ORIGINAL ARTICLE

siRNA-Targeting Transforming Growth Factor-b Type I Receptor Reduces Wound Scarring and Extracellular Matrix Deposition of Scar Tissue Yi-Wen Wang1,2, Nien-Hsien Liou3, Juin-Hong Cherng4, Shu-Jen Chang5, Kuo-Hsing Ma3, Earl Fu4, Jiang-Chuan Liu3 and Niann-Tzyy Dai6 Hypertrophic scarring is related to persistent activation of transforming growth factor-b (TGF-b)/Smad signaling. In the TGF-b/Smad signaling cascade, the TGF-b type I receptor (TGFBRI) phosphorylates Smad proteins to induce fibroblast proliferation and extracellular matrix deposition. In this study, we inhibited TGFBRI gene expression via TGFBRI small interfering RNA (siRNA) to reduce fibroblast proliferation and extracellular matrix deposition. Our results demonstrate that downregulating TGFBRI expression in cultured human hypertrophic scar fibroblasts significantly suppressed cell proliferation and reduced type I collagen, type III collagen, fibronectin, and connective tissue growth factor (CTGF) mRNA, and type I collagen and fibronectin protein expression. In addition, we applied TGFBRI siRNA to wound granulation tissue in a rabbit model of hypertrophic scarring. Downregulating TGFBRI expression reduced wound scarring, the extracellular matrix deposition of scar tissue, and decreased CTGF and a-smooth muscle actin mRNA expression in vivo. These results suggest that TGFBRI siRNA could be applied clinically to prevent hypertrophic scarring. Journal of Investigative Dermatology (2014) 134, 2016–2025; doi:10.1038/jid.2014.84; published online 27 March 2014

INTRODUCTION Hypertrophic scarring usually develops during the healing process after skin trauma or severe burn injury (Gauglitz et al., 2011). Scarring leads to a deformed appearance and contracted neogenetic tissue, which result in physiological and psychological problems for patients. Annually, millions of people suffer these discomforts (Bayat et al., 2003). Because traditional management seldom provides satisfactory outcomes, it is crucial to develop an effective strategy to prevent hypertrophic scarring. 1

Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei City, Taiwan, Republic of China; 2Burn Center, Tri-Service General Hospital, Taipei City, Taiwan, Republic of China; 3Department of Biology and Anatomy, National Defense Medical Center, Taipei City, Taiwan, Republic of China; 4School of Dentistry, National Defense Medical Center, Taipei City, Taiwan, Republic of China; 5Department of Dentistry, National Yang-Ming University, Taipei City, Taiwan, Republic of China and 6Department of Plastic and Reconstructive Surgery, Tri-Service General Hospital, Taipei City, Taiwan, Republic of China Correspondence: Niann-Tzyy Dai, Department of Plastic and Reconstructive Surgery, Tri-Service General Hospital, No. 325, Section 2, Cheng-Gung Road, Nei-Hu DistrictTaipei 114, Taiwan City, Republic of China. E-mail: [email protected] Abbreviations: CTGF, connective tissue growth factor; ECM, extracellular matrix; Hhsf, human hypertrophic scar fibroblasts; SEI, scar elevation index; a-SMA, a-smooth muscle actin; TGF-b, transforming growth factor-b; TGFBRI, transforming growth factor-b type I receptor; TGFBRII, transforming growth factor-b type II receptor; siRNA, small interfering RNA; VSS, Vancouver scar scale Received 12 September 2013; revised 9 January 2014; accepted 21 January 2014; accepted article preview online 13 February 2014; published online 27 March 2014

2016 Journal of Investigative Dermatology (2014), Volume 134

In the proliferative phase of the wound healing process, an important element to promote wound healing is the transforming growth factor-b (TGF-b)/Smad signaling (Leask and Abraham, 2004; Cutroneo, 2007). TGF-bI stimulates the expression of many fibrogenic genes, including those for type I and III collagen, fibronectin, and connective tissue growth factor (CTGF) (Ellis et al., 2003; Leask et al., 2003). When TGF-b binds to the TGF-b type II receptor (TGFBRII), this complex immediately recruits and activates the TGF-b type I receptor (TGFBRI). The activated TGFBRI subsequently phosphorylates Smad proteins, which trigger the transcription of downstream target genes, causing proliferation of fibroblasts and production of extracellular matrix (ECM) (Leask and Abraham, 2004). However, persistent activation of TGF-b/ Smad signaling, even when the wound has healed, causes overproliferation of fibroblasts and excessive production of ECM (e.g., collagen, fibronectin), resulting in hypertrophic scarring (Sarrazy et al., 2011; Profyris et al., 2012). Fibroblasts derived from hypertrophic scars have been shown to have increased expression levels of TGF-b and TGF-b receptor proteins (Schmid et al., 1998; Chin et al., 2001). Various studies have tried to suppress wound scarring via blocking TGF-b/Smad signaling, e.g., by using anti-TGF-b antibody (Shah et al., 1994; Cordeiro et al., 1999; Mead et al., 2003; Lu et al., 2005) or downregulating the gene expression of TGF-bI (Brown et al., 1995; Cordeiro et al., 2003). However, many kinds of cells energetically produce TGF-b in the wound healing process (Leask and Abraham, 2004), making it difficult to block a large amount of TGF-b in the environment of wound repair. Therefore, other effective & 2014 The Society for Investigative Dermatology

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

targets for inhibiting the downstream activity of TGF-b/Smad signaling could be Smad3 or TGF-b receptors, including TGFBRI, TGFBRII, and TGFBRIII. In fact, wound scarring has been shown to be reduced by downregulating expression of Smad3 (Sekiguchi et al., 2007; Wang et al., 2007; Lee et al., 2010) or TGFBRII protein expression (Nakamura et al., 2004; Chu et al., 2008). In addition, overexpression of dominant negative mutant TGFBRII has been used to inhibit TGF-b binding to normal TGF-b receptors (Reid et al., 2007). Interestingly, TGF-b signaling pathways can be activated by mild expression of the kinase-deficient TGFBRII, and the enhancement of TGFb signaling depends on TGFBRI kinase (Denton et al., 2005). Furthermore, TGFBRI, but not TGFBRII, protein levels were shown to be higher in fibroblasts derived from keloid scar tissues compared with those from normal skin tissues (Tsujita-Kyutoku et al., 2005). Moreover, overexpression of TGFBRI increased type I collagen protein levels in a dose-dependent manner, whereas the overexpression of TGFBRII did not have the same effect (Pannu et al., 2004). These results suggest that TGFBRI is a vital determinant of fibrogenic effects, but little attention has been given to TGFBRI. Given that TGFBRI is a crucial mediator of TGF-b/Smad signaling, we hypothesized that inhibiting TGFBRI expression to block TGF-b/Smad signaling would decrease hypertrophic scarring. An effective strategy for specifically knocking down a target is RNA interference, which has been widely applied to knock down genes of interest without changing the host DNA or leading to long-term effects (Dominska and Dykxhoorn, 2010). Therefore, we chose small interfering RNA (siRNA) to silence TGFBRI gene expression. In this study, we transfected TGFBRI siRNA into cultured human hypertrophic scar fibroblasts, resulting in suppression of cell proliferation and ECM expression. In addition, we used a rabbit model of hypertrophic scarring to inject TGFBRI siRNA into granulated tissue in the late proliferative and early remodeling phases of wound healing. Our results show that TGFBRI siRNA reduced ECM deposition and scar contracture in vivo. These findings suggest that TGFBRI siRNA could be applied clinically to prevent hypertrophic scarring. RESULTS

TGFBRI siRNA (P ¼ 0.455). TGFBRI mRNA levels from cells treated with scrambled siRNA were normalized to 1 (Figure 1a). The TGFBRI protein expression level (confirmed by western blot and immunofluorescence staining at day 5 after siRNA transfection) was lower in TGFBRI siRNAtreated hHSFs than that in scrambled siRNA-treated cells (Figure 1b, c). Phosphorylated Smad2/Smad3 protein expression levels (confirmed by western blot on day 5 after siRNA transfection) were lower in TGFBRI siRNA-treated hHSFs than those in scrambled siRNA-treated cells (Figure 1d). These results indicate that TGFBRI siRNA inhibits TGFBRI expression and suppresses TGF-b/Smad signaling in primary cultures of hHSFs. TGFBRI siRNA suppresses cell proliferation in human hypertrophic scar fibroblasts

As TGF-b/Smad signaling has an important role in promoting fibroblast proliferation, and excess proliferation results in hypertrophic scarring (Profyris et al., 2012), we hypothesized that blocking TGF-b/Smad signaling with TGFBRI siRNA would inhibit fibroblast proliferation. To test this hypothesis, we transfected hHSFs with TGFBRI siRNA or scrambled siRNA and either stimulated them with 2 ng ml  1 TGF-b1 or did not. In vitro CCK-8 assays showed that all doses of TGFBRI siRNA significantly suppressed cell proliferation on days 7 and 10. Moreover, 60 nM TGFBRI siRNA reduced proliferation significantly more than that by 15 nM TGFBRI siRNA on day 10 (P ¼ 0.0102), but its effect was not significantly different from that of 150 nM TGFBRI siRNA at any time point (Supplementary Figure Sb online). Cell proliferation in the absence of TGF-bI stimulation did not differ significantly between 60 nM TGFBRI siRNA-treated and scrambled siRNAtreated hHSFs at days 3, 7, and 10. However, cell proliferation in the presence of TGF-bI stimulation was suppressed significantly more at days 7 and 10 in TGFBRI siRNA-treated hHSFs than that in scrambled siRNA-treated cells (Po0.05) (Figure 1e). These results indicate that with TGF-bI stimulation, the proliferation of hHSFs was suppressed by TGFBRI siRNA. We also treated hHSFs with 1 mM TGFBRI inhibitor (LY-364947), and found that it inhibited cell proliferation to an extent comparable to that of 60 nM TGFBRI siRNA (Figure 1f).

TGFBRI siRNA inhibits TGFBRI expression

To confirm siRNA transfection efficiency, human hypertrophic scar fibroblasts (hHSFs) were transfected with 60 nM FAMlabeled scrambled siRNA for 4 hours. More than 90% of cells displayed green fluorescent particles (Supplementary Figure Sa online). Following the same procedure, hHSFs were transfected with 15, 60, and 150 nM TGFBRI siRNA or scrambled siRNA. TGFBRI mRNA expression levels in TGFBRI siRNAtreated hHSFs (confirmed by quantitative real-time PCR analysis on day 3 after siRNA transfection) were significantly lower at each dose of TGFBRI siRNA than the expression levels in the scrambled siRNA-treated cells (15 nM: reduced 65%, Po0.001; 60 nM: reduced 89%, Po0.001; 150 nM: reduced 90%, Po0.001). Moreover, the effect of 60 nM TGFBRI siRNA was significantly different from that of 15 nM TGFBRI siRNA (Po0.001), but not from the effect of 150 nM

Downregulating TGFBRI reduces ECM production

As TGF-b/Smad signaling induces ECM deposition, which promotes hypertrophic scarring (Leask and Abraham, 2004), we wanted to determine the effect of TGFBI siRNA on ECM production. Thus, we inhibited TGF-b/Smad signaling by transfecting hHSFs with 60 nM TGFBRI siRNA or with 1 mM TGFBRI inhibitor (LY-364947), with or without TGF-bI stimulation, and measured the mRNA expression of four markers of ECM production: type I collagen, type III collagen, fibronectin, and CTGF. As control, hHSFs were treated with scrambled siRNA, with or without TGF-bI stimulation. mRNA expression was analyzed by real-time PCR at 72 hours after siRNA transfection. TGF-bI upregulated mRNA expression of type I collagen, type III collagen, fibronectin, and CTGF, and these effects were blocked by www.jidonline.org 2017

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

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Figure 1. Transforming growth factor-b 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 ¼ 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 ¼ 3), scale bar ¼ 50 mm. (d) TGFBRI siRNA decreased phosphorylated Smad2/Smad3 protein expression (n ¼ 5). (e) hHSFs were transfected with TGFBRI siRNA or scrambled siRNA, with or without TGF-b1 stimulation (n ¼ 5). Cell proliferation in the absence of transforming growth factor-b (TGF-bI) 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-bI stimulation was suppressed significantly more at days 7 and 10 in TGFBRI siRNA-treated hHSFs than in scrambled siRNA-treated cells (Po0.05) (f). hHSFs were treated with 1 mM TGFBRI inhibitor or DMSO, with or without TGF-b1 stimulation (n ¼ 3). Values are mean±SEM, *Po0.05, ***Po0.001. TGFBRI inhibitor inhibited cell proliferation to a comparable extent as 60 nM TGFBRI siRNA.

TGFBRI siRNA or TGFBRI inhibitor (Figure 2a). To examine collagen production by fibroblasts, we measured type I collagen concentration in the medium of treated cells at day 7 after siRNA transfection. TGFBRI siRNA significantly reduced the amount of type I collagen secreted into the culture medium (about 40%, Po0.01) (Figure 2b). Immunofluorescence staining for fibronectin showed that TGFBRI siRNA decreased fibronectin expression in ECM of hHSFs at day 6 after transfection (Figure 2c). These results show that TGFBRI siRNA reduces ECM production in the presence of TGF-b1. Because Smad3 is the downstream of TGF-b/Smad signaling, we further investigated fibrosis-related gene expression by inhibiting Smad3 activity. We treated hHSFs with 3, 5, and 10 mM SIS3, a Smad3 inhibitor. Higher doses of SIS3 (5 and 10 mM) decreased expression of all fibrosis-related genes tested, but treatment with a lower dose (3 mM SIS3) elevated the expression of type I and III collagen genes (Figure 2d). This 2018 Journal of Investigative Dermatology (2014), Volume 134

result suggests that TGF-b/Smad signaling may have a biphasic effect on type I and III collagen production. Downregulating TGFBRI alter fibrosis-related gene expression in vivo

The rabbit ear hypertrophic scarring model has similar properties to human skin hypertrophic scarring (Morris et al., 1997), indicating that this model is suitable for investigating the effect of TGFBRI siRNA in hypertrophic scar prevention. To ensure the presence of siRNA in the granulation tissue of rabbit ears, FAM-labeled scrambled siRNA was injected into the granulation tissue, which was collected 4 hours after injection, cryosectioned, and viewed for green fluorescence (Supplementary Figure Sc online). To examine the short-term changes in fibrosis-related gene expression in vivo, we injected a single dose of 240 pmol TGFBRI siRNA into the wound granulation tissue of rabbit ears 2 weeks after wounding, and measured the expression of TGFBRI and five

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

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Figure 2. Downregulating transforming growth factor-b type I receptor (TGFBRI) reduces extracellular matrix (ECM) production. Human hypertrophic scar fibroblasts (hHSFs) were transfected with 1 mM TGFBRI inhibitor, 60 nM TGFBRI siRNA, or scrambled siRNA, with or without transforming growth factor-b (TGF-b1) 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-b1 were normalized to 1. Data are expressed as fold change; values are mean±SEM (n ¼ 5), *Po0.05, **Po0.01, ***Po0.001 (compared with cells treated with scrambled siRNA, with TGF-b1 stimulation); #Po0.001 (compared with cells treated with scrambled siRNA, without TGF-b1 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 ¼ 100 mm. (d) hHSFs were treated with Smad3 inhibitor (SIS3) or DMSO (vehicle), with TGF-b1 stimulation (n ¼ 3). mRNA levels from cells treated with DMSO and stimulated with TGF-b1 were normalized to 1.

fibrogenic genes. Two days after injecting TGFBRI siRNA, the gene expressions of TGFRBI, CTGF, and a-smooth muscle actin (a-SMA) were about 40%, Po0.05, 99%, Po0.01, and 78%, Po0.01, respectively, which were lower than those in the control group (Figure 3a TGFRBI, CTGF, and a-SMA panel). These results suggest that downregulating TGFBRI

may decrease scar hypertrophy and contracture. However, after a single TGFBRI siRNA treatment, gene expression of type I collagen, type III collagen, and fibronectin increased about 2.3-fold (Po0.01), 1.3-fold (P ¼ 0.228), and 1.9-fold (P ¼ 0.132), respectively. These results are similar to the ECM gene expression of 3 mM SIS3-treated hHSFs. www.jidonline.org 2019

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

TGFBRI siRNA attenuates hypertrophic scarring

To enhance transfection efficiency, we injected siRNA three times into the granulation tissue (at 2, 3, and 4 weeks after wounding). TGFBRI protein expression level was lower in the TGFBRI siRNA-treated group compared with that in the vehicle-treated control group (confirmed by immunofluorescence staining at 6 weeks after wounding, n ¼ 3) (Figure 4d). TGFBRI siRNA treatment of rabbit ears at 2, 3, and 4 weeks after wounding improved the appearance of the repaired wound at 6, 10, and 14 weeks (Figure 3b). Repaired wounds were evaluated by the Vancouver scar scale (VSS) and visual analog scale (VAS). Total VSS scores were significantly lower in the TGFBRI siRNA-treated group compared with those in the vehicle-treated control group at all time points after wounding (2.75±0.31 vs. 5.56±0.32 at 6 weeks, Po0.01; 3.88±0.49 vs. 6.81±0.45 at 10 weeks, Po0.01; 4.88±0.44 vs. 6.93±0.53 at 14 weeks, Po0.05) (Figure 3c). VAS scores were significantly higher in the TGFBRI siRNA-treated group compared with those in the control group at all time points after wounding. (63.54±7.26 vs. 38.44±4.11 at 6 weeks, Po0.01; 62.29±4.64 vs. 38.73±4.48 at 10 weeks, Po0.01; 62.92±4.72 vs. 47.24±4.35 at 14 weeks, Po0.05) (Figure 3d). These data suggest that TGFBRI siRNA attenuates hypertrophic scarring in vivo. Collagen deposition was determined in scar tissues collected at 6, 10, and 14 weeks after wounding, and stained with Masson’s trichrome stain. Microscopic examination showed that the collagen deposition area decreased more in the TGFBRI siRNA-treated group than that in the control group at all time points (Figure 4a,b). Furthermore, collagen fibers in the control group were arranged in circular and irregular patterns, a feature that was improved in the TGFBRI siRNAtreated group (Figure 4b). All tissue sections were evaluated by the scar elevation index (SEI), the ratio of the scar area over normal skin dermis, which is used to quantify scar hypertrophy (Kloeters et al., 2007). The rabbit ear wounds treated with TGFBRI siRNA had lower SEI than the vehicle-treated control group at all time points after wounding (1.38±0.036 vs. 1.89±0.095 at 6 weeks, Po0.01; 1.38±0.05 vs. 1.74±0.11 at 10 weeks, Po0.01; and 1.47±0.06 vs. 1.69±0.09 at 14 weeks, Po0.05) (Figure 4c). These data show that TGFBRI siRNA reduces TFGBRI protein expression, collagen deposition, and scar contracture, thus attenuating hypertrophic scarring in vivo. DISCUSSION In this study, we demonstrated that downregulating TGFBRI by RNA interference decreased expression of genes for CTGF and a-SMA, resulting in attenuation of wound scarring in a rabbit hypertrophic scar model. Furthermore, we found that downregulating the TGFBRI by TGFBRI siRNA in hHSFs stimulated with TGF-bI reduced phosphorylated Smad2/Smad3, fibroblast proliferation, as well as expression of type 1 collagen, type 3 collagen, fibronectin, and CTGF. Our in vitro results suggest that our in vivo results are due to silencing of TGFBRI expression, leading to the repression of TGF-b/Smad signaling, and finally to a decrease in cell proliferation and ECM deposition in hHSF. The current study shows that the effect 2020 Journal of Investigative Dermatology (2014), Volume 134

of TGFBRI siRNA is similar to that of a TGFBRI inhibitor in scar prevention (Boys et al., 2012). These results indicate that TGFBRI has a critical role in hypertrophic scarring and adequately explain high expression levels of TGFBRI in fibroblasts derived from hypertrophic scars (Schmid et al., 1998; Chin et al., 2001). Our results are also consistent with those of a previous study showing an increase of collagen accumulation and dermal fibrosis in conditional TGFBRI knockin mice (Sonnylal et al., 2007). Our results show that TGFBRI siRNA and a TGFBRI inhibitor had similar inhibitory effects on cell proliferation and gene expression of downstream factors. However, the TGFBRI inhibitor had a greater inhibitory effect on CTGF expression than TGFBRI siRNA, which may have been caused by the effect of the TGFBRI inhibitor on other receptor serine/ threonine kinases (Vogt et al., 2011). These results highlight the advantage of TGFBRI siRNA as specifically targeting mRNA, which directly controls protein expression. Another advantage is that the TGFBRI inhibitor needs to be continually applied to maintain a mM concentration level, but 60 nM TGFBRI siRNA only needs to be applied once to achieve a similar effect in our in vitro experiment. As siRNA has the capacity to specifically knock down genes of interest and as using a lower dose can reduce latent side effects, it can be used for developing novel therapeutics. In the rabbit hypertrophic scarring model, we made a large wound (18 mm  18 mm) that provided a chronic form of hypertrophic scarring, but this model has some limitations. The delayed wound healing in the rabbit ear made the cartilage bare, which occasionally leads to cartilage necrosis and hypertrophy. Although the deformed subdermal cartilaginous layer increases the complexity of measuring an elevated scar, SEI assessments did not include the area of the hypertrophied cartilage. We observed the effects of TGFBRI siRNA in wound scarring up to 14 weeks. Differences between the TGFBRI siRNA-treated wounds and those of the control group decreased over time, as SEI scores decreased gradually in the control group but did not change in the TGFBRI siRNAtreated group. This decrease in SEI score in the control group may be explained by the contraction of scar tissue during the remodeling phase, the later period of the wound healing process. This contraction is due to myofibroblasts, actin-rich cells that attach to collagen at several points, which make the scar contract and decrease its surface area (Profyris et al., 2012). Because a-SMA can increase fibroblast contractile activity (Hinz et al., 2001), inhibition of TGFBRI expression and TGF-b/Smad signaling may decrease scar contractility by reducing the expression of a-SMA mRNA. Our in vivo results show that CTGF and a-SMA gene expressions were significantly lower in the TGFBRI siRNA-treated group than in the control group. This result is consistent with a report that TGF-bI-induced scar contraction was inhibited by blocking the function of TGF-bI (Zhang et al., 2009). Taken with previous results, our data suggest that TGFBRI siRNA treatment reduces scar contractility. Downregulating TGFBRI in hHSFs reduced expression of genes for type I collagen, type III collagen, and fibronectin. However, expression of these genes increased after a single

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

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Figure 3. Transforming growth factor-b 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 ¼ 3), *Po0.05, **Po0.01. (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 ¼ 5 mm. (c) Repaired wounds were evaluated by the Vancouver scar scale (VSS) and (d) visual analog scale (VAS). Values are mean±SEM, *Po0.05, **Po0.01.

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Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

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Figure 4. Transforming growth factor-b 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-mm-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 ¼ 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 ¼ 100 mm. (c) All tissue sections were evaluated by the scar elevation index (SEI). Values are mean±SEM, *Po0.05, **Po0.01. (d) Immunofluorescence staining for TGFBRI in cryosections of scar tissue, scale bar ¼ 500 mm.

TGFBRI siRNA treatment applied to wound granulation tissue, which was inconsistent with our in vitro results (Figure 2a). These results may have occured due to the complexity of the in vivo environment and the intricacy of the TGF-b/Smad signaling pathway. When the TGF-b/Smad signaling pathway was blocked, other minor growth factors (e.g., IGF-I or platelet-derived growth factors), which can also promote ECM expression (Burger et al., 1998; Edmondson et al., 2003; Takasao et al., 2012), may have been increased to facilitate wound healing during the proliferation phase of the wound healing process. Moreover, cell division is faster in the proliferation phase; thus, one dose of TGFBRI siRNA cannot 2022 Journal of Investigative Dermatology (2014), Volume 134

fully block the TGF-b/Smad signaling pathway in vivo. The different outcomes of the in vivo and in vitro studies, particularly for ECM gene expression, may also be partially explained by our in vitro results with the Smad3 inhibitor SIS3 (Figure 2d). At higher doses (5 and 10 mM), SIS3 decreased fibrosis-related gene expression, whereas treatment with a lower dose (3 mM SIS3) elevated type I and III collagen gene expression. This result suggests that incomplete suppression of the TGF-b/Smad signaling pathway may upregulate ECM production. Many studies have demonstrated that downregulating TGFBRII or overexpressing mutated TGFBRII can reduce

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

hypertrophic scarring. Regarding receptor interactions, TGFBRII can form complexes with both TGFBRI and ALK-1, a type I receptor that can be activated by TGF-b, inducing an opposite effect to the canonical TGF-b signaling pathway (Goumans et al. 2003). This characteristic suggests a competitive relationship between TGFBRI and ALK-1 in determining the direction of the TGF-b signaling pathway. Therefore, TGFBRII seems to have a signal receptor role rather than a direction-determining role in the TGF-b signaling pathway. The direction of the TGF-b signaling pathway may be implicated in the ratio of TGFBRI to TGFBRII. This ratio was higher in fibrotic tissues than in normal controls and was correlated with collagen over-production (Pannu et al., 2004). A consequence of this phenomenon is that higher TGFBRI protein levels increase the opportunity for heterodimeric TGFBRI/TGFBRII complex formation rather than for the formation of ALK-1/TGFBRII complexes. These results imply that TGFBRI has an important role in the fibrotic phenotype of fibroblasts. TGF-b1 and its receptors are closely related to the pathological process of dermal scarring and to several fibrotic diseases such as renal fibrosis and pulmonary fibrosis (Shi-Wen et al., 2006; Pohlers et al., 2009). For example, several inhibitors of TGFBRI have been shown to supress fibrotic processes (de Gouville et al., 2005; Kim et al., 2008; Shen et al., 2013). Our finding that TGFBRI siRNA decreases TGFBRI expression suggests that TGFBRI siRNA can be used in fibrosis prevention. The siRNA applied in our study was the chemically modified double-stranded duplexe Stealth siRNA, which increases effectiveness and stability in in vivo application. Stealth siRNA has been used in some published animal studies (Wilson et al., 2007; Wang et al., 2009). In addition, the transfection reagent used in our study was composed of simulated viral cell-penetrating peptides, which provide high efficiency siRNA delivery to mammalian cells (Wang et al., 2010). However, this reagent was cytotoxic and inhibited cell proliferation in our in vitro model, lowering cell proliferation in siRNA-treated experiments (for both scrambled siRNA- and TGFBRI siRNA-treated groups) compared with that in TGFBRI inhibitor-treated experiments (Figure 1e, f). Therefore, a highly efficient and minimally cytotoxic transfection method is needed for future clinical applications. In summary, this study demonstrates that downregulating the TGFBRI by TGFBRI siRNA inhibited TGF-b/Smad signaling, reduced fibroblast proliferation and ECM production, and finally attenuated wound scarring in a rabbit hypertrophic scar model. Our results suggest that canonical TGF-b signaling is important for the phenotype of hypertrophic scar fibroblasts and that inhibition of TGFBRI expression by TGFBRI siRNA is a potential clinical strategy for preventing hypertrophic scarring. MATERIALS AND METHODS Culture of human hypertrophic scar fibroblasts Primary cultured hHSFs were established from five skin samples (patient information see Supplementary Table S1 online). All of these skin samples were discarded scar tissues from scar revision surgery. The sample collection protocol was approved by the Tri-Service

General Hospital Institutional Review Board, was conducted according to the Declaration of the Helsinki Guidelines, and written informed consent was obtained from each donor. Specimens were processed according to the protocol described by Rnjak et al., 2009. The resulting cells were then maintained in DMEM supplemented with 10% fetal bovine serum. Fibroblasts between passages three and six were collected for subsequent studies.

siRNA transfection The design of TGFBRI Stealth siRNA was based on the program BLOCK-iT RNAi Designer (Invitrogen, Carlsbad, CA) and NCBIBLAST. Three selected siRNA duplexes (sense 50 -GACAUCUAUG CAAUGGGCUUAGUAU-30 , 50 -GCAUCUCACUCAUGUUGAUG GUCUA-30 , 50 -AGUAAGACAUGAUUCAGCCACAGAU-30 ) cover different regions of the human TGFBRI mRNA (NM_004612.2). These three siRNA duplexes were also designed to match rabbit TGFBRI mRNA (XM_002708153.1). The designed TGFBRI siRNA sequences were custom synthesized by Invitrogen. A non-specific scrambled siRNA (50 -UUCUCCGAACGUGUCACGUTT-30 ) (MDbio, Taipei, Taiwan) was used as a control. TGFBRI siRNAs (a mixture containing three types of TGFBRI siRNA duplexes) and scrambled siRNA were each transfected into fibroblasts with the PepMute transfection kit (SignaGen, Rockville, MD). The final siRNA concentration was 60 nM, depending on the optimal test. After 24 hours, the medium was replaced with DMEM, 5% fetal bovine serum with or without additional TGF-bI 2 ng ml  1 (Invitrogen). Five hHSF strains were treated using the same process.

RNA isolation, reverse transcription, and quantitative real-time PCR Gene expression of treated cells was analyzed after siRNA transfection or 1 mM TGFBRI inhibitor (LY-364947) (Calbiochem, San Diego, CA), or 3, 5, 10 mM Smad3 inhibitor (SIS3) (Calbiochem) treatment. Total RNA was isolated using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). RNA was then converted to cDNA with the MultiScript High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Gene expression levels were measured for TGFBRI and glyceraldehyde 3-phosphate dehydrogenase by quantitative realtime PCR using QuantiFast Probe Assay kit (Qiagen). For Type I collagen, Type III collagen, fibronectin, CTGF, and a-SMA gene expression levels were quantified by using appropriate primers (see Supplementary Table S2 online) with Power SYBR Green PCR master mix (Applied Biosystems, Warrington, UK), and GAPDH gene expression served as an internal control. TaqMan real-time PCR and SYBR Green quantitative real-time PCR were performed and analyzed in the LightCycler 480 System (Roche Diagnostics, Penzberg, Germany).

Western blotting At day 5 after siRNA transfection, treated cells were analyzed for expression of TGFBRI, Smad2, Smad3, as well as phosphorylated Smad2 and Smad3 protein. Cells were treated with TGF-bI 2 ng ml  1 for 30 minutes to trigger TGF-b/Smad signaling, then lysed in Pro-Prep protein extraction solution (Intron, Seoul, Korea) with protease and phosphatase inhibitors (Roche Applied Science). Protein extracts were applied to a 4–20% gradient gel (Bio-Rad, Hercules, CA) and then transferred to polyvinylidene difluoride membranes. Polyvinylidene difluoride membranes were immunoblotted with rabbit anti-human TGFBRI antibody (GeneTex, San Antonio, TX), rabbit anti-human www.jidonline.org 2023

Y-W Wang et al. TGFBRI siRNA Reduces Wound Scarring

phospho Smad2 and Smad3 antibody, rabbit anti-human Smad2, and Smad3 antibody (Cell Signaling Technology, Beverly, MA), with mouse anti-GAPDH, a-tubulin antibody (GeneTex, San Antonio, TX) as the internal control. Appropriate secondary antibody conjugated with horseradish peroxidase was incubated with membrane to activate chemiluminescent substrate (Visual protein, Taipei, Taiwan). Protein expression levels were detected and quantitated by UVP BioImaging System (UVP, Upland, CA).

Fibroblast proliferation assay Fibroblast proliferation was quantified using Cell Counting Kit-8 (Boster Biological Technology, Wuhan, China). In brief, hHSFs derived from five skin samples were seeded at 5  103 cells per well in triplicate in 96-well plates and treated with TGFBRI siRNAs 60 nM or TGBFRI inhibitor 1 mM. One day after treatment, the medium was changed to culture medium with or without additional TGF-bI 2 ng ml  1. The medium was refreshed every 2 days. At days 3, 7, and 10, 10 ml of CCK-8 solution in 90 ml of DMEM without phenol red was added into each well. After 1 hour of incubation, absorbance was recorded at 450 nm.

ELISA For determining type I collagen production in the supernatant fluid of fibroblasts after TGFBRI siRNA transfection, the siRNA-transfected fibroblasts were incubated in culture medium with additional TGF-bI 2 ng ml  1. The medium was refreshed every other day. We collected the medium from day 5 to day 7 during the experimental period. The supernatants were measured by ELISA using the human collagen Type I ELISA kit (BlueGene, Shanghai, China). Absorbance was recorded at 450 nm against standard curve by spectrophotometer (Bio-Rad). All measurements were done in triplicate, performed in five hHSF strains, and expressed in mg per ml.

of each ear. The wounds were covered with Vaseline gauze and fixed with CoBan (3M Healthcare, St Paul, MN) for 2 weeks without dressing change unless a wound became infected. These procedures would form about 5-mm-thickgranulation tissue on the edges of the lesion side at day 14. Eighty-eight wounds were divided into TGFBRI siRNA-treatment and control groups. In the TGFBRI siRNA-treatment group, the granulation sites of each wound were injected with 240 pmol of TGFBRI siRNAs in 40 ml transfection buffer prepared with 2.5 ml of transfection reagent at 2, 3, and 4 weeks after wounding. Control groups were injected with vehicle alone. The repaired wounds were evaluated by the VSS and VAS (Fearmonti et al., 2010) at 6, 10, and 14 weeks after skin-defect surgery. The evaluation criteria for VSS include pliability, height, vascularity, and pigmentation. The evaluator intraclass correlation coefficient of VSS is 0.865 (one evaluator was involved), and that of VAS is 0.834 (three evaluators were involved, performed in a fully blinded fashion). Animals were then euthanized by overdose of anesthetics to collect scar specimens. Scar specimens were fixed with 10% formalin, embedded in paraffin, sectioned at 5-mm thickness onto glass slides, and stained with Masson’s trichrome stain (Sigma-Aldrich, St Louis, MO). Hypertrophied dermis was evaluated using the SEI (Kloeters et al., 2007). Scar specimens were also prepared as 40-mm-thick cryosections for TGFBRI immunofluorescence staining.

Statistics Statistical differences were calculated using either paired t-test or Student’s t-test. P-values o0.05 were considered statistically significant.

CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS

Immunofluorescence staining hHSFs on 6-well culture dishes were fixed with 0.1% acetic acid in ethanol for 10 min. Cells were stained by the indirect method using goat anti-human TGFBRI antibody, anti-goat IgG-conjugated Alexa ` 594 (Ana Spec, San Jose, CA), rabbit anti-human fibronectin FluorO (Santa Cruz Biotechnology, Santa Cruz, CA), and rhodamine-conjugated goat anti-rabbit antibody (Jackson IR, West Grove, PA). The nuclei were counterstained with Hoechst (Ana Spec).

Animal model To evaluate the outcomes of treatment for a long period, we chose a chronic scarring model based on the study byMorris et al., (1997). In that study, a hypertrophic scar from a 1.5 cm  4.5–7.5 cm wound persisted for 288 days. To reduce animal use, we modified the wound size to 1.8 cm  1.8 cm, and found in our pilot study that hypertrophic scars can persist for at least 100 days. This wound size allowed us to make four full-thickness skin-defect wounds in each ear. Adult New Zealand white rabbits were purchased from the Animal Health Research Institute (New Taipei City, Taiwan). All surgical approaches and procedures were approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center. Six-monthold rabbits (3–3.5 kg) were sedated with an intramuscular injection of zoletil (1 mg kg  1) plus xylazine (3 mg kg  1) and anesthetized by inhalation using isoflurane 1.5–4%. Full-thickness skin-defect wounds with removal of the perichondrium were shaped on the concave side 2024 Journal of Investigative Dermatology (2014), Volume 134

This research was supported by grants from the Tri-Service General Hospital (TSGH-C100-157, TSGH-C102-006-008-013-S02) and National Defense Medical Center (MAB102-05), Taiwan. We are grateful to the Medical Research Office of the Tri-Service General Hospital for their support in instrument use. We appreciate the technical assistance and animal care provided by the staff of the Instrument Center and Animal Center at National Defense Medical Center. We especially thank Claire Baldwin, Elizabeth Earl Phillips, and Daniel Steve Villarreal for English language editing and their helpful comments. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid

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siRNA-targeting transforming growth factor-β type I receptor reduces wound scarring and extracellular matrix deposition of scar tissue.

Hypertrophic scarring is related to persistent activation of transforming growth factor-β (TGF-β)/Smad signaling. In the TGF-β/Smad signaling cascade,...
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