Function-Blocking RHAMM Peptides Attenuate Fibrosis and Promote Antifibrotic Adipokines in a Bleomycin-Induced Murine Model of Systemic Sclerosis

Open AccessPublished:November 23, 2020DOI:https://doi.org/10.1016/j.jid.2019.11.032
      Systemic sclerosis a chronic, fibrotic disorder associated with high disease-specific mortality and morbidity. Cutaneous manifestations include dermal thickening and obliteration of dermal adipose tissue. Accumulation of low-molecular-weight hyaluronan, which signals through the receptor for hyaluronan-mediated motility, RHAMM, leads to progressive fibrosis and is correlated with increased severity of systemic sclerosis. The purpose of this study is to test the efficacy of two function-blocking RHAMM peptides, NPI-110 and NPI-106, in reducing skin fibrosis in a bleomycin-induced mouse model of systemic sclerosis. NPI-110 reduced visible measures of fibrosis (dermal thickness and collagen production, deposition, and organization) and profibrotic gene expression (Tgfb1, c-Myc, Col1a1, Col3a1). NPI-110 treatment also increased the expression of the antifibrotic adipokines perilipin and adiponectin. Both RHAMM peptides strongly reduced dermal RHAMM expression, predicting that dermal fibroblasts are peptide targets. Transcriptome and cell culture analyses using Rhamm−/− and Rhamm-rescued dermal fibroblasts reveal a TGFβ1/RHAMM/MYC signaling axis that promotes fibrogenic gene expression and myofibroblast differentiation. RHAMM function‒blocking peptides suppress this signaling and prevent TGFβ1-induced myofibroblast differentiation. These results suggest that inhibiting RHAMM signaling will offer a treatment method for cutaneous fibrosis in systemic sclerosis.

      Abbreviations:

      BLM (bleomycin), DAT (dermal adipose tissue), HA (hyaluronan), LMW-HA (low-molecular-weight hyaluronan), SSc (systemic sclerosis)

