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• W2024059588 abstract "The nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-γ, originally identified as a key mediator of adipogenesis, is expressed widely and implicated in diverse biological responses. Both natural and synthetic agonists of PPAR-γ abrogated the stimulation of collagen synthesis and myofibroblast differentiation induced by transforming growth factor (TGF)-β in vitro. To characterize the role of PPAR-γ in the fibrotic process in vivo, the synthetic agonist rosiglitazone was used in a mouse model of scleroderma. Rosiglitazone attenuated bleomycin-induced skin inflammation and dermal fibrosis as well as subcutaneous lipoatrophy and counteracted the up-regulation of collagen gene expression and myofibroblast accumulation in the lesioned skin. Rosiglitazone treatment reduced the induction of the early-immediate transcription factor Egr-1 in situ without also blocking the activation of Smad2/3. In both explanted fibroblasts and skin organ cultures, rosiglitazone prevented the stimulation of collagen gene transcription and cell migration elicited by TGF-β. Rosiglitazone-driven adipogenic differentiation of both fibroblasts and preadipocytes was abrogated in the presence of TGF-β; this effect was accompanied by the concomitant down-regulation of cellular PPAR-γ mRNA expression. Collectively, these results indicate that rosiglitazone treatment attenuates inflammation, dermal fibrosis, and subcutaneous lipoatrophy via PPAR-γ in a mouse model of scleroderma and suggest that pharmacological PPAR-γ ligands, widely used as insulin sensitizers in the treatment of type-2 diabetes mellitus, may be potential therapies for scleroderma. The nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-γ, originally identified as a key mediator of adipogenesis, is expressed widely and implicated in diverse biological responses. Both natural and synthetic agonists of PPAR-γ abrogated the stimulation of collagen synthesis and myofibroblast differentiation induced by transforming growth factor (TGF)-β in vitro. To characterize the role of PPAR-γ in the fibrotic process in vivo, the synthetic agonist rosiglitazone was used in a mouse model of scleroderma. Rosiglitazone attenuated bleomycin-induced skin inflammation and dermal fibrosis as well as subcutaneous lipoatrophy and counteracted the up-regulation of collagen gene expression and myofibroblast accumulation in the lesioned skin. Rosiglitazone treatment reduced the induction of the early-immediate transcription factor Egr-1 in situ without also blocking the activation of Smad2/3. In both explanted fibroblasts and skin organ cultures, rosiglitazone prevented the stimulation of collagen gene transcription and cell migration elicited by TGF-β. Rosiglitazone-driven adipogenic differentiation of both fibroblasts and preadipocytes was abrogated in the presence of TGF-β; this effect was accompanied by the concomitant down-regulation of cellular PPAR-γ mRNA expression. Collectively, these results indicate that rosiglitazone treatment attenuates inflammation, dermal fibrosis, and subcutaneous lipoatrophy via PPAR-γ in a mouse model of scleroderma and suggest that pharmacological PPAR-γ ligands, widely used as insulin sensitizers in the treatment of type-2 diabetes mellitus, may be potential therapies for scleroderma. Excessive collagen accumulation in the skin and lungs, the hallmark of systemic sclerosis (SSc), can lead to organ dysfunction, failure, and death.1Jimenez S Derk C Following the molecular pathways toward an understanding of the pathogenesis of systemic sclerosis.Ann Intern Med. 2004; 140: 37-50Crossref PubMed Google Scholar The pathogenesis of fibrosis remains incompletely understood.2Varga J Abraham D Systemic sclerosis: a prototypic multisystem fibrotic disorder.J Clin Invest. 