Screening for Inhibitors of YAP Nuclear Localization Identifies Aurora Kinase A as a Modulator of Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFß (Transforming Growth Factor ß) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Diagnostic Accuracy of Endobronchial Optical Coherence Tomography for the Microscopic Diagnosis of Usual Interstitial Pneumonia.

Rationale: Early, accurate diagnosis of interstitial lung disease (ILD) informs prognosis and therapy, especially in idiopathic pulmonary fibrosis (IPF). Current diagnostic methods are imperfect. High-resolution computed tomography has limited resolution, and surgical lung biopsy (SLB) carries risks of morbidity and mortality. Endobronchial optical coherence tomography (EB-OCT) is a low-risk, bronchoscope-compatible modality that images large lung volumes in vivo with microscopic resolution, including subpleural lung, and has the potential to improve the diagnostic accuracy of bronchoscopy for ILD diagnosis. Objectives: We performed a prospective diagnostic accuracy study of EB-OCT in patients with ILD with a low-confidence diagnosis undergoing SLB. The primary endpoints were EB-OCT sensitivity/specificity for diagnosis of the histopathologic pattern of usual interstitial pneumonia (UIP) and clinical IPF. The secondary endpoint was agreement between EB-OCT and SLB for diagnosis of the ILD fibrosis pattern. Methods: EB-OCT was performed immediately before SLB. The resulting EB-OCT images and histopathology were interpreted by blinded, independent pathologists. Clinical diagnosis was obtained from the treating pulmonologists after SLB, blinded to EB-OCT. Measurements and Main Results: We enrolled 31 patients, and 4 were excluded because of inconclusive histopathology or lack of EB-OCT data. Twenty-seven patients were included in the analysis (16 men, average age: 65.0 yr): 12 were diagnosed with UIP and 15 with non-UIP ILD. Average FVC and DlCO were 75.3% (SD, 18.5) and 53.5% (SD, 16.4), respectively. Sensitivity and specificity of EB-OCT was 100% (95% confidence interval, 75.8-100.0%) and 100% (79.6-100%), respectively, for both histopathologic UIP and clinical diagnosis of IPF. There was high agreement between EB-OCT and histopathology for diagnosis of ILD fibrosis pattern (weighted κ: 0.87 [0.72-1.0]). Conclusions: EB-OCT is a safe, accurate method for microscopic ILD diagnosis, as a complement to high-resolution computed tomography and an alternative to SLB.

Selective YAP/TAZ inhibition in fibroblasts via dopamine receptor D1 agonism reverses fibrosis.

Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gαs-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice.

An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4+ regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT.

A Small-Molecule CD4-Mimetic Compound Protects Bone Marrow-Liver-Thymus Humanized Mice From HIV-1 Infection.

Background: Small-molecule CD4-mimetic compounds (CD4mc) inhibit human immunodeficiency virus (HIV-1) entry by blocking binding to the CD4 receptor and by premature triggering of the viral envelope glycoprotein (Env) spike. Methods: The efficacy of a CD4mc in protecting bone marrow-liver-thymus (BLT) humanized mice from vaginal HIV-1 challenge was evaluated. Results: Intravaginal application of the CD4mc JP-III-48, either before or simultaneously with virus challenge, protected BLT humanized mice from HIV-1JR-CSF infection in a dose- dependent manner. Conclusion: The direct antiviral effects of a CD4mc prevent HIV-1 infection in a murine model of sexual transmission.

Therapeutic Efficacy of Vectored PGT121 Gene Delivery in HIV-1-Infected Humanized Mice.

Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required.IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice.

The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis.

Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-ß, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.

ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis.

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified. Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression is increased by transforming growth factor (TGF)-ß1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-ß1-induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10-sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

High sensitivity HPLC method for determination of the allysine concentration in tissue by use of a naphthol derivative.

Common to all fibrotic and metastatic diseases is the uncontrollable remodeling of tissue that leads to the accumulation of fibrous connective tissue components such as collagen and elastin. Build-up of fibrous tissue occurs through the cross-linking of collagen or elastin monomers, which is initiated through the oxidation of lysine residues to form α-aminoadipic-δ-semialdehyde (allysine). To provide a measure of the extent of collagen oxidation in disease models of fibrosis or metastasis, a rapid, sensitive HPLC method was developed to quantify the amount of allysine present in tissue. Allysine was reacted with sodium 2-naphthol-7-sulfonate under conditions typically applied for acid hydrolysis of tissues (6M HCl, 110°C, 24h) to prepare AL-NP, a fluorescent bis-naphthol derivative of allysine. High performance liquid chromatography was applied for analysis of allysine content. Under optimal reaction and detection conditions, successful separation of AL-NP was achieved with excellent analytical performance attained. Good linear relationship (R2=0.994) between peak area and concentration for AL-NP was attained for 0.35-175pmol of analyte. A detection limit of 0.02pmol in the standard sample with a 20µL injection was achieved for AL-NP, with satisfactory recovery from 88 to 100% determined. The method was applied in the quantification of allysine in healthy and fibrotic mouse lung tissue, with the fibrotic tissue showing a 2.5 fold increase in the content of allysine.

Inhibition of CTGF ameliorates peritoneal fibrosis through suppression of fibroblast and myofibroblast accumulation and angiogenesis.

Peritoneal fibrosis (PF) is a serious complication in various clinical settings, but the mechanisms driving it remain to be fully determined. Connective tissue growth factor (CTGF) is known to regulate fibroblast activities. We therefore examined if CTGF inhibition has anti-fibrotic effects in PF. PF was induced by repetitive intraperitoneal injections of chlorhexidine gluconate (CG) in mice with type I pro-collagen promoter-driven green fluorescent protein (GFP) expression to identify fibroblasts. FG-3019, an anti-CTGF monoclonal antibody, was used to inhibit CTGF. CG-induced PF was significantly attenuated in FG-3019-treated mice. CG challenges induced marked accumulations of proliferating fibroblasts and of myofibroblasts, which were both reduced by FG-3019. Levels of peritoneal CTGF expression were increased by CG challenges, and suppressed in FG-3019-treated mice. FG-3019 treatment also reduced the number of CD31+ vessels and VEGF-A-positive cells in fibrotic peritoneum. In vitro studies using NIH 3T3 fibroblasts and peritoneal mesothelial cells (PMCs) showed that CTGF blockade suppressed TGF-ß1-induced fibroblast proliferation and myofibroblast differentiation, PMC mesothelial-to-mesenchymal transition, and VEGF-A production. These findings suggest that the inhibition of CTGF by FG-3019 might be a novel treatment for PF through the regulation of fibroblast and myofibroblast accumulation and angiogenesis.

The autotaxin-lysophosphatidic acid pathway emerges as a therapeutic target to prevent liver cancer.

Using transcriptome meta-analysis, we recently identified the autotaxin (ATX)-lysophosphatidic acid (LPA) pathway as a regulator of hepatocellular carcinoma (HCC) risk in human cirrhosis patients. Pharmacological targeting of this pathway reduced fibrosis progression and HCC development in animals, identifying ATX-LPA signaling as a novel chemoprevention strategy for cirrhosis and HCC.

Type I collagen-targeted PET probe for pulmonary fibrosis detection and staging in preclinical models.

Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 = 0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.

Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease.

The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency γ-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex+ T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.

Lysophosphatidic acid signaling through its receptor initiates profibrotic epithelial cell fibroblast communication mediated by epithelial cell derived connective tissue growth factor.

The expansion of the fibroblast pool is a critical step in organ fibrosis, but the mechanisms driving expansion remain to be fully clarified. We previously showed that lysophosphatidic acid (LPA) signaling through its receptor LPA1 expressed on fibroblasts directly induces the recruitment of these cells. Here we tested whether LPA-LPA1 signaling drives fibroblast proliferation and activation during the development of renal fibrosis. LPA1-deficient (LPA1-/-) or -sufficient (LPA1+/+) mice were crossed to mice with green fluorescent protein expression (GFP) driven by the type I procollagen promoter (Col-GFP) to identify fibroblasts. Unilateral ureteral obstruction-induced increases in renal collagen were significantly, though not completely, attenuated in LPA1-/-Col-GFP mice, as were the accumulations of both fibroblasts and myofibroblasts. Connective tissue growth factor was detected mainly in tubular epithelial cells, and its levels were suppressed in LPA1-/-Col-GFP mice. LPA-LPA1 signaling directly induced connective tissue growth factor expression in primary proximal tubular epithelial cells, through a myocardin-related transcription factor-serum response factor pathway. Proximal tubular epithelial cell-derived connective tissue growth factor mediated renal fibroblast proliferation and myofibroblast differentiation. Administration of an inhibitor of myocardin-related transcription factor/serum response factor suppressed obstruction-induced renal fibrosis. Thus, targeting LPA-LPA1 signaling and/or myocardin-related transcription factor/serum response factor-induced transcription could be promising therapeutic strategies for renal fibrosis.

Molecular Liver Cancer Prevention in Cirrhosis by Organ Transcriptome Analysis and Lysophosphatidic Acid Pathway Inhibition.

Cirrhosis is a milieu that develops hepatocellular carcinoma (HCC), the second most lethal cancer worldwide. HCC prediction and prevention in cirrhosis are key unmet medical needs. Here we have established an HCC risk gene signature applicable to all major HCC etiologies: hepatitis B/C, alcohol, and non-alcoholic steatohepatitis. A transcriptome meta-analysis of >500 human cirrhotics revealed global regulatory gene modules driving HCC risk and the lysophosphatidic acid pathway as a central chemoprevention target. Pharmacological inhibition of the pathway in vivo reduced tumors and reversed the gene signature, which was verified in organotypic ex vivo culture of patient-derived fibrotic liver tissues. These results demonstrate the utility of clinical organ transcriptome to enable a strategy, namely, reverse-engineering precision cancer prevention.

An Autotaxin/Lysophosphatidic Acid/Interleukin-6 Amplification Loop Drives Scleroderma Fibrosis.

OBJECTIVE: We previously implicated the lipid mediator lysophosphatidic acid (LPA) as having a role in dermal fibrosis in systemic sclerosis (SSc). The aim of this study was to identify the role of the LPA-producing enzyme autotaxin (ATX), and to connect the ATX/LPA and interleukin-6 (IL-6) pathways in SSc. METHODS: We evaluated the effect of a novel ATX inhibitor, PAT-048, on fibrosis and IL-6 expression in the mouse model of bleomycin-induced dermal fibrosis. We used dermal fibroblasts from SSc patients and control subjects to evaluate LPA-induced expression of IL-6, and IL-6-induced expression of ATX. We next evaluated whether LPA-induced ATX expression is dependent on IL-6, and whether baseline IL-6 expression in fibroblasts from SSc patients is dependent on ATX. Finally, we compared ATX and IL-6 expression in the skin of patients with SSc and healthy control subjects. RESULTS: PAT-048 markedly attenuated bleomycin-induced dermal fibrosis when treatment was initiated before or after the development of fibrosis. LPA stimulated expression of IL-6 in human dermal fibroblasts, and IL-6 stimulated fibroblast expression of ATX, connecting the ATX/LPA and IL-6 pathways in an amplification loop. IL-6 knockdown abrogated LPA-induced ATX expression in fibroblasts, and ATX inhibition attenuated IL-6 expression in fibroblasts and the skin of bleomycin-challenged mice. Expression of both ATX and IL-6 was increased in SSc skin, and LPA-induced IL-6 levels and IL-6-induced ATX levels were increased in fibroblasts from SSc patients compared with controls. CONCLUSION: ATX is required for the development and maintenance of dermal fibrosis in a mouse model of bleomycin-induced SSc and enables 2 major mediators of SSc fibrogenesis, LPA and IL-6, to amplify the production of each other. Our results suggest that concurrent inhibition of these 2 pathways may be an effective therapeutic strategy for dermal fibrosis in SSc.