      Introduction

      Systemic sclerosis (SSc) is a chronic, progressive disorder characterized by extensive cutaneous and visceral fibrosis (
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      ). Although rare, SSc has the highest mortality of all rheumatologic conditions (
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      ;
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      ). A cardinal feature of SSc is fibrosis of the skin and visceral organs resulting from the excessive deposition of extracellular matrix components, such as collagen (
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      ;
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      ). Patients face the devastating sequelae of diffuse fibrosis, including renal failure, gastroesophageal reflux, interstitial lung disease, and pulmonary arterial hypertension (
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      Systemic sclerosis.
      ;
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      • Avvedimento E.V.
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      Scleroderma.
      ). Although not life threatening, progressive cutaneous fibrosis presents significant morbidity. Patients experience physical limitations with speaking, eating, and social distress with loss of facial expression (
      • Nakayama A.
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      ;
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      ).
      In addition to dermal thickening, the loss of dermal adipose tissue (DAT) is seen in both patients with SSc and animal models of cutaneous fibrosis (
      • Fleischmajer R.
      • Damiano V.
      • Nedwich A.
      Scleroderma and the subcutaneous tissue.
      ;
      • Ohgo S.
      • Hasegawa S.
      • Hasebe Y.
      • Mizutani H.
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      Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF-β1.
      ;
      • Wu M.
      • Melichian D.S.
      • Chang E.
      • Warner-Blankenship M.
      • Ghosh A.K.
      • Varga J.
      Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.
      ). DAT lies below the reticular dermis and is histologically and metabolically distinct from subcutaneous adipose tissue. In humans, DAT surrounds the pilosebaceous units, and in rodents, DAT lies between the reticular dermis and panniculus carnosus (
      • Driskell R.R.
      • Jahoda C.A.
      • Chuong C.M.
      • Watt F.M.
      • Horsley V.
      Defining dermal adipose tissue.
      ) (Supplementary Figure S1). Subcutaneous adipose depots are relatively stable, but DAT expands and involutes with each hair cycle and participates in unique nonmetabolic functions such as immune surveillance, hair growth, and wound healing (
      • Chen S.X.
      • Zhang L.J.
      • Gallo R.L.
      Dermal white adipose tissue: a newly recognized layer of skin innate defense.
      ;
      • Zwick R.K.
      • Guerrero-Juarez C.F.
      • Horsley V.
      • Plikus M.V.
      Anatomical, physiological, and functional diversity of adipose tissue.
      ). DAT loss in SSc is a result of both adipocyte apoptosis and cellular reprogramming into activated myofibroblasts (
      • Driskell R.R.
      • Jahoda C.A.
      • Chuong C.M.
      • Watt F.M.
      • Horsley V.
      Defining dermal adipose tissue.
      ). This trans-differentiation and loss of antifibrotic adipokines enhance the fibrotic environment of scleroderma (
      • Marangoni R.G.
      • Lu T.T.
      The roles of dermal white adipose tissue loss in scleroderma skin fibrosis.
      ;
      • Yamashita T.
      • Lakota K.
      • Taniguchi T.
      • Yoshizaki A.
      • Sato S.
      • Hong W.
      • et al.
      An orally-active adiponectin receptor agonist mitigates cutaneous fibrosis, inflammation and microvascular pathology in a murine model of systemic sclerosis.
      ).
      Hyaluronan (HA) is ubiquitous in the extracellular matrix but accumulates to the highest levels within the skin. HA regulates the equilibrium between fibrosis and adipogenesis, and its metabolism is altered in SSc (
      • Freitas J.P.
      • Filipe P.
      • Emerit I.
      • Meunier P.
      • Manso C.F.
      • Guerra Rodrigo F.
      Hyaluronic acid in progressive systemic sclerosis.
      ;
      • Levesque H.
      • Baudot N.
      • Delpech B.
      • Vayssairat M.
      • Gancel A.
      • Lauret P.
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      Clinical correlations and prognosis based on hyaluronic acid serum levels in patients with progressive systemic sclerosis.
      ;
      • Scheja A.
      • Akesson A.
      • Hørslev-Petersen K.
      Serum levels of aminoterminal type III procollagen peptide and hyaluronan predict mortality in systemic sclerosis.
      ). Native, high-molecular-weight HA (>500 kDa) maintains homeostasis as a hydrophilic, viscoelastic macromolecule, providing hydration and suppression of inflammation and fibrosis (
      • Petrey A.C.
      • de la Motte C.A.
      Hyaluronan, a crucial regulator of inflammation.
      ). With tissue injury, ROS, nitrogen species, and hyaluronidases released from damaged cells depolymerize high-molecular-weight HA into proinflammatory, low-molecular-weight fragments (<500 kDa) (
      • Chapple I.L.
      Reactive oxygen species and antioxidants in inflammatory diseases.
      ;
      • Yamazaki K.
      • Fukuda K.
      • Matsukawa M.
      • Hara F.
      • Yoshida K.
      • Akagi M.
      • et al.
      Reactive oxygen species depolymerize hyaluronan: involvement of the hydroxyl radical.
      ). The persistent accumulation of low-molecular-weight HA (LMW-HA) leads to unremitting inflammation and chronic fibrosis. Higher LMW-HA serum levels are correlated with increased cutaneous involvement and severity of systemic disease in patients with SSc (
      • Scheja A.
      • Akesson A.
      • Hørslev-Petersen K.
      Serum levels of aminoterminal type III procollagen peptide and hyaluronan predict mortality in systemic sclerosis.
      ). LMW-HA fragments are profibrotic (
      • Bollyky P.L.
      • Bogdani M.
      • Bollyky J.B.
      • Hull R.L.
      • Wight T.N.
      The role of hyaluronan and the extracellular matrix in islet inflammation and immune regulation.
      ;
      • Misra S.
      • Hascall V.C.
      • Markwald R.R.
      • Ghatak S.
      Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer.
      ;
      • Savani R.C.
      • Hou G.
      • Liu P.
      • Wang C.
      • Simons E.
      • Grimm P.C.
      • et al.
      A role for hyaluronan in macrophage accumulation and collagen deposition after bleomycin-induced lung injury.
      ) and antiadipogenic (
      • Park B.G.
      • Lee C.W.
      • Park J.W.
      • Cui Y.
      • Park Y.S.
      • Shin W.S.
      Enzymatic fragments of hyaluronan inhibit adipocyte differentiation in 3T3-L1 pre-adipocytes.
      ;
      • Savani R.C.
      • Wang C.
      • Yang B.
      • Zhang S.
      • Kinsella M.G.
      • Wight T.N.
      • et al.
      Migration of bovine aortic smooth muscle cells after wounding injury. The role of hyaluronan and RHAMM.
      ). HA signals through a variety of receptors, but receptor for HA-mediated motility, RHAMM is chronically elevated in fibrotic and inflammatory diseases and demonstrates preferential binding to LMW-HA (
      • Hauser-Kawaguchi A.
      • Luyt L.G.
      • Turley E.
      Design of peptide mimetics to block pro-inflammatory functions of HA fragments.
      ;
      • Tolg C.
      • Poon R.
      • Fodde R.
      • Turley E.A.
      • Alman B.A.
      Genetic deletion of receptor for hyaluronan-mediated motility (Rhamm) attenuates the formation of aggressive fibromatosis (desmoid tumor).
      ;
      • Tolg C.
      • Hamilton S.R.
      • Zalinska E.
      • McCulloch L.
      • Amin R.
      • Akentieva N.
      • et al.
      A RHAMM mimetic peptide blocks hyaluronan signaling and reduces inflammation and fibrogenesis in excisional skin wounds.
      ). Blocking the HA-binding functions of RHAMM suppresses skin (
      • Tolg C.
      • Hamilton S.R.
      • Zalinska E.
      • McCulloch L.
      • Amin R.
      • Akentieva N.
      • et al.
      A RHAMM mimetic peptide blocks hyaluronan signaling and reduces inflammation and fibrogenesis in excisional skin wounds.
      ) and lung fibrosis (
      • Cui Z.
      • Liao J.
      • Cheong N.
      • Longoria C.
      • Cao G.
      • DeLisser H.M.
      • et al.
      The receptor for hyaluronan-mediated motility (CD168) promotes inflammation and fibrosis after acute lung injury.
      ;
      • Markasz L.
      • Savani R.C.
      • Sedin G.
      • Sindelar R.
      The receptor for hyaluronan-mediated motility (RHAMM) expression in neonatal bronchiolar epithelium correlates negatively with lung air content.
      ).
      Management for SSc consists of oral immunosuppressant agents and symptomatic management (
      • Denton C.P.
      Advances in pathogenesis and treatment of systemic sclerosis.
      ;
      • Rossi D.
      • Zanatta E.
      • Marson P.
      • Sciascia S.
      • Polito P.
      • Roccatello D.
      • et al.
      How I treat patients with systemic sclerosis in clinical practice.
      ). With no approved disease-modifying treatments, there remains an urgent need for the development of effective targeted therapeutic interventions. Function-blocking RHAMM peptides have been developed (
      • Hauser-Kawaguchi A.
      • Luyt L.G.
      • Turley E.
      Design of peptide mimetics to block pro-inflammatory functions of HA fragments.
      ) that present a potential treatment option for cutaneous fibrosis. On the basis of the HA:RHAMM-binding sequence, these either bind to and sequester LMW-HA fragments (NPI-106) or bind directly to RHAMM to sterically prevent its association with LMW-HA (NPI-110) (
      • Hauser-Kawaguchi A.
      • Luyt L.G.
      • Turley E.
      Design of peptide mimetics to block pro-inflammatory functions of HA fragments.
      ). In this study, we investigate the efficacy of these two classes of function-blocking RHAMM peptides (NPI-110 and NPI-106) in reducing fibrosis and promoting antifibrotic adipokine expression in a mouse model of bleomycin (BLM)-induced SSc.

      Results

      The BLM-induced SSc mouse model (
      • Yamamoto T.
      • Takagawa S.
      • Katayama I.
      • Yamazaki K.
      • Hamazaki Y.
      • Shinkai H.
      • et al.
      Animal model of sclerotic skin. I: local injections of bleomycin induce sclerotic skin mimicking scleroderma.
      ) recapitulates the phases of scleroderma that are contributed to by HA (
      • Yoshizaki A.
      • Iwata Y.
      • Komura K.
      • Ogawa F.
      • Hara T.
      • Muroi E.
      • et al.
      CD19 regulates skin and lung fibrosis via toll-like receptor signaling in a model of bleomycin-induced scleroderma.
      ). Female mice were used because this parallels SSc where there is a predilection for women, although the disease phenotype is more severe in men (
      • Peoples C.
      • Medsger Jr., T.A.
      • Lucas M.
      • Rosario B.L.
      • Feghali-Bostwick C.A.
      Gender differences in systemic sclerosis: relationship to clinical features, serologic status and outcomes.
      ;
      • Wielosz E.
      • Majdan M.
      • Dryglewska M.
      • Suszek D.
      Comparison of clinical and serological parameters in female and male patients with systemic sclerosis.
      ).

       Peptide NPI-110 reduces dermal thickness, collagen density, and hydroxyproline content

      Mouse skin biopsy samples were visualized with H&E and Masson’s Trichrome (Sigma-Aldrich, St. Louis, MO) to determine whether peptide treatment ameliorated BLM-induced fibrosis (Figure 1a). BLM treatment caused a 25% increase in dermal thickness (P < 0.0001, Figure 1d) and a 14% loss of DAT compared with that in the control (P = 0.001, Supplementary Figure S2a). Both peptides blunted dermal collagen accumulation and caused a 6% reduction of dermal thickness compared with BLM treatment (NPI-110, P = 0.004; NPI-106, P = 0.006, Figure 1d); however, neither peptide ameliorated the loss of visible DAT (Supplementary Figure S2a).
      Figure thumbnail gr1
      Figure 1Peptide NPI-110 reduces dermal thickness and collagen density. Paraffin-processed histology sections of mice from four treatment groups stained for (a) Masson’s Trichrome and (b) Picrosirius Red visualized under (c) polarized light to highlight tissue architecture and collagen distribution. Comparison of (d) dermal thickness and (e) area fraction of collagen is from image analysis of Picrosirius Red slides. Histochemistry was verified using RT-PCR for (f) Col1a1 and (g) Col3a1 mRNA. Values represent the mean and SEM from three random measurements in three sections per mouse and 12 mice per treatment group. Analysis of dermal thickness was performed using the Kruskal‒Wallis test with post-hoc Mann‒Whitney U test. All other experiments were tested with one-way ANOVA and posthoc LSD test for significance. ∗P < 0.05, ∗∗∗P < 0.0001. Bar = 100 μm. BLM, bleomycin; LSD, least significant difference.
      Collagen bundling was investigated with Picrosirius Red (Polysciences, Warrington, PA) staining (
      • Lattouf R.
      • Younes R.
      • Lutomski D.
      • Naaman N.
      • Godeau G.
      • Senni K.
      • et al.
      Picrosirius Red Staining: a useful tool to appraise collagen networks in normal and pathological tissues.
      ). Collagen in lesional skin treated with BLM stained intensely red, indicating densely packed collagen fibrils, whereas peptide treatment resulted in a weaker staining that showed a less dense collagen network (Figure 1b). Viewed under polarized light, collagen fibers in BLM-treated skin demonstrated strong birefringence with fibers appearing white. Both peptides caused attenuation of this birefringence (Figure 1c). BLM treatment increased the area fraction of collagen by 24% compared to the controls (Figure 1e, P = 0.001). NPI-110 decreased the BLM-induced increase in collagen (P = 0.03), but NPI-106 did not have a significant effect (P = 0.53). To assess whether NPI-110 also reduces skin collagen content, hydroxyproline assays were performed (
      • Cui H.
      • Ge J.
      • Xie N.
      • Banerjee S.
      • Zhou Y.
      • Liu R.M.
      • et al.
      miR-34a promotes fibrosis in aged lungs by inducing alveolarepithelial dysfunctions [published correction appears in Am J Physiol Lung Cell Mol Physiol 2018;314(2):L332].
      ). BLM treatment increased skin hydroxyproline content by three folds above control levels (Supplementary Figure S3, P < 0.0001). NPI-110 reduced this BLM-induced increase by two folds (P < 0.001), although the levels remained slightly higher than those in the control skin (P < 0.001). These findings suggest that NPI-110 treatment reduced dermal thickness as well as collagen density and content in BLM-induced SSc, whereas NPI-106 was less effective.

       Peptides NPI-110 and NPI-106 reduce Col1a1 mRNA expression and restore the control Col1a1-to-Col3a1 ratio

      Clinically, SSc is characterized by increased expression of type I collagen and reduced expression of type III collagen (
      • Fleischmajer R.
      • Dessau W.
      • Timpl R.
      • Krieg T.
      • Luderschmidt C.
      • Wiestner M.
      Immunofluorescence analysis of collagen, fibronectin, and basement membrane protein in scleroderma skin.
      ;
      • Søndergaard K.
      • Heickendorff L.
      • Risteli L.
      • Risteli J.
      • Zachariae H.
      • Stengaard-Pedersen K.
      • et al.
      Increased levels of type I and III collagen and hyaluronan in scleroderma skin.
      ). RT-PCR analysis confirmed the increased expression of collagen I expression in the BLM-induced SSc model. BLM treatment resulted in a 1.22-fold increase in Col1a1 mRNA expression compared with that in the control (P = 0.03, Figure 1f). NPI-110 and NPI-106 decreased Col1a1 mRNA expression by 11 folds (P < 0.0001) and 14 folds (P < 0.0001), respectively (Figure 1f). Conversely, BLM treatment reduced Col3a1 mRNA expression (Figure 1g, P < 0.0001), whereas NPI-110 restored Col3a1 mRNA expression (Figure 1g, P = 0.012). Unexpectedly, NPI-106 further decreased Col3a1 expression relative to BLM treatment (Figure 1g, P < 0.0001). Nevertheless, both NPI-110 and NPI106 reduced the Col1a1-to-Col3a1 ratio to the control (NPI-106, Supplementary Figure S4b, P = 0.017) or below the control (NPI-110, Supplementary Figure S2b, P < 0.0001) levels. These results, combined with the collagen density (Figure 1e) show that NPI-110 and NPI-106 strongly suppress dermal fibrosis, detected by collagen organization and expression of collagen subtypes. Because the loss of dermal adipogenesis is causally linked to fibrosis in SSc and NPI-110 promotes adipocyte differentiation (
      • Bahrami S.B.
      • Tolg C.
      • Peart T.
      • Symonette C.
      • Veiseh M.
      • Umoh J.U.
      • et al.
      Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.
      ), we next assessed peptide effects on the expression of antifibrotic adipokines.

       Peptides NPI-110 and NPI-106 treatment increases the expression of antifibrotic adipokines

      Expression of adiponectin, a circulating adipokine that has antifibrotic functions (
      • Marangoni R.G.
      • Masui Y.
      • Fang F.
      • Korman B.
      • Lord G.
      • Lee J.
      • et al.
      Adiponectin is an endogenous anti-fibrotic mediator and therapeutic target.
      ,
      • Yamauchi T.
      • Iwabu M.
      • Okada-Iwabu M.
      • Kadowaki T.
      Adiponectin receptors: a review of their structure, function and how the work.
      ), was first assessed. Reduced DAT thickness in BLM-injected skin resulted from a significant decrease in adiponectin expression (Figure 2a–c, P < 0.0001). NPI-110 treatment significantly reversed this effect by restoring adiponectin protein expression to baseline levels (Figure 2b and c, P < 0.0001). However, there was no significant change with NPI-106 treatment (P = 0.06). Plin expression was also measured because it is highly expressed in adipocytes (
      • Sohn J.H.
      • Lee Y.K.
      • Han J.S.
      • Jeon Y.G.
      • Kim J.I.
      • Choe S.S.
      • et al.
      Perilipin 1 (Plin1) deficiency promotes inflammatory responses in lean adipose tissue through lipid dysregulation.
      ), is linked to fibrosis in other tissues (
      • Najt C.P.
      • Senthivinayagam S.
      • Aljazi M.B.
      • Fader K.A.
      • Olenic S.D.
      • Brock J.R.
      • et al.
      Liver-specific loss of perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.
      ), and is regulated by Rhamm (
      • Bahrami S.B.
      • Tolg C.
      • Peart T.
      • Symonette C.
      • Veiseh M.
      • Umoh J.U.
      • et al.
      Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.
      ;
      • Rossi D.
      • Zanatta E.
      • Marson P.
      • Sciascia S.
      • Polito P.
      • Roccatello D.
      • et al.
      How I treat patients with systemic sclerosis in clinical practice.
      ). BLM treatment reduced Plin mRNA expression by seven folds, and both NPI-110 and NPI-106 significantly increased Plin mRNA expression (Figure 2d, P = 0.03). In contrast to its effects on collagen and antifibrotic adipokines, BLM did not detectably sustain inflammation in this mouse model because macrophage numbers did not change (Supplementary Figure S5), as has been previously reported (
      • Sun C.
      • Chen S.Y.
      RGC32 promotes bleomycin-induced systemic sclerosis in a murine disease model by modulating classically activated macrophage function.
      ).
      Figure thumbnail gr2
      Figure 2Peptide NPI-110 treatment increases the expression of antifibrotic adipokines. Paraffin-processed skin sections of mice from all the four treatment groups on day 28 stained with (a) anti-adiponectin antibody and quantification of immunohistochemical staining for adiponectin in the (b) dermis and (c) DAT. Values represent the mean and SEM from three random measurements in three sections per mouse. (d) RT-PCR analysis of Plin expression of five mice per treatment group and five technical replicates. Analysis was performed with one-way ANOVA and posthoc LSD test for significance. ∗P < 0.05, ∗∗∗P < 0.0001. Bar = 100 μm. BLM, bleomycin; DAT, dermal adipose tissue; LSD, least significant difference.

       Transcriptome analysis of peptide function

      To identify the molecular mechanisms for the antifibrotic effects of NPI-110 and NPI-106, the expression of 84 fibrosis-associated and 84 adipogenesis-associated genes were profiled. NPI-110 significantly reduced the expression of 22 of the 84 fibrosis genes, including Myc, which was decreased by 196 fold (Figure 3a and Supplementary Table S1). Adipogenesis pathway analysis showed that most profibrosis genes were downregulated (Supplementary Table S2).
      Figure thumbnail gr3
      Figure 3Transcriptional profiles of NPI-110 and NPI-106. (a) Selected data of 29 fibrosis-associated genes profiled by RT-PCR that are either upregulated or downregulated by NPI-110 (blue) or NPI-106 (orange) with fold expression relative to BLM-treated group. (b) The top 12 IPA-predicted affected canonical pathways with NPI-110 treatment showing the percentage of overlapping upregulated (green) and downregulated (red) genes in each pathway. The yellow line indicates the IPA-predicted P-value of the overlap. (c) The top 5 IPA-identified upstream regulators based on the ranking of IPA-generated P-values of the overlap. (d) Model of the top predicted signaling pathway. BLM, bleomycin; IPA, ingenuity pathway analysis.
      These transcriptional profiles were analyzed using Ingenuity Pathway Analysis bioinformatics software. Ingenuity pathway analysis identified hepatic fibrosis and/or stellate cell activation as the top function out of 12 pathways that NPI-110 and 106 downregulated (Figure 3b). The top upstream regulators of these pathways were Tgfβ1 and BLM (Figure 3c). A model depicting a putative Tgfβ1/Rhamm/Myc signaling pathway was constructed on the basis of these analyses (Figure 3d). The changes in Tgfβ1 (Figure 4a–d) and c-Myc (Figure 4e–h) expression were then confirmed by RT-PCR. Tgfb1 mRNA expression was not significantly upregulated in the BLM-treatment group and showed no significant changes with peptide treatment (Figure 4d). Immunohistochemical analysis of Tgfβ1 protein expression, using an antibody that detects both latent and active forms of this cytokine, was performed to determine whether changes in the protein expression occurred independently of the mRNA levels. BLM treatment resulted in significantly increased levels of Tgfβ1 protein expression in the dermis (P = 0.04) and DAT (P < 0.001) (Figure 4a–c). NPI-110 treatment significantly reduced Tgfβ1 protein expression by 67% in DAT (P = 0.03) and to baseline levels in the dermis (P < 0.0001) (Figure 4b and c). However, NPI-106 did not decrease Tgfβ1 protein expression in either tissue layer. c-Myc mRNA expression was increased by two folds (P = 0.03) with BLM treatment and significantly downregulated by NPI-110 and NPI-106 (Figure 4h). Immunohistochemical analyses demonstrated a 1.8-fold increase in c-Myc protein expression with BLM treatment (P = 0.031) (Figure 4e–g), and this was significantly reduced by both peptides to baseline levels (NPI-110, P = 0.013; NPI-106, P = 0.002). Rhamm expression was next quantified in BLM-treated skin (Figure 5a–c). Rhamm mRNA expression was increased by 1.5 fold in the BLM-treatment group compared with that in the control (P = 0.03). NPI-110 reduced Rhamm mRNA expression to baseline level (P = 0.03), and NPI-106 treatment resulted in a further 2.8-fold reduction in the expression compared with that in the control (P = 0.003) (Figure 5c). Rhamm protein expression was increased by 18.3 fold by BLM (P < 0.0001) and reduced to baseline level by NPI-110 (P < 0.0001) and NPI-106 (P < 0.0001) (Figure 5a and b). Rhamm protein expression was not quantified separately in the dermis and DAT because these layers were difficult to consistently distinguish in the immunofluorescent-stained slides. These results suggest that NPI-110 and NPI-106 are suppressing a Tgfβ1/Rhamm/Myc signaling pathway in dermal cells, and a culture model was used to probe these associations.
      Figure thumbnail gr4
      Figure 4Peptides reduce Tgfβ1 and Myc expression. Paraffin-processed skin sections of mice from all the four treatment groups on day 28 stained for (a) anti-Tgfβ1 antibody or (e) anti‒c-Myc antibody. Quantification of Tgfβ1 (b, c) protein and (d) mRNA expression as well as c-Myc (f, g) protein and (h) mRNA expression. Values represent the mean and SEM from three random measurements in three sections per mouse. RT-PCR analysis of (e) Tgfb1 and (h) c-Myc mRNA expression of five mice per treatment group and five technical replicates. Analysis was performed with one-way ANOVA and posthoc LSD test for significance. ∗P < 0.05, ∗∗P < 0.001, ∗∗P < 0.0001. ∗P < 0.05, ∗∗P < 0.001, ∗∗P < 0.0001. Bar = 100 μm. BLM, bleomycin; LSD, least significant difference; n.s., not significant.
      Figure thumbnail gr5
      Figure 5Peptides reduce RHAMM expression. (a) Paraffin-processed skin sections of mice from all the four treatment groups on day 28 stained for RHAMM. (b) Quantification of Rhamm protein and (c) RT-PCR of Rhamm mRNA expression. Values represent the mean and SEM from three random measurements in three sections per mouse, five mice per treatment group, and five technical replicates. Western blot assays of (d) Rhamm and (e) Myc protein expression in Rhamm-rescued dermal fibroblasts treated with Tgfβ1 ± NPI-110. Gapdh was used as a loading control. Analysis was performed with one-way ANOVA and posthoc LSD test for significance. ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001. Bar = 100 μm. BLM, bleomycin; LSD, least significant difference.

       Rhamm is required for Tgfβ1-induced increase in Myc expression and myofibroblast differentiation, which is blocked by NPI-110

      The blocking of dermal Rhamm expression by NPI-110 and NPI-106 predicted that dermal fibroblasts are the peptide targets. Rhamm−/− and Rhamm-rescued dermal fibroblasts were therefore used as culture models. The use of Rhamm-rescued dermal fibroblasts permitted a direct assessment of Rhamm-regulated functions that impact fibroblast contribution to fibrosis. Furthermore, regulated re-expression of Rhamm in null fibroblasts resulted in constitutive cell surface Rhamm display (
      • Tolg C.
      • Hamilton S.R.
      • Nakrieko K.A.
      • Kooshesh F.
      • Walton P.
      • McCarthy J.B.
      • et al.
      Rhamm-/- fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair.
      ), which is required for RHAMM peptide response (
      • Bahrami S.B.
      • Tolg C.
      • Peart T.
      • Symonette C.
      • Veiseh M.
      • Umoh J.U.
      • et al.
      Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.
      ). NPI-110 reduced Rhamm protein expression (Figure 5d) and blocked the Tgfβ1-induced increase in Myc protein expression only in Rhamm-rescued dermal fibroblasts (Figure 5e). Notably, Myc levels were unaffected by Tgfβ1 and NPI-110 in the fibroblasts lacking Rhamm expression (Figure 6e). These results show that Rhamm-expressing dermal fibroblasts are NPI-110 targets, and the culture model appropriately replicates in vivo effects of these peptides.
      Figure thumbnail gr6
      Figure 6Peptides block Tgfβ1-induced myofibroblast differentiation. (a) Immunohistochemical staining for smooth muscle actin in Rhamm-rescued dermal fibroblasts treated with Tgfβ1 ± NPI-110. (b) Quantification of the number of cells exhibiting smooth muscle actin‒positive fibrils. Values represent the mean and SEM from five random measurements for three replicates. (c) Western blot assay of smooth muscle actin (ACTA2) in Rhamm-rescued dermal fibroblasts treated with Tgfβ1 ± NPI-110. (d) Immunohistochemical staining for Acta2 in a 10T1/2 fibroblast line grown in conditions preventing Rhamm protein expression and treated with Tgfβ1 ± NPI-110. (e) Western blot assay of 10T1/2 fibroblasts for Rhamm and Myc protein expression. (f) Model of proposed myofibroblast target of peptides that result in fibrosis reduction. Gapdh was used as a loading control for western blots. Analysis was performed with one-way ANOVA and posthoc LSD test for significance. ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001. Bar = 100 μm. BLM, bleomycin; LMW-HA, low-molecular-weight hyaluronan; LSD, least significant difference; Tgfβr, Tgfβ receptor.
      An important profibrotic function of Tgfβ1 is the promotion of myofibroblast differentiation. The addition of Tgfβ1 to Rhamm-rescued fibroblasts significantly increased the number of cells with prominent smooth muscle actin (Acta2) fibers used as a measure of myofibroblast differentiation (Figure 6a and b, P < 0.05). This effect required Rhamm expression because Tgfβ1 did not alter Acta2 organization in either 10T fibroblasts that do not export Rhamm (Figure 6d) or Rhamm−/− dermal fibroblasts (Supplementary Figure S5a). NPI-110 strongly blocked Tgfβ1-induced Accta2 fibers in Rhamm-rescued dermal fibroblasts (Figure 6a and b, P < 0.0001) to levels below those of the control (P < 0.001) but had no effect on either Tgfβ1-treated 10T fibroblasts (Figure 6e) or Rhamm−/− dermal fibroblasts (Supplementary Figure S5a). None of these treatments affected the total cellular level of Acta2 (Figure 6c and Supplementary Figure S5b). These results show that Rhamm expression is necessary for the Tgfβ1-induced increase in Myc and myofibroblast differentiation as modeled in Figure 6f.

      Discussion

      Two Rhamm function‒blocking peptides NPI-110 and NPI-106 were tested for their ability to restore normal tissue homeostasis and to alter dermal collagen mRNA expression; total collagen production, detected by hydroxyproline levels; and deposition of dermal collagen. The differential effects of these two peptides are likely explained by their distinct mechanisms for blocking Rhamm signaling. Fragmentation of high-molecular-weight HA during tissue inflammation produces a variety of polymer sizes. These LMW-HA fragments bind to Rhamm receptors with a varying affinity (
      • Cyphert J.M.
      • Trempus C.S.
      • Garantziotis S.
      Size Matters: molecular weight specificity of hyaluronan effects in cell biology.
      ;
      • Tolg C.
      • Telmer P.
      • Turley E.
      Specific sizes of hyaluronan oligosaccharides stimulate fibroblast migration and excisional wound repair.
      ). NPI-106 binds directly to HA, and this interaction is likely to preferentially occur to a specific range of LMW-HA (
      • Skurikhin E.G.
      • Pershina O.V.
      • Reztsova A.M.
      • Ermakova N.N.
      • Khmelevskaya E.S.
      • Krupin V.A.
      • et al.
      Modulation of bleomycin-induced lung fibrosis by pegylated hyaluronidase and dopamine receptor antagonist in mice.
      ). Fragments outside of this range may retain signaling through Rhamm, and signaling is not completely blocked. In contrast, NPI-110 binds directly to Rhamm preventing all sizes of fragments from signaling through Rhamm (
      • Bahrami S.B.
      • Tolg C.
      • Peart T.
      • Symonette C.
      • Veiseh M.
      • Umoh J.U.
      • et al.
      Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.
      ). Both peptides interfere with extracellular Rhamm functions because they have not been modified to facilitate direct penetration through the plasma membrane. However, intracellular Rhamm functions such as gene transcription (
      • Meier C.
      • Spitschak A.
      • Abshagen K.
      • Gupta S.
      • Mor J.M.
      • Wolkenhauer O.
      • et al.
      Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.
      ) may also be indirectly modified by peptides.
      Transcriptome analyses and peptide inhibition studies suggest that one peptide target is the Tgfβ1/Myc profibrotic signaling pathway. A well-established downstream target of Tgfβ1, c-Myc, is one of the three members of the Myc family of proto-oncogenes involved in a broad range of cellular functions, including cellular proliferation, differentiation, and fibrosis (
      • Chen H.
      • Liu H.
      • Qing G.
      Targeting oncogenic Myc as a strategy for cancer treatment.
      ;
      • Katoh M.
      Multi-layered prevention and treatment of chronic inflammation, organ fibrosis and cancer associated with canonical WNT/β-catenin signalling activation (review).
      ;
      • Liu T.
      • Zhou Y.
      • Ko K.S.
      • Yang H.
      Interactions between Myc and mediators of inflammation in chronic liver diseases.
      ). In our study, RT-PCR demonstrated an increase in c-Myc mRNA and protein with BLM treatment, which was suppressed by both NPI-110 and NPI-106. MYC activity is regulated by either its elevated expression or phosphorylation (
      • Alvarez E.
      • Northwood I.C.
      • Gonzalez F.A.
      • Latour D.A.
      • Seth A.
      • Abate C.
      • et al.
      Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation Characterization of the phosphorylation of c-myc and c-jun proteins by an rpidermal growth factor receptor threonine 669 protein kinase.
      ;
      • Sears R.
      • Nuckolls F.
      • Haura E.
      • Taya Y.
      • Tamai K.
      • Nevins J.R.
      Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability.
      ), which determine its target genes. For example, Myc phosphorylation is required for the transcription of genes involved in oxidative stress (
      • Benassi B.
      • Fanciulli M.
      • Fiorentino F.
      • Porrello A.
      • Chiorino G.
      • Loda M.
      • et al.
      c-Myc phosphorylation is required for cellular response to oxidative stress.
      ), whereas Myc overexpression is responsible for its increased activity in Tgfβ1-driven fibrosis (
      • Gabay M.
      • Li Y.
      • Felsher D.W.
      MYC activation is a hallmark of cancer initiation and maintenance.
      ;
      • Shen Y.
      • Miao N.
      • Wang B.
      • Xu J.
      • Gan X.
      • Xu D.
      • et al.
      c-Myc promotes renal fibrosis by inducing integrin αv-mediated transforming growth factor-β signaling.
      ). HA treatment of quiescent NIH/3T3 cells induced c-Myc‒stimulated cellular proliferation (
      • Moon S.O.
      • Lee J.H.
      • Kim T.J.
      Changes in the expression of c-myc, RB and tyrosine-phosphorylated proteins during proliferation of NIH 3T3 cells induced by hyaluronic acid.
      ), further suggesting that HA and HA receptors, such as Rhamm, are critically involved in the regulation of Myc activity.
      We propose a model where Rhamm integrates and activates a Tgfβ1/Myc signaling complex that promotes the expression of fibrogenic genes. We and others have previously shown that extracellular Rhamm acts as a coreceptor for GF receptors (
      • Du Y.C.
      • Chou C.K.
      • Klimstra D.S.
      • Varmus H.
      Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis.
      ;
      • Hatano H.
      • Shigeishi H.
      • Kudo Y.
      • Higashikawa K.
      • Tobiume K.
      • Takata T.
      • et al.
      RHAMM/ERK interaction induces proliferative activities of cementifying fibroma cells through a mechanism based on the CD44-EGFR.
      ;
      • Zhang S.
      • Chang M.C.
      • Zylka D.
      • Turley S.
      • Harrison R.
      • Turley E.A.
      The hyaluronan receptor RHAMM regulates extracellular-regulated kinase.
      ) and propose a similar function for Tgfβ receptor, which NPI-110 and NPI-106 block to inhibit Myc expression. Intracellular Rhamm may also participate in this signaling pathway. Intracellular Rhamm performs multiple functions (
      • Maxwell C.A.
      • McCarthy J.
      • Turley E.
      Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions?.
      ), including participation in transcriptional complexes (
      • Meier C.
      • Spitschak A.
      • Abshagen K.
      • Gupta S.
      • Mor J.M.
      • Wolkenhauer O.
      • et al.
      Association of RHAMM with E2F1 promotes tumour cell extravasation by transcriptional up-regulation of fibronectin.
      ). We predict that this intracellular nuclear function is important in Tgfβ1-regulated MYC expression. Blocking Tgfβ1, Myc (
      • Chen C.
      • Peng Y.
      • Peng Y.
      • Peng J.
      • Jiang S.
      miR-135a-5p inhibits 3T3-L1 adipogenesis through activation of canonical Wnt/β-catenin signaling.
      ;
      • Ouyang D.
      • Xu L.
      • Zhang L.
      • Guo D.
      • Tan X.
      • Yu X.
      • et al.
      MiR-181a-5p regulates 3T3-L1 cell adipogenesis by targeting Smad 7 and Tcf712.
      ), and RHAMM (
      • Liu X.F.
      • Bera T.K.
      • Kahue C.
      • Escobar T.
      • Fei Z.
      • Raciti G.A.
      • et al.
      ANKRD26 and its interacting partners TRIO, GPS2, HMMR, and DIPA regulate adipogenesis in 3T3-L1 cells.
      ) signaling suppresses adipogenesis and production of antifibrotic adipokines such as adiponectin. Thus, the profibrotic Tgfβ1/Rhamm/Myc signaling pathway likely targets both adipocytes and fibroblasts to affect tissue fibrosis by multiple mechanisms.
      In summary, we posit that Rhamm function‒blocking peptides inhibit dermal fibrosis by blocking Tgfβ1 and Myc signaling primarily as a result of Rhamm expression downregulation, which then inactivates the pathway (Figure 6f). Inhibition of Tgfβ1/Myc signaling releases the inhibition of Pparg target gene expression in particular the expression of antifibrotic adipokines, adiponectin, and Plin. This signaling regulation is particularly relevant to dermal fibrosis. For example, the mRNA expression of both adiponectin and the adiponectin receptors is reduced in primary SSc fibroblasts (
      • Fang F.
      • Liu L.
      • Yang Y.
      • Tamaki Z.
      • Wei J.
      • Marangoni R.G.
      • et al.
      The adipokine adiponectin has potent anti-fibrotic effects mediated via adenosine monophosphate-activated protein kinase: novel target for fibrosis therapy.
      ). Together with adiponectin, Pparg further suppresses Tgfβ1-induced fibrosis (
      • Ghosh A.K.
      • Bhattacharyya S.
      • Lakos G.
      • Chen S.J.
      • Mori Y.
      • Varga J.
      Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.
      ;
      • Wu M.
      • Melichian D.S.
      • Chang E.
      • Warner-Blankenship M.
      • Ghosh A.K.
      • Varga J.
      Rosiglitazone abrogates bleomycin-induced scleroderma and blocks profibrotic responses through peroxisome proliferator-activated receptor-gamma.
      ). Thus, Rhamm function‒blocking peptides may represent a method of switching the rigid fibrotic cellular program toward adipogenesis in scleroderma and possibly in other fibrotic diseases.

      Materials and Methods

       Study design and samples

      A total of 48 6-week-old female C57BL/6J mice were randomized into four treatment groups: (i) BLM group: 50 μl of 1 μg/μl BLM sulfate in 0.9% saline injected subcutaneously to the dorsal back skin on alternate days for 28 days; (ii) control group: 50 μl of 0.9% saline injected on alternate days; (iii) BLM injection as in group 1 with 3 mg/kg of NPI-110 (peptide sequence 644KLKDENSQLKSEVSK) every fourth day after BLM injections; and (iv) BLM injection as in group 1 with 3 mg/kg of NPI-106 (peptide sequence RGGRGRRR) every fourth day after BLM injections. Mice were killed on day 28. Full-thickness excisional dorsal back skin biopsies were obtained and fixed in 10% neutral buffered formalin for histology sections or were snap frozen. Samples used for mRNA analyses and immunohistochemical analyses were obtained from separate mice. Peptides were synthesized and purified to >95% purity (gift from L Luyt, Western University, London, Ontario, Canada). Mouse studies were conducted by Stelic MC (Tokyo, Japan) with institutional ethics approval and in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). Rhamm−/− and Rhamm-rescued dermal fibroblasts were obtained and cultured as previously described (
      • Tolg C.
      • Hamilton S.R.
      • Nakrieko K.A.
      • Kooshesh F.
      • Walton P.
      • McCarthy J.B.
      • et al.
      Rhamm-/- fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair.
      ). 10T1/2 fibroblasts were purchased from ATCC (Manassas, VA).

       Immunohistochemical analyses

      The 4-μm paraffin-processed histology slides were stained with H&E, Masson’s Trichrome, and Picrosirius Red according to the manufacturers’ instructions. Dermal thickness was measured from the dermal‒epidermal junction to the dermal‒adipose junction, and DAT thickness was measured from the dermal‒adipose junction to the adipose‒panniculus carnosus junction. Three measurements from three skin sections per mouse and 12 mice per treatment group were obtained.
      Picrosirius Red slides were scanned using the Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) with a circular polarizer, and images were quantified using National Institutes of Health ImageJ 1.47 (Rockville, MD). The area fraction of collagen was calculated as the total number of pixels in collagen fibrils over the total dermis area.
      For immunostaining, paraffin-processed histology slides were rehydrated. Endogenous biotin was quenched with 3% hydrogen peroxide, and slides were blocked with 3% BSA for 2 hours before incubation with the primary antibody (adiponectin, Abcam [Cambridge, United Kingdom], 1:600; TGFβ1, Abcam, 1:50; c-Myc, Abcam, 5 μg/ml dilution; S100A8/MAC387, Abcam, 1:300) overnight at 4 °C. Bound antibodies were visualized with biotin-conjugated anti-rabbit secondary IgG (Vector Laboratories, Burlingame, CA). Staining was amplified with streptavidin-horseradish peroxidase (Abcam) and visualized using DAKO Liquid DAB Substrate+ Chromogen system (Agilent Technologies, Santa Clare, CA). Quantification was performed using ImageJ 1.47 with image deconvolution using the H-DAB setting. The number of positive pixels per mm2 was recorded separately for the dermis and DATs after excluding the epidermis and hair follicles for three sections per slide, three slides per mouse, and three mice per group.
      For immunofluorescence staining, paraffin-processed histology slides were deparaffinized and rehydrated. Antigen retrieval was performed using 10 mM of sodium citrate buffer, pH 6.0, using a microwave for 18 minutes. Nonspecific antibody binding was blocked by incubation with PBS and/or 3% BSA for 60 minutes, and slides were then incubated with Rhamm antibody (Abcam, 1:50) overnight at 4 °C. All slides were washed using PBS and then incubated with Alexa 488‒conjugated goat anti-rabbit (A32731, Invitrogen, Carlsbad, CA) for 2 hours at room temperature. Slides were mounted with ProLong Gold Antifade Moutant with DAPI (Thermo Fisher Scientific, Waltham, MA). Quantification was performed in ImageJ 1.47 using the Split Channel algorithm with negative control used as the threshold. The intensity was detected using the Pixel Measurement algorithm.

       Immunofluorescence assays

      Dermal fibroblasts were subcultured onto glass coverslips at 50% subconfluence overnight in DMEM and 10% fetal calf serum and then treated with 2 μg/ml of TGFβ1 with or without 2 μg/ml of peptide NPI-110 for 24 hours. Coverslips were washed in PBS, fixed in 3% paraformaldehyde for 10 minutes at room temperature, and incubated in PBS with 3% BSA to block nonspecific binding sites. Coverslips were incubated with smooth muscle actin antibody (Abcam, 1:1,000) for 1 hour at room temperature, then incubated with Alexa 488‒conjugated anti-rabbit IgG (Abcam, 1:250), and then mounted and examined as described earlier.

       Western blot assays

      Dermal fibroblasts were subcultured and then harvested in a radioimmunoprecipitation assay buffer. Protein was separated using 5–12% SDS-PAGE, transferred to a nitrocellulose membrane, and then incubated with either Myc (Abcam, anti-mouse monoclonal, 1:500), Rhamm (Abcam, anti-rabbit monoclonal, 1:500), or Acta2 (Abcam, anti-rabbit monoclonal, 1:5,000) antibody. Bound antibodies were detected by Enhanced Chemiluminescent. Gapdh (Invitrogen, anti-mouse monoclonal, 1:1,000) was used as a loading control.

       Hydroxyproline assays

      Hydroxyproline was quantified in tissue sections using a hydroxyproline colorimetric assay kit (BioVision, Milpitas, CA). A total of 20 paraffin-processed tissue sections per treatment group were scraped into pressure-tight Teflon capped vials and hydrolyzed in 100 μl of 6N hydrochloric acid at 95 °C for 16 hours. A total of 10-μl samples were micropipetted into 96-well plates and dried overnight. Hydroxyproline was quantified using chloramine T and p-dimethylaminobenzaldehyde as per the manufacturer’s instructions, was measured at 560 nm in a microplate reader, and was quantified against a standard curve of hydroxyproline. Values were standardized by calculating tissue section area using QuPath (University of Edinburgh).

       RT-PCR

      RNA was extracted from skin samples using the Trizol-chloroform liquid‒liquid extraction method (
      • Rio D.C.
      • Ares Jr., M.
      • Hannon G.J.
      • Nilsen T.W.
      Purification of RNA using TRIzol (TRI reagent).
      ). The RNA pellet was then resuspended in UltraPure RNAse-free distilled water and quantified using NanoDrop One/One (Thermo Fisher Scientific). SuperScript VILO cDNA Mastermix (Thermo Fischer Scientific) was used to generate cDNA. RT-PCR was then performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) with cDNA and forward and reverse primer (Supplementary Table S3). Reactions were run using the Strategene Mx3000P system (Agilent Technologies) and analyzed using 2-ΔΔCt method normalized to Gapdh (
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method.
      ).

       Pathway analysis

      The expression of 84 fibrosis (Supplementary Table S1) and 84 adipogenesis (Supplementary Table S2) pathway‒focused genes were profiled using the 96-well RT2 Profiler PCR Plate Array (Qiagen, Hilden, Germany) with one technical replicate from one mouse per treatment group. Data were analyzed with the SABiosciences PCR array data analysis software using the 2-ΔΔCt method normalized to Gapdh and compared with those of the BLM-treated mouse. Ingenuity Pathway Analysis Software (Qiagen Bioinformatics, Hilden, Germany) was used to compare the transcriptional profiles of NPI-110 and NPI-106 treatment. Core analysis was performed using fold-expression values of each assayed gene from the RT2 Profiler Fibrosis and Adipogenesis arrays with strict criteria for a fold change >1.5 for upregulated and downregulated genes. Causal pathway analysis was performed for canonical pathways and upstream regulators.

       Statistical analysis

      Data recorded in Excel were imported into SPSS Statistics 25 (IBM, Armonk, NY) and tested for normality using the Shapiro‒Wilk test. Statistical analysis was performed for parametric data using one-way ANOVA to compare the means among all the four treatment groups and posthoc analysis with Fisher’s least significant difference test. Nonparametric data were analyzed using the Kruskal‒Wallis test with posthoc Mann‒Whitney U for repeated comparisons. Significance was set at P < 0.05.

       Data availability statement

      Datasets related to this article are available by contacting the corresponding author.

      ORCIDs

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We would like to thank Caroline O’Neil and Hao Yin for their assistance with Picrosirius Red staining and polarized light microscopy. This research was supported by the Frederik Banting and Charles Best Canada Graduate Scholarship (KW) and the Breast Cancer Society of Canada (EAT). This study was presented in part at the 72nd annual meeting of the Canadian Society of Plastic Surgeons.
      Mouse work was completed in Minami-Kamata in Ota City, Tokyo, Japan. All subsequent molecular analysis was completed in London, Ontario, Canada.

      Author Contributions

      Conceptualization: KW, EAT, AY; Data Curation: KW; Formal Analysis: KW, SK, AS, KM, VML; Funding Acquisition: EAT; Investigation: KW, SK, AS, KM, VML; Methodology: KW, SK, EAT, VML; Supervision: EAT; Writing - Original Draft Preparation: KW, SK, VML, EAT; Writing - Review and Editing: KW, SK, VML, EAT

      Supplementary Materials

      Figure thumbnail fx1
      Supplementary Figure S1Comparative anatomy of DAT in human and mouse skin. Schematic diagram of layers within human and mouse skin illustrating the differential location of the DAT layer. In human skin, DAT form cones surrounding the hair follicular unit and is directly superficial to but distinct from the subcutaneous adipose tissue. In mouse skin, the DAT is separated from the subcutaneous adipose layer by the panniculus carnosus, which is absent in humans. DAT, dermal adipose tissue.
      Figure thumbnail fx2
      Supplementary Figure S2Peptide NPI-110 treatment reduces the hydroxyproline content of BLM-treated skin. Analysis of hydroxyproline in control, BLM-treated, and BLM + NPI-110 peptide‒treated (BLM + NPI-110) tissue sections. Values are standardized to tissue section area (mm2) and represent the mean and SEM. Analysis was performed with one-way ANOVA and posthoc LSD test for significance. ∗∗P < 0.001, ∗∗∗P < 0.0001. BLM, bleomycin; LSD, least significant difference.
      Figure thumbnail fx3
      Supplementary Figure S3BLM reduces DAT thickness and increases the Col1a1-to-Col3a1 ratio. (a) Analysis of DAT measured in tissue sections. (b) RT-PCR analysis of the Col1a1-to-Col3a1 ratio in five mice per treatment group and five technical replicates from each of the four treatment groups on day 28. Values represent the mean and SEM. The analysis of DAT thickness was performed using Kruskal‒Wallis test with posthoc Mann‒Whitney U. All other experiments were performed with one-way ANOVA and posthoc LSD test for significance. ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.0001. BLM, bleomycin; DAT, dermal adipose thickness; LSD, least significant difference.
      Figure thumbnail fx4
      Supplementary Figure S4BLM treatment does not increase the number of macrophages. (a) Paraffin-processed skin sections from control and BLM-treated mice on day 28 stained for anti-S100A8/MAC387 antibody. Quantification of positive staining cells in the (a) dermis and (b) DAT. Values represent the mean and SEM from three random measurements in three sections per mouse. Analysis was performed with one-way ANOVA and posthoc LSD test for significance. BLM, bleomycin; DAT, dermal adipose thickness; LSD, least significant difference; n.s., not significant.
      Figure thumbnail fx5
      Supplementary Figure S5Rhamm−/− dermal fibroblasts do not respond to Tgfβ1. (a) Rhamm−/− dermal fibroblast cultures were stained for smooth muscle actin (Acta2) after treatment with Tgfβ1 ± NPI-110 peptide mimetic. Quantification of the number of cells exhibiting smooth muscle actin‒positive fibrils. (b) Values represent the mean and SEM from five random measurements for three replicates. (c) Western blot assays showing smooth muscle actin protein expression in Rhamm−/− dermal fibroblasts, with GAPDH used as a loading control. (d) Western blot assays of Rhamm protein expression in Rhamm-rescued and Rhamm−/− dermal fibroblasts. Bar = 100 μm. ns, no significance.

      Supplementary Data

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