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Localized scleroderma (LS) is an autoimmune disease characterized by inflammation and fibrosis, leading to severe cutaneous manifestations such as skin hardening, tightness, discoloration, and other textural changes that may result in disability. While LS shares similar histopathologic features and immune-fibroblast interactions with systemic sclerosis (SSc), its molecular mechanisms remain understudied. Endothelial cells (EC) are known to play a crucial role in SSc but have not been investigated in LS. Single-cell RNA sequencing (scRNA-seq) now allows for detailed examination of this cell type in the primary organ of interest for scleroderma, the skin. In this study, we analyzed skin-isolated cells from 27 LS patients (pediatric and adult) and 17 healthy controls using scRNA-seq. Given the known role of EC damage as an initial event in SSc and the histologic and clinical skin similarities to LS, we focused primarily on endothelial cells. Our analysis identified eight endothelial subclusters within the dataset, encompassing both disease and healthy samples. Interaction analysis revealed that signaling from diseased endothelial cells was predicted to promote fibrosis through SELE interaction with FGFBP1 and other target genes. We also observed high levels of JAG in arterial endothelial cells and NOTCH in capillary endothelial cells, indicating the activation of a signaling pathway potentially responsible for epidermal abnormalities and contributing to LS pathogenesis. In summary, our scRNA-seq analysis identified potential disease-propagating endothelial cell clusters with upregulated pathways in LS skin, highlighting their importance in disease progression.

Juvenile localized and systemic scleroderma are rare autoimmune diseases which cause significant disability and morbidity in children. The mechanisms driving juvenile scleroderma remain unclear, necessitating further cellular and molecular level studies. The Visium CytAssist spatial transcriptomics (ST) platform, which preserves the spatial location of cells and simultaneously sequences the whole transcriptome, was employed to profile the histopathological slides from skin lesions of juvenile scleroderma patients. (1) Spatial domains were identified from ST data and exhibited strong concordance with the pathologist's annotations of anatomical structures. (2) The integration of paired ST data and single-cell RNA sequencing (scRNA-seq) from the same patients validated the comparable accuracy of the two platforms and facilitated the estimation of cell type composition in ST data. (3) The pathologist-annotated immune infiltrates, such as perivascular immune infiltrates, were clearly delineated by the ST analysis, underscoring the biological relevance of the findings. This is the first study utilizing spatial transcriptomics to investigate skin lesions in juvenile scleroderma patients. The validity of the ST data was corroborated by gene expression analyses and the pathologist's assessments. Integration with scRNA-seq data facilitated the cell type-level analysis and validation. Analyses of immune infiltrates through combined ST data and pathological review enhances our understanding of the pathogenesis of juvenile scleroderma.

Adipose tissue transplantation shows great therapeutic potential in reversing localized scleroderma-associated skin fibrosis. Brown adipose tissue (BAT) can specifically secrete various cytokines against fibrosis, but its therapeutic potential in improving skin fibrosis has not yet been demonstrated. In this study, we have demonstrated the superior therapeutic efficacy of BAT transplantation for sclerotic skin by transplanting two distinct types of adipose tissue. In comparison to the white adipose tissue (WAT) group, mice treated with BAT transplantation exhibited a significant reduction in dermal thickness. BAT transplantation effectively reverses skin sclerosis through mechanisms involving inflammation reduction, promotion of angiogenesis, inhibition of myofibroblast accumulation, and collagen deposition. This therapeutic effect can be attributed to its unique paracrine effects. Furthermore, transcriptome sequencing (RNA-Seq) revealed upregulation of pathways associated with lipogenesis and fatty acid metabolism in BAT while downregulating pathways are related to transforming growth factor β(TGF-β), epithelial-mesenchymal transition (EMT), and inflammatory response. These findings suggest that BAT transplantation holds great promise as a novel approach for localized scleroderma treatment., (© 2023 Federation of American Societies for Experimental Biology.)

Background: Inflammation and fibrosis of the skin are characteristics of localized scleroderma (LS). Emerging evidence has demonstrated that exosomes from human adipose tissue-derived mesenchymal stem cells (ADSC-Exo) could alleviate skin fibrosis., Objective: The impact and potential mechanism of ADSC-Exo on LS fibrosis was examined., Methods: ADSC-Exo was isolated and identified. The effects of ADSC-Exo on the abilities of proliferation and migration of LS-derived fibroblasts (LSFs) were assessed by CCK-8 and scratch assays, respectively. qRT-PCR, western blot, and immunofluorescence were conducted to detect LSFs stimulated with ADSC-Exo, ADSC-Exo Anti-let-7a-5p , let-7a-5p mimic/TGF-βR1 shRNA virus, and negative controls. The impact of ADSC-Exo on C57BL/6j LS mice was evaluated by photographic morphology, hematoxylin-eosin (H&E), Masson's trichrome, and immunohistochemical staining., Results: The verified ADSC-Exo limited the proliferation and migration of LSFs and reduced the expression of COL1, COL3, α-SMA, TGF-βR1, and p-Smad2/ 3 in vitro and in vivo. TGF-βR1 knockdown and let-7a-5p mimic in LSFs reduced the expression of COL1, COL3, α-SMA, and p-Smad2/3. However, compared with the ADSC-Exo NC group, the dermal thickness was increased, collagen arrangement was disordered, and α-SMA and TGF-βR1 levels were increased after exposure to ADSC-Exo Anti-let-7a-5p ., Conclusions: In this study, it might show that ADSC-Exo may successfully prevent LSF bioactivity, collagen deposition, and myofibroblast trans-differentiation. Additionally, we confirmed that let-7a-5p in ADSC-Exo could directly target TGF-R1 to control the Smad pathway and reduce fibrosis in LSFs. Our work offered a brand-new therapeutic approach and clarified the unique mechanism for the clinical management of LS., Competing Interests: Declaration of Competing Interest The authors have declared no competing interests., (Copyright © 2023 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Localized scleroderma (LS) is an autoimmune disease with both inflammatory and fibrotic components causing an abnormal deposition of collagen in the skin and underlying tissue, often leading to disfigurement and disability. Much of its pathophysiology is extrapolated from systemic sclerosis (SSc) since the histopathology findings in the skin are nearly identical. However, LS is critically understudied. Single-cell RNA sequencing (scRNA seq) technology provides a novel way to obtain detailed information at the individual cellular level, overcoming this barrier. Here, we analyzed the affected skin of 14 patients with LS (pediatric and adult) and 14 healthy controls. Fibroblast populations were the focus, since they are the main drivers of fibrosis in SSc. We identified 12 fibroblast subclusters in LS, which overall had an inflammatory gene expression (IFN and HLA-associated genes). A myofibroblast-like cluster (SFRP4/PRSS23) was more prevalent in LS subjects and shared many upregulated genes expressed in SSc-associated myofibroblasts, though it also had strong expression of CXCL9/10/11, known CXCR3 ligands. A CXCL2/IRF1 cluster identified was unique to LS, with a robust inflammatory gene signature, including IL-6, and according to cell communication analysis are influenced by macrophages. In summary, potential disease-propagating fibroblasts and associated gene signatures were identified in LS skin via scRNA seq.

Scleroderma is an autoimmune disease mainly characterized by progressive fibrosis of the skin. There are two types of scleroderma, namely localized scleroderma (LS) and systemic sclerosis (SSc); skin lesions in both types of scleroderma are histologically identical. Progressive skin sclerosis induces psychological and ecological burden for scleroderma patients. However, there is no effective treatment for scleroderma due to its unclear etiology. Aryl hydrocarbon receptor (AhR) is recognized as an environmental chemical effector that can respond to ultraviolet radiation, which has been demonstrated to participate in the pathogenesis of SSc in our previous study. In this study, we verify whether the anti-fibrosis effect of ultraviolet A1 (UVA1) phototherapy could be partially induced through Ficz/AhR/MAPK signaling activation for fibrotic lesions in both SSc and LS patients. This is the first study to show the association between the AhR pathway and the anti-fibrotic mechanism of UVA1 phototherapy, which provides additional evidence of the role of AhR in the fibrotic mechanism of systemic scleroderma from different perspectives. Ficz and other AhR agonists may replace UVA1 phototherapy as anti-fibrotic agents in scleroderma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Competing Interests: Competing interests: RS reports receiving funds for the following activities: research support: GlaxoSmithKline, Genentech/Roche, Novartis, Corbus Pharmaceuticals, Cytori Therapeutics, Evidera, Actelion Pharmaceuticals, ChemoCentryx, Boehringer Ingelheim Pharmaceuticals, Forbius, InfaRx, Sanofi, Kadmon Corporation; consulting: GlaxoSmithKline, Janssen Pharmaceuticals, Sanofi Aventis, ChemoCentryx, Forbius, CSL Behring; DEO reports receiving funds from Pfizer and personal fees from Astra Zeneca, outside the submitted work; JKG reports receiving funds for the following activities: research support: Corbus Pharmaceuticals, Cumberland Pharmaceuticals, and Eicos Sciences, outside the submitted work.

Fibrosis is a common pathophysiological response of many tissues and organs subjected to chronic injury. Despite the diverse aetiology of keloid, lacaziosis and localized scleroderma, the process of fibrosis is present in the pathogenesis of all of these three entities beyond other individual clinical and histological distinct characteristics. Fibrosis was studied in 20 samples each of these three chronic cutaneous inflammatory diseases. An immunohistochemical study was carried out to explore the presence of α-smooth muscle actin (α-SMA) and vimentin cytoskeleton antigens, CD31, CD34, Ki67, p16; CD105, CD163, CD206 and FOXP3 antigens; and the central fibrotic cytokine TGF-β. Higher expression of vimentin in comparison to α-SMA in all three lesion types was found. CD31- and CD34-positive blood vessel endothelial cells were observed throughout the reticular dermis. Ki67 expression was low and almost absent in scleroderma. p16-positive levels were higher than ki67 and observed in reticular dermis of keloidal collagen in keloids, in collagen bundles in scleroderma and in the external layers of the granulomas in lacaziosis. The presence of α-actin positive cells and rarely CD34 positive cells, observed primarily in keloids, may be related to higher p16 antigen expression, a measure of cell senescence. Low FOXP3 expression was observed in all lesion types. CD105-positive cells were mainly found in perivascular tissue in close contact with the adventitia in keloids and scleroderma, while, in lacaziosis, these cells were chiefly observed in conjunction with collagen deposition in the external granuloma layer. We did not find high involvement of CD163 or CD206-positive cells in the fibrotic process. TGF-β was notable only in keloid and lacaziosis lesions. In conclusion, we have suggested vimentin to be the main myofibroblast general marker of the fibrotic process in all three studied diseases, while endothelial-to-mesenchymal transition (EndoMT) and mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role., (© 2022 Company of the International Journal of Experimental Pathology (CIJEP).)

The role and potential molecular mechanism of inflammatory cells in pediatric localized scleroderma are poorly investigated. In this study, we first investigated the profiling of inflammatory cells in skin samples from pediatric localized scleroderma. Among them, CD4+ T-cells were up-regulated. Co-culture dermal fibroblasts with CD4+ T-cells promoted fibrosis of fibroblasts. Candidate lncRNAs were further explored by lncRNAs-seq between the normal skin tissues and pediatric localized scleroderma tissues, and the lncRNAs-seq between fibroblasts co-cultured with CD4+ T lymphocytes and control fibroblasts. By comparing the two datasets, we identified eight up-regulated and three down-regulated lncRNAs, which were the potential lncRNAs for the phenotype of pediatric localized scleroderma. Here, we identified the CD4+ T-cells infiltration in pediatric localized scleroderma and potential lncRNAs for the treatment of pediatric localized scleroderma., (© 2022 Wiley Periodicals LLC.)

Background/purpose: Localized scleroderma (LS) is a rare disease leading to progressive hardening and induration of the skin and subcutaneous tissues. LS is responsive to UVA-1 phototherapy, though its exact mechanism of action dermal fibrosis is yet to be fully elucidated. We aimed to investigate the molecular changes induced by UVA-1 rays in human primary fibroblasts cultures., Methods: A total of 16 LS patients were treated with medium-dose UVA-1 phototherapy. At baseline, during and after therapy, Localized Scleroderma Assessment Tool, Dermatology Life Quality Index and lesions' staging and mapping were performed along with high-frequency ultrasound (HFUS) examination for dermal thickness assessment. Gene expression analysis for 23 mRNA transcripts, in vitro UVA-1 irradiation and viability tests were realized on lesional fibroblasts' primary cultures, before and 3 months after therapy., Results: The dermal thickness, the LoSCAT and the DLQI progressively decreased starting from the last phototherapy session up to the 6 and 9 month follow-ups (-57% and -60%, respectively). Molecular gene analysis (rt-PCR) revealed that UVA-1 phototherapy exerts multiple effects: the activation of specific anti-fibrotic pathways (e.g., overexpression of CTHRC1 and metalloproteases 1, 2, 7, 8, 9, 12, suppression of TIMP-1), the downregulation of peculiar pro-fibrotic pathways (e.g., downregulation of TGF-ß, TGF-ßrII, Grb2, SMAD 2/3, TNRSF12A, CTGF) through a significant overexpression of IL-1ß; the stabilization of collagen synthesis acting on genes COL1A1, COL3A1, COL8A1, COL10A1, COL12A1., Conclusion: UVA-1 phototherapy adds significant benefits in local tissue remodeling, rebalancing the alteration between pro-fibrotic and anti-fibrotic pathways; these changes can be well monitored by HFUS., (© 2022 The Authors. Photodermatology, Photoimmunology & Photomedicine published by John Wiley & Sons Ltd.)

Objectives: Determine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time., Methods: A total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions., Results: Gene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up., Conclusions: Skin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design., Competing Interests: Competing interests: MDM reports support from Mitsubishi-Tanabe, Boehringer Ingelheim, EICOS and Corbus to her institution outside the submitted work, and fees from Medtelligence, Actelion Pharma, Mitsubishi-Tanabe, Boehringer Ingelheim and EICOS to her outside the submitted work. SA reports grant support from Momenta, Boehringer Ingelheim, Janssen, and the Scleroderma Research Foundation to his institution outside the submitted work; consulting fees from Boehringer Ingelheim, Novartis, Corbus, CSL Behring, Abbvie, AstraZeneca to him outside the submitted work; honorarium from Boehringer Ingelheim for lectures and payment from the American College of Rheumatology to him outside the submitted work. BS, MAL, WRS, GS, MW, TMT, JC and CPV have no relevant competing interests to declare., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo . We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application., Methods: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression., Results: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses., Conclusions: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc., Competing Interests: Competing interests: SH is an employee of Capella Bioscience, which holds a patent for CBS004 (GB1911188.9)., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Scleroderma refers to a group of chronic fibrotic immune-mediated diseases of unknown etiology. Characterizing epigenetic changes in childhood-onset scleroderma, systemic sclerosis or localized scleroderma, has not been previously performed. The aim of this study was to assess DNA methylation differences and similarities between juvenile systemic sclerosis (jSSc) and juvenile localized scleroderma (jLS) compared to matched healthy controls. Genome-wide DNA methylation changes in peripheral blood mononuclear cell samples were assessed using the MethylationEPIC array followed by bioinformatic analysis and limited functional assessment. We identified a total of 105 and 144 differentially methylated sites compared to healthy controls in jSSc and jLS, respectively. The majority of differentially methylated sites and genes represented were unique to either jSSc or jLS suggesting a different underlying epigenetic pattern in both diseases. Among shared differentially methylated genes, methylation levels in a CpG site in FGFR2 can distinguish between LS and healthy PBMCs with a high accuracy. Canonical pathway analysis revealed that inflammatory pathways were enriched in genes differentially methylated in jSSc, including STAT3, NF-κB, and IL-15 pathways. In contrast, the HIPPO signaling pathway was enriched in jLS. Our data also suggest a potential role for NOTCH3 in both jSSc and jLS, and revealed a number of transcription factors unique to each of the two diseases. In summary, our data revealed important insights into jSSc and jLS and suggest a potentially novel epigenetic diagnostic biomarker for LS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fibronectin (FN) circulating in the blood and produced by cells provides the basis of the extracellular matrix (ECM) formed in healing acute wounds. The time-dependent deposition of FN by macrophages, its synthesis by fibroblasts and myofibroblasts, and later degradation in the remodeled granulation tissue are a prerequisite for successful healing of wounds. However, the pattern of FN expression and deposition in skin lesions is disturbed. The degradation of the ECM components including FN in varicose veins prevails over ECM synthesis and deposition. FN is inconspicuous in the fibrotic lesions in lipodermatosclerosis, while tenascin-C containing FN-like peptide sequences are prominent. FN is produced in large amounts by fibroblasts at the edge of venous ulcers but FN deposition at the wound bed is impaired. Both the proteolytic environment in the wounds and the changed function of the ulcer fibroblasts may be responsible for the poor healing of venous ulcers. The aim of this review is to describe the current knowledge of FN pathophysiology in chronic venous diseases. In view of the fact that FN plays a crucial role in organizing the ECM, further research focused on FN metabolism in venous diseases may bring results applicable to the treatment of the diseases.

Sclerosing polycystic adenosis (SPA) is a rare salivary gland disease. Histologically it resembles a low-grade ductal carcinoma in situ or sclerosing adenosis of the breast, characterized by lobular proliferation of ducts with apocrine cellular features surrounded by fibrosclerotic stroma. Although SPA is typically benign, recurrence is not uncommon, and cases with a malignant component have been documented. Thus, complete excision is desirable but preoperative diagnosis is challenging. A 12-year-old boy presented with a painless mass in the right neck. We identified a well-demarcated mass in the right parotid region measuring approximately 2 cm using cervical echography and magnetic resonance (MR) imaging. Fine-needle aspiration (FNA) revealed two cell types. There were loosely cohesive clusters of polymorphic epithelioid cells with irregular nuclei and abundant vacuolated cytoplasm containing zymogen granules. Some of these cells were binuclear. The other cell types represented normal ductal cells. The original cytological diagnosis was Warthin tumor. Right parotidectomy was performed. Histologically, we observed proliferation of ducts with granular, vacuolated, zymogen granules, and apocrine-like features in the cytoplasm with hyalinizing sclerotic stroma and some binuclear cells. Four years after parotidectomy, there has been no recurrence or malignant transformation.Cytological diagnosis of SPA is challenging on FNA specimens since SPA is a very rare entity of the salivary gland that can mimic other salivary gland neoplasms. A mixture of apocrine-like cells and sebaceous-like cells, nuclear pleomorphism, and zymogen granules can help to diagnose this rare lesion during the initial cytological diagnosis., (© 2019 Wiley Periodicals, Inc.)

Background: Localized Scleroderma (LoS) encompasses a group of idiopathic skin conditions characterized by (sub)cutaneous inflammation and subsequent development of fibrosis. Currently, lack of accurate tools enabling disease activity assessment leads to suboptimal treatment approaches., Objective: To investigate serum concentrations of cytokines and chemokines implicated in inflammation and angiogenesis in LoS and explore their potential to be utilized as biomarker of disease activity. Additionally, to investigate the implication of potential biomarkers in disease pathogenesis., Methods: A 39-plex Luminex immuno-assay was performed in serum samples of 74 LoS and 22 Healthy Controls. The relation between a validated clinical measure of disease activity (mLoSSI) and serum analytes was investigated. Additionally, gene and protein expression were investigated in circulating cells and skin biopsies., Results: From the total of 39, 10 analytes (CCL18, CXCL9, CXCL10, CXCL13, TNFRII, Galectin-9, TIE-1, sVCAM, IL-18, CCL19) were elevated in LoS serum. Cluster analysis of serum samples revealed CCL18 as most important analyte to discriminate between active and inactive disease. At individual patient level, CCL18 serum levels correlated strongest with mLoSSI-scores (r s  = 0.4604, P < 0.0001) and in longitudinal measures CCL18 concentrations normalised with declining disease activity upon treatment initiation. Additionally, CCL18 was elevated in LoS serum, and not in (juvenile) dermatomyositis or spinal muscular atrophy. Importantly, CCL18 gene and protein expression was increased at the inflammatory border of cutaneous LoS lesions, with normal expression in unaffected skin and circulating immune cells., Conclusion: CCL18 is specific for disease activity in LoS thereby providing relevance as a biomarker for this debilitating disease., (Copyright © 2019. Published by Elsevier Ltd.)

Purpose of Review: To describe diverse neurologic and neuroradiologic presentations of two rare, immunologically mediated skin conditions: Sweet disease and localized scleroderma (morphea)., Recent Findings: Core syndromes of neuro-Sweet disease (NSD) are steroid responsiveness, recurrent meningitis, and encephalitis. Focal neurologic, neuro-vascular, and neuro-ophthalmologic syndromes have been reported recently in NSD. A variety of steroid-sparing treatments and biologics have been used for relapsing NSD. Localized craniofacial scleroderma is associated with seizures, headaches, and, less commonly, focal deficits and cognitive decline. Immunosuppressive therapy may be required in patients with disease progression; some refractory cases have responded to IL-6 inhibition. Our review provides an up-to-date reference for neurologists faced with a patient with a history or skin findings consistent with Sweet disease or localized scleroderma. We hope that it will stimulate collaborative studies aimed at unraveling the pathogenesis of these disorders, better characterization of their neurologic manifestations, and discovery of optimal therapeutic solutions.

Background: Fibrosis is thought to be the main pathophysiology of scleroderma, and myofibroblasts play the main role in abnormal fibrotic pathologies. Altered distribution of dermal dendritic cells (DDCs) and vascular abnormalities has been reported to relate to the pathogenesis of scleroderma., Objective: To investigate fibrotic pathogenesis of morphea (localized scleroderma) by demonstrating the relative expression and distribution of DDCs and myofibroblasts, we performed immunohistochemical stains using several relevant antibodies., Methods: Skin lesions of 50 patients with morphea and age-, sex- and site-matched normal skin of 50 subjects were evaluated for the following antibodies: CD34, factor XIIIa (FXIIIa), smooth muscle actin (SMA), CD31 and vascular cell adhesion molecule-1 (VCAM-1)., Results: CD34 stromal stain was significantly lower in patients than controls (P = 0.000), while FXIIIa, SMA and VCAM-1 stains were significantly higher in patients than controls (P = 0.043, P = 0.000 and P = 0.027, respectively). In subtype analysis within patients, CD34 stromal stain showed decreasing trends with increasing disease extent and increasing fibrosis, respectively. CD34 stromal stain showed an inverse correlation and mutually exclusive spatial expression pattern with SMA stain (r = -0.286, P = 0.044). The inverse relationship was maintained in each dermal layer analysis, upper and lower dermis (r = -0.397, P = 0.004 and r = -0.281, P = 0.048, respectively)., Conclusions: Mutually exclusive staining patterns of CD34 stromal and SMA stains suggest a phenotypic change of CD34+ DDCs into SMA+ myofibroblasts with increasing disease extent and fibrosis in morphea. Degree of loss of CD34+ DDCs can be a useful marker in predicting the extent and severity of morphea., (© 2018 European Academy of Dermatology and Venereology.)

Purpose of Review: Dermal white adipose tissue (DWAT) is distinct from subcutaneous white adipose tissue and is lost in scleroderma skin fibrosis. The roles of DWAT loss in scleroderma skin fibrosis have not been well understood, and here we discuss recent findings that begin to provide insight into the multiple mechanisms involved., Recent Findings: The DWAT loss in part reflects the direct contribution of DWAT cells to the fibrotic tissue, with the reprogramming of adipocytes to myofibroblasts. The DWAT contains reparative adipose-derived stromal cells and expresses antifibrotic cytokines such as adiponectin, and the loss of these skin-protective mechanisms with DWAT