2007; 117: 557-567Crossref PubMed Scopus (920) Google Scholar Although inflammation is a prevalent early feature, its precise role in fibrosis remains controversial, and anti-inflammatory therapies are generally ineffective in reversing or slowing the progression of the process.3Charles C Clements P Furst DE Systemic sclerosis: hypothesis-driven treatment strategies.Lancet. 2006; 367: 1683-1691Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar Therefore, there is an urgent need for anti-fibrotic therapies. The fibroblast is the key effector cell driving the fibrotic process in SSc. In response to extracellular cues such as transforming growth factor (TGF)-β, fibroblasts become activated with increased collagen production, expression of cell surface receptors for growth factors, secretion of cytokines and chemokines, resistance to apoptosis induction, and myofibroblast differentiation.4Abraham D Eckes B Rajkumar V Krieg T New developments in fibroblast and myofibroblast biology: implications for fibrosis and scleroderma.Curr Rheumatol Rep. 2007; 9: 136-143Crossref PubMed Scopus (129) Google Scholar In light of the key role of TGF-β in the pathogenesis of fibrosis, therapeutic strategies to block its production, activity, or intracellular signaling are under investigation.5Trojanowska M Varga J Molecular pathways as novel therapeutic targets in systemic sclerosis.Curr Opin Rheumatol. 2007; 19: 568-573Crossref PubMed Scopus (34) Google Scholar However, because the potent anti-inflammatory and immunosuppressive activities of TGF-β are physiologically important, global TGF-β blockade could be complicated by spontaneous autoimmunity. Indeed, mice lacking TGF-β die at an early age from severe inflammation.6Dang H Geiser A Letterio J Nakabayashi T Kong L Fernandes G Talal N SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-beta knockout mice.J Immunol. 1995; 155: 3205-3212PubMed Google Scholar Therefore, an ideal anti-fibrotic strategy targeting TGF-β must selectively abrogate fibrotic responses without disrupting its important immunosuppressive activities.Rosiglitazone is an insulin-sensitizing agent widely in type 2 diabetes mellitus that exerts its biological effects in part via the peroxisome proliferator activated receptor (PPAR)-γ.7Yki-Järvinen H Thiazolidinediones.N Engl J Med. 2004; 351: 1106-1118Crossref PubMed Scopus (1880) Google Scholar Originally identified in adipose tissue, PPAR-γ is one of a family of closely related nuclear receptors and ligand-activated transcription factors with a primary role in adipogenesis.8Rosen ED Spiegelman B PPARgamma: a nuclear regulator of metabolism, differentiation, and cell growth.J Biol Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar In addition to adipocytes, PPAR-γ is expressed in macrophages, vascular endothelial and smooth muscle cells, and fibroblasts. The PPAR-γ receptor acts as a lipid sensor that can be activated by fatty acids, eicosanoids, and related endogenous products of metabolism.9Lehrke M Lazar MA The many faces of PPARgamma.Cell. 2005; 123: 993-999Abstract Full Text Full Text PDF PubMed Scopus (1135) Google Scholar A majority of experimental studies of PPAR-γ have used the natural ligand 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2), or synthetic ligands such as rosiglitazone. In the absence of ligand, PPAR-γ is complexed to the retinoid X receptor (RXR) and co-repressors, preventing its binding to DNA. Upon receptor ligation, co-repressors are displaced from the PPAR-γ/RXR complex and co-activators such as p300 are recruited, allowing sequence-specific binding to conserved PPAR-γ response elements (PPREs) in target gene promoters.10Heikkinen S Auwerx J Argmann CA PPARgamma in human and mouse physiology.Biochim Biophys Acta. 2007; 1771: 999-1013Crossref PubMed Scopus (173) Google Scholar Ligands of PPAR-γ exert a broad range of proliferative, anti-inflammatory, and repair activities in addition to adipogenesis and insulin sensitization.11Michalik L Wahli W Involvement of PPAR nuclear receptors in tissue injury and wound repair.J Clin Invest. 2006; 116: 598-606Crossref PubMed Scopus (178) Google Scholar Recent studies have shown that PPAR-γ inhibits basal and stimulated collagen synthesis in vitro and in vivo, suggesting a novel biological role in connective tissue homeostasis.12Ghosh AK Bhattacharyya S Lakos G Chen SJ Mori Y Varga J Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.Arthritis Rheum. 2004; 50: 1305-1318Crossref PubMed Scopus (180) Google Scholar, 13Burgess H Daugherty L Thatcher T Lakatos H Ray D Redonnet M Phipps R Sime P PPARgamma agonists inhibit TGF-beta induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis.Am J Physiol. 2005; 288: L1146-L1153Crossref PubMed Scopus (264) Google Scholar, 14Milam J Keshamouni V Phan S Hu B Gangireddy S Hogaboam C Standiford T Thannickal V Reddy R PPAR-{gamma} agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis.Am J Physiol. 2008; 294: L891-L901Crossref PubMed Scopus (162) Google Scholar Ligand inhibition of TGF-β-dependent fibrotic responses was mediated via the PPAR-γ receptor and involved antagonistic cross talk with the intracellular TGF-β/Smad signal transduction pathway.12Ghosh AK Bhattacharyya S Lakos G Chen SJ Mori Y Varga J Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.Arthritis Rheum. 2004; 50: 1305-1318Crossref PubMed Scopus (180) Google ScholarBecause fibroblast activation by TGF-β is a key pathogenetic event in SSc, blockade of TGF-β signaling by PPAR-γ could be a novel therapeutic approach to pathological fibrogenesis. In the present studies, therefore, we investigated the effect of rosiglitazone, the most potent pharmacological PPAR-γ agonist, in bleomycin-induced scleroderma. The results indicate that rosiglitazone ameliorated the development of fibrosis in this mouse model of scleroderma. The anti-fibrotic effect of rosiglitazone involved blockade of TGF-β-induced fibroblast activation, as well as attenuation of the inflammatory response. Furthermore, rosiglitazone counteracted the development of subcutaneous adipose atrophy and loss of local PPAR-γ expression. Together, these findings indicate that ligands of PPAR-γ, already in clinical use for the treatment of type 2 diabetes mellitus, block fibrotic TGF-β responses without triggering aberrant immunity, suggesting their potential as novel anti-fibrotic agents in SSc.Materials and MethodsAnimals and Experimental ProtocolsSix- to eight-week-old female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) were used in these studies. The protocols were institutionally approved by the Northwestern University Animal Care and Use Committee. Mice were housed in autoclaved cages and fed sterile food and water. Filter-sterilized bleomycin [20 μg/mouse dissolved in phosphate-buffered saline (PBS)] (Mayne Pharma, Paramus, NJ) or PBS was administered by daily subcutaneous injections into the shaved backs of mice using a 27-gauge needle.15Takagawa S Lakos G Mori Y Yamamoto T Nishioka K Varga J Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.J Invest Dermatol. 2003; 121: 41-50Crossref PubMed Scopus (100) Google Scholar Rosiglitazone (5 mg/kg) (GlaxoSmithKline, King of Prussia, PA) and the irreversible PPAR-γ antagonist GW9662 (Biomol International, Plymouth Meeting, PA) were administrated by daily intraperitoneal injection. At the end of each experiment, mice were sacrificed and lesional skin was processed for analysis. Each experimental group consisted of at least five mice.Histochemical AnalysisLesional skin tissue was embedded in paraffin, and consecutive 4-μm serial sections were stained with hematoxylin and eosin (H&E). To evaluate collagen content and organization in the lesional skin, deparaffinized sections were stained with Picrosirius Red and viewed under a polarized light microscope.16Lakos G Takagawa S Chen SJ Ferreira AM Han G Masuda K Wang XJ DiPietro LA Varga J Targeted disruption of TGF-beta/Smad3 signaling modulates skin fibrosis in a mouse model of scleroderma.Am J Pathol. 2004; 165: 203-217Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar Dermal thickness, defined as the distance between the epidermal-dermal junction and the dermal-adipose layer junction, and the adipose layer, defined as the distance between the dermal-adipose junction and the muscle, was determined in H&E-stained sections at ×100 microscopic magnification. For localizing tissue lipids, samples immersed in freezing medium (Triangle Biomedical Sciences, Durham, NC) were sectioned (7 μm) using a CM 1900 UV cryostat (Leica Microsystems, Bannockburn, IL) and stained with Oil Red O.17Ramírez-Zacarías J Castro-Muñozledo F Kuri-Harcuch W Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O.Histochemistry. 1992; 97: 493-497Crossref PubMed Scopus (817) Google Scholar Mast cells were identified by Astra blue staining and quantified by counting in six random fields under high magnification.15Takagawa S Lakos G Mori Y Yamamoto T Nishioka K Varga J Sustained activation of fibroblast transforming growth factor-beta/Smad signaling in a murine model of scleroderma.J Invest Dermatol. 2003; 121: 41-50Crossref PubMed Scopus (100) Google Scholar To detect apoptosis, terminal dUTP nick-end labeling (TUNEL) assays were performed on paraffin-embedded tissues using an in situ cell death detection kit (Roche Molecular Biochemicals, Indianapolis, IN).Immunohistochemistry and ImmunofluorescenceFour-μm sections from lesional skin were deparaffinized, rehydrated, and immersed in TBS-T buffer (Tris-buffered saline and 0.1% Tween 20), and treated with target retrieval solution (DAKO, Carpinteria, CA) at 95°C for 10 minutes. Tissue embedded in freezing medium (Triangle Biomedical Sciences) was sectioned (7 μm) in a CM 1900 UV cryostat. Primary antibodies against CD3 (BD Pharmingen, San Diego, CA); Mac-3 (BD Pharmingen), α-smooth muscle actin (Sigma-Aldrich, St. Louis, MO), TGF-β1 and Egr-1 (both from Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Smad2 (Cell Signaling, Danvers, MA), PPAR-γ (Santa Cruz), or I-Ad (BD Pharmingen) were used. Bound antibodies were detected using secondary antibodies from the Histomouse-Max kit (Zymed Laboratories, San Francisco, CA) and the DakoCytomation Envision + System-HRP (3,3′-diaminobenzidine tetrahydrochloride) according to the manufacturers' instructions. Substitution of the primary antibody with isotype-matched irrelevant IgG served as negative controls. After counterstaining with hematoxylin, sections were mounted with Permount (Fisher Scientific, Pittsburgh, PA) and viewed under an Olympus BH-2 microscope (Olympus, Tokyo, Japan). Fibroblasts were identified by their spindle-shaped morphology. The number of immunopositive cells was determined at ×400 magnification by two blinded observers. Immunofluorescence intensity was quantitated by image analysis.Quantification of Tissue Levels of Collagen and TGF-β1Lesional skin tissues were homogenized in 0.5 mol/L glacial acetic acid and centrifuged at 10,000 rpm for 15 minutes. Supernatant were then assayed for soluble collagen using Sircol colorimetric assays (Biocolor, Newton Abbey, UK), and TGF-β1 was quantitated by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN).RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction 9 (qPCR)Total RNA was isolated from frozen skin tissue using Trizol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy mini spin columns (Qiagen, Valencia, CA). Reverse transcription was performed in a DNA thermal cycler (2720 Thermal Cycler; Applied Biosystems, Foster City, CA) using a reverse transcription system kit (Promega, Madison, WI). Real-time qPCR was performed using SYBR Green Master Mix (Applied Biosystems) in an ABI Prism 7700 sequence detection system (Applied Biosystems). The oligonucleotide primers used for qPCR are listed in Table 1. The results of real-time qPCR analysis are expressed as -fold change in mRNA levels relative to 18S RNA or GAPDH levels in each sample.Table 1Primers Used for Quantitative Real-Time PCRGenecDNA sequence F, forward; R, reverseCOL1A1F: 5′-CCTGAGTCAGCAGATTGAGAA-3′; R: 5′-ACTGAACTTGACCGTACACCAGTACTCTCCGCTCTTCAA-3′COL1A2F: 5′-CCGTGCTTCTCAGAACATCA-3′; R: 5′-CTTGCCCCATTCATTTGTCT-3′α-SMAF: 5′-CAGCGGGCATCCACGAA-3′; R: 5′-GCCACCGATCCAGACAGA-3′TGF-β1F: 5′-TACAGCAAGGTCCTTGCCCT-3′; R: 5′-GCAGCACGGTGACGCC-3′PPAR-γ1F: 5′-GAGTGTGACGACAAGATTTG-3′; R: 5′-GGTGGGCCAGAATGGCATCT-3′PPAR-γ2F: 5′-TCTGGGAGATTCTCCTATTGA-3′; R: 5′-GGTGGGCCAGAATGGCATCT-3′Egr-1F: 5′-TTTGCCTCCGTTCCACCTGC-3′; R: 5′-TGCCAACTTGATGGTCATGCGC-3′18S rRNAF: 5′-TTCGAACGTCTGCCCTATCA-3′; R: 5′-ATGGTAGGCACGGCGACTA-3′ Open table in a new tab Flow Cytometry and Determination of Adiponectin, Cytokine, and Chemokine LevelsPeripheral blood was obtained at day 5 of bleomycin injections via cardiac puncture with citrate solution (100 mmol/L Na Citrate, 130 mmol/L glucose, pH 6.5) as anticoagulant, and mononuclear cells were purified by density gradient centrifugation. Spleens were removed and triturated with the end of a 3-ml syringe, and filtered through 70-μm nylon mesh (BD Bioscience, Bedford, MA). Single cell suspensions of peripheral blood mononuclear cells or spleen cells were analyzed by flow cytometry using antibodies against I-Ad (MHC-II)-FITC, CD86-PE, or CD-11b-APC, along with mouse IgG1-PE, IgG2a/2b-FITC, and IgG-APC (all from BD Pharmingen) as isotype controls. Events were collected on a DakoCytomation (DAKO, Fort Collins, CO) by gating for live cells based on forward and side scatter, and data were analyzed using Summit software V4.3 (DAKO). Serum levels of adiponectin, and the inflammatory mediators interleukin (IL)-1β and MCP-1 were determined at 7 or 28 days after the start of bleomycin injection by ELISA (R&D).Collagen Transcription AssaysCultures of dermal fibroblasts were established by explantation of skin biopsies from newborn Col1a2-luc transgenic mice. These mice harbor a construct of the mouse proα2(I) collagen gene promoter containing 17 kb 5′ of the transcription start site, fused to a luciferase reporter gene, that drives high levels of reporter gene expression in fibroblasts.18Bou-Gharios G Garrett LA Rossert J Niederreither K Eberspaecher H Smith C Black C Crombrugghe B A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice.J Cell Biol. 1996; 134: 1333-1344Crossref PubMed Scopus (135) Google Scholar When the fibroblasts reached confluence, cultures were incubated in serum-free media containing 0.1% bovine serum albumin and indicated concentrations of rosiglitazone, followed 30 minutes later by 10 ng/ml of TGF-β1 (Amgen, Thousand Oaks, CA). At the end of a further 48 hours of incubation, cultures were harvested and cell lysates were assayed for their luciferase activities.19Ghosh A Bhattacharyya S Varga J The tumor suppressor p53 abrogates Smad-dependent collagen gene induction in mesenchymal cells.J Biol Chem. 2004; 279: 47455-47463Crossref PubMed Scopus (52) Google ScholarSkin Organ CulturesTriplicate 3-mm punch biopsies were obtained from the back skin of 12-week-old female C57BL/6 mice. The tissues were incubated in 96-well plates (one tissue piece per 150 μl of culture medium) in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 1% vitamins, 100 U/ml penicillin/streptomycin, 2 mmol/L l-glutamine with rosiglitazone (10 μmol/L) in presence or absence of TGF-β1 (10 ng/ml). After 48 hours, conditioned media were collected and newly synthesized soluble collagens (types I to IV) were quantified by Sircol colorimeteric assays. Total RNA was isolated from skin homogenates using Trizol reagent and subjected to real-time qPCR analysis.In Vitro Adipogenic Differentiation AssaysTo assess adipogenic differentiation, mouse 3T3L1 preadipocyte (Zen-Bio, Research Triangle Park, NC) were incubated in either preadipocyte maintenance medium or DM2 adipogenic differentiation medium (both from Zen-Bio). Low-passage (three to four) dermal fibroblasts established from the skin of newborn BALB/c mice were seeded in 12-well plates (105 cells/well), and allowed to attach overnight. Cultures were then incubated in Dulbecco's modified Eagle's medium or DM2 in the presence or absence of rosiglitazone (10 μmol/L) and/or TGF-β2 (5 ng/ml) (Genzyme, Framingham, MA) for up to 5 days. To assess intracellular lipid accumulation, cells were stained with Oil Red O (0.5% Oil Red O dye in 60% isopropanol) for 30 minutes followed by gentle rinse before microscopic visualization. Lipid-containing cells were enumerated in 10 random microscopic fields for each experimental condition, and experiments were repeated at least three times.For immunocytochemistry, cultures were washed in PBS and fixed in 4% paraformaldehyde for 10 minutes followed by methanol for permeabilization, and stained with antibodies specific for leptin (Santa Cruz Biotechnology), fatty acid binding protein-4 (FABP4) (R&D), and α-smooth muscle actin (Sigma-Aldrich). Slides were overlaid with secondary horseradish peroxidase-conjugated anti-rabbit IgG for 30 minutes, and 3,3′-diaminobenzidine tetrahydrochloride was used for chromogenic localization of antibody. Sections were counterstained with hematoxylin, and images were obtained by digital capture. Fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG was used as secondary antibody of α-smooth muscle actin. Nuclei were identified with 4′-6-diamidino-2-phenylindole. Images were obtained by digital capture in a Nikon Eclipse TE200 microscope (Fryer Co., Huntley, IL) and viewed under ×200 magnification.In Vitro Cell Migration AssaysThe modulation of cell migration in vitro was analyzed by monolayer wound-healing assays.14Milam J Keshamouni V Phan S Hu B Gangireddy S Hogaboam C Standiford T Thannickal V Reddy R PPAR-{gamma} agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis.Am J Physiol. 2008; 294: L891-L901Crossref PubMed Scopus (162) Google Scholar Briefly, primary skin fibroblasts from C57BL/6 mice were grown to early confluence. Scratch wounds were induced in monolayers using standard p1000 pipette tips, followed by incubation of the cultures in media with 10 ng/ml of mitomycin C (Sigma-Aldrich) to block cell proliferation. Rosiglitazone (2 to 20 μmol/L) and TGF-β1 (10 ng/ml) were added and incubation continued for a further 24 hours. The wounds were monitored at intervals by phase contrast microscopy. Wound gap length was measured at six different sites in each sample at indicated times, and experiments were repeated multiple times with similar results.Statistical AnalysisResults are expressed as the means ± SD or ± SEM. Mann-Whitney's U-test (in vivo studies) or Student's t-test (in vitro studies) was used for comparison between two groups. Values <0.05 were considered statistically significant.ResultsEffects of Rosiglitazone on Skin Inflammation and FibrosisTo evaluate the effects of rosiglitazone in a mouse model of scleroderma, BALB/c mice given daily injections of subcutaneous bleomycin for up to 28 days were treated with intraperitoneal rosiglitazone. Bleomycin induced an early and transient accumulation of inflammatory cells in the skin that peaked on day 7 after initiation of treatment, and was most prominent in the deep dermis and subcutaneous adipose tissue (Figure 1A, and data not shown). Concurrent treatment with rosiglitazone significantly reduced the inflammatory cell infiltration (Figure 1A, bottom). Pretreatment of the mice with the selective PPAR-γ antagonist GW9662 abrogated the effects of rosiglitazone, indicating that anti-inflammatory response was mediated via activation of cellular PPAR-γ. Immunohistochemical analysis showed that mice treated with rosiglitazone plus bleomycin for 7 days had reduced accumulation of Mac-3-positive cells in the lesional skin (Figure 1B), whereas the numbers of infiltrating CD3-positive cells and mast cells were primarily unchanged (data not shown). The enhanced local expression of MHC class-II (I-Ad), a hallmark of classical monocyte/macrophage activation, was substantially attenuated by administration of rosiglitazone (Figure 1C).After 28 days of bleomycin injections, a considerable increase in dermal thickness and accumulation of densely packed collagen bundles was evident in the lesional skin (Figure 2A, top). At the same time, the subcutaneous adipose layer showed striking atrophy, and was virtually replaced by acellular densely packed connective tissue. Mice treated with rosiglitazone showed significant expansion of the subcutaneous adipose layer with accumulation of intracellular lipids, as demonstrated by strong staining with Oil Red O (Figure 2B). When rosiglitazone was administered together with bleomycin, dermal fibrosis was attenuated and collagen bundles were loosely packed and randomly oriented. Quantitative evaluation showed that whereas dermal thickness of the dermis was >50% increased in bleomycin-injected mice compared with PBS-injected control mice, rosiglitazone attenuated the increase (P < 0.05). Furthermore, the integrity of the subcutaneous adipose layer was partially preserved in these mice (Figure 2A, bottom).Figure 2Rosiglitazone prevents skin fibrosis and subcutaneous adipose atrophy. Lesional skin was examined after 28 days. A: H&E stain. Top: a, control; b, bleomycin; c, rosiglitazone; d, bleomycin plus rosiglitazone. Representative photomicrographs. Arrows indicate the extent of the subcutaneous adipose layer. Bottom: Thick ness of the dermis and subcutaneous adipose tissue. Results represent the means ± SD from five mice per group, *P < 0.05. B: Oil Red O stain. a, control; b, bleomycin; c, rosiglitazone; d, bleomycin plus rosiglitazone; Scale bar = 100 μm. Original magnifications: ×100 (B, insets); ×400 (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Staining with Picrosirius Red was used to investigate the deposition and organization of collagenous matrix in the skin. In bleomycin-injected mice, the dermis and subcutaneous tissue showed dense collagen accumulation with strong red birefringence (indicative of highly cross-linked mature fibers), whereas mice given rosiglitazone together with bleomycin showed weaker birefringence (Figure 3A). To examine the effects of rosiglitazone on collagen gene expression in vivo, mRNA in the lesional skin was quantified by real-time PCR. The results showed a threefold to sixfold increase in the levels of COL1A1 and COL1A2 mRNA in mice treated with bleomycin compared with PBS-treated control mice (Figure 3B). Concurrent treatment with rosiglitazone markedly attenuated the up-regulation of collagen mRNA (P < 0.01). The expression of α-smooth muscle actin, a marker for identifying myofibroblasts that play crucial roles in pathological fibrogenesis, was determined by immunohistochemistry. After 28 days of bleomycin, a twofold increase in α-smooth muscle actin was noted in the lesional dermis and subcutaneous layers (Figure 3C, top). Concurrent treatment of the mice with rosiglitazone significantly reduced the number of α-smooth muscle actin-positive fibroblastic cells (Figure 3C, bottom). Plasma levels of adiponectin, an adipocyte-specific circulating cytokine, were increased by ∼40% in rosiglitazone-treated mice compared with controls (5.3 ± 0.4 μg versus 3.7 ± 0.4 μg, P < 0.05). Detection of in situ cell death demonstrated a modest increase in the number of TUNEL-positive fibroblastic cells in the lesional dermis at 28 days after bleomycin injections were started; the increase in apoptotic cells was abrogated in rosiglitazone-treated mice (data not shown).Figure 3Rosiglitazone attenuates collagen deposition and myofibroblast accumulation. Lesional skin (dermis plus subcutaneous adipose tissue) was examined after 28 days. A: Picrosirius Red stain (viewed under polarized microscopy). a, control; b, bleomycin; c, rosiglitazone; d, bleomycin plus rosiglitazone; e, GW9662; f, bleomycin plus rosiglitazone plus GW9662. Representative photomicrographs. Arrows indicate the extent of the dermis. B: Real-time qPCR analysis. Results are normalized for 18S RNA and represent the means ± SD of duplicate determinations from three mice per group, *P < 0.01. C, Top: α-Smooth muscle actin immunohistochemistry. a, control; b, bleomycin; c, rosiglitazone; d, bleomycin plus rosiglitazone. Bottom: The proportion of α-smooth muscle actin-positive fibroblastic cells was quantified from at least six separate fields from five mice per group. The results indicate the means ± SD. *P < 0.05. Scale bars: 100 μm (A); 50 μm (C); 20 μm (C, inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Reduced Mononuclear Cell ActivationPeripheral blood and spleen mononuclear cells were obtained on day 5 after treatment was initiated, and analyzed by flow cytometry. The results indicated that both the frequency and the absolute numbers of I-Ad-positive mononuclear cells were modestly increased in the circulation of bleomycin-injected mice compared with control mice, and rosiglitazone treatment was associated with a nearly 30% reduction (Figure 4A). Similar results were seen when spleen cells were analyzed (Figure 4B). C" @default.
• W2024059588 created "2016-06-24" @default.
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• W2024059588 date "2009-02-01" @default.
• W2024059588 modified "2023-10-14" @default.
• W2024059588 title "Rosiglitazone Abrogates Bleomycin-Induced Scleroderma and Blocks Profibrotic Responses Through Peroxisome Proliferator-Activated Receptor-γ" @default.
• W2024059588 cites W1494611500 @default.
• W2024059588 cites W1497601632 @default.
• W2024059588 cites W1580962701 @default.
• W2024059588 cites W1633340712 @default.
• W2024059588 cites W1812573520 @default.
• W2024059588 cites W1964410726 @default.
• W2024059588 cites W1964738051 @default.
• W2024059588 cites W1965726485 @default.
• W2024059588 cites W1970779494 @default.
• W2024059588 cites W1971585190 @default.
• W2024059588 cites W1973401836 @default.
• W2024059588 cites W1983203756 @default.
• W2024059588 cites W1985602371 @default.
• W2024059588 cites W1992213652 @default.
• W2024059588 cites W1996267589 @default.
• W2024059588 cites W1998424002 @default.
• W2024059588 cites W2004861205 @default.
• W2024059588 cites W2006367961 @default.
• W2024059588 cites W2008683618 @default.
• W2024059588 cites W2009995147 @default.
• W2024059588 cites W2013900793 @default.
• W2024059588 cites W2017070582 @default.
• W2024059588 cites W2023974524 @default.
• W2024059588 cites W2026356057 @default.
• W2024059588 cites W2032283922 @default.
• W2024059588 cites W2033204507 @default.
• W2024059588 cites W2045113312 @default.
• W2024059588 cites W2046833545 @default.
• W2024059588 cites W2048851534 @default.
• W2024059588 cites W2062761869 @default.
• W2024059588 cites W2069649414 @default.
• W2024059588 cites W2071007459 @default.
• W2024059588 cites W2072762682 @default.
• W2024059588 cites W2080269439 @default.
• W2024059588 cites W2081741824 @default.
• W2024059588 cites W2088770687 @default.
• W2024059588 cites W2113313714 @default.
• W2024059588 cites W2114597310 @default.
• W2024059588 cites W2118820170 @default.
• W2024059588 cites W2158957820 @default.
• W2024059588 cites W2159347821 @default.
• W2024059588 cites W2160854440 @default.
• W2024059588 cites W2166508420 @default.
• W2024059588 cites W2167248947 @default.
• W2024059588 cites W2171370326 @default.
• W2024059588 cites W4240449862 @default.
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