) inhibits TGFBRI expression and cell proliferation. (a) Human hypertrophic scar fibroblasts (hHSFs) transfected with TGFBRI siRNA 15, 60, 150 nM, which downregulated TGFBRI gene expression (confirmed by quantitative reverse transcription-PCR analysis at day 3) (n=3). (b–e) hHSFs transfected with TGFBRI siRNA 60 nM or scrambled siRNA 60 nM. (b, c) TGFBRI siRNA reduced TGFBRI protein expression level (n=3), scale bar=50 μm. (d) TGFBRI siRNA decreased phosphorylated Smad2/Smad3 protein expression (n=5). (e) hHSFs were transfected with TGFBRI siRNA or scrambled siRNA, with or without TGF-β1 stimulation (n=5). Cell proliferation in the absence of transforming growth factor-β (TGF-βI) stimulation did not differ significantly between TGFBRI siRNA-treated and scrambled siRNA-treated hHSFs at days 3, 7, and 10. However, cell proliferation in the presence of TGF-βI stimulation was suppressed significantly more at days 7 and 10 in TGFBRI siRNA-treated hHSFs than in scrambled siRNA-treated cells (P<0.05) (f). hHSFs were treated with 1 μM TGFBRI inhibitor or DMSO, with or without TGF-β1 stimulation (n=3). Values are mean±SEM, *P<0.05, ***P<0.001. TGFBRI inhibitor inhibited cell proliferation to a comparable extent as 60 nM TGFBRI siRNA. Journal of Investigative Dermatology 2014 134, 2016-2025DOI: (10.1038/jid.2014.84) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Downregulating transforming growth factor-β type I receptor (TGFBRI) reduces extracellular matrix (ECM) production. Human hypertrophic scar fibroblasts (hHSFs) were transfected with 1 μM TGFBRI inhibitor, 60 nM TGFBRI siRNA, or scrambled siRNA, with or without transforming growth factor-β (TGF-β1) stimulation. (a) Gene expression was analyzed 72 hours after siRNA transfection or TGFBRI inhibitor treatment. mRNA levels from cells transfected with scrambled small interfering RNA (siRNA) and stimulated with TGF-β1 were normalized to 1. Data are expressed as fold change; values are mean±SEM (n=5), *P<0.05, **P<0.01, ***P<0.001 (compared with cells treated with scrambled siRNA, with TGF-β1 stimulation); #P<0.001 (compared with cells treated with scrambled siRNA, without TGF-β1 stimulation). (b) Medium of treated cells was analyzed 7 days after siRNA transfection for secreted type I collagen. (c) Fibronectin immunofluorescence staining shows less fibronectin in TGFBRI siRNA-transfected fibroblasts than in control cells 6 days after transfection; scale bar=100 μm. (d) hHSFs were treated with Smad3 inhibitor (SIS3) or DMSO (vehicle), with TGF-β1 stimulation (n=3). mRNA levels from cells treated with DMSO and stimulated with TGF-β1 were normalized to 1. Journal of Investigative Dermatology 2014 134, 2016-2025DOI: (10.1038/jid.2014.84) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Transforming growth factor-β type I receptor (TGFBRI) small interfering RNA (siRNA) attenuates hypertrophic scarring. (a) TGFBRI siRNA 240 pmol or vehicle was injected into wound granulation tissue of rabbit ears 2 weeks after wounding. Two days later, granulation tissues were analyzed for fibrosis-related gene expression. Data are expressed as fold change between groups; values are mean±SEM (n=3), *P<0.05, **P<0.01. (b, c) TGFBRI siRNA or vehicle was injected into the wound granulation tissue of rabbit ears at 2, 3, and 4 weeks after wounding. (b) Representative photomicrographs of repaired tissue at 6, 10, and 14 weeks after wounding, scale bar=5 mm. (c) Repaired wounds were evaluated by the Vancouver scar scale (VSS) and (d) visual analog scale (VAS). Values are mean±SEM, *P<0.05, **P<0.01. Journal of Investigative Dermatology 2014 134, 2016-2025DOI: (10.1038/jid.2014.84) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Transforming growth factor-β type I receptor (TGFBRI) small interfering RNA (siRNA) reduces collagen deposition in vivo. TGFBRI siRNA was injected into the wound granulation tissue of rabbit ears at 2, 3, and 4 weeks after wounding. Scar tissues were harvested at 6, 10, and 14 weeks after wounding, and 5-μm-thick sections were stained with Masson’s trichrome stain. (a, b, d) Representative photomicrographs of repaired tissue. (a) Cross-sections of whole-scar tissue at 6, 10, and 14 weeks after wounding, scale bar=2 mm. (b) Zoomed image of sections in a, showing collagen fibers arranged in a circular pattern in vehicle-treated controls, but in a linear pattern in TGFBRI siRNA-treated cells, scale bar=100 μm. (c) All tissue sections were evaluated by the scar elevation index (SEI). Values are mean±SEM, *P<0.05, **P<0.01. (d) Immunofluorescence staining for TGFBRI in cryosections of scar tissue, scale bar=500 μm. Journal of Investigative Dermatology 2014 134, 2016-2025DOI: (10.1038/jid.2014.84) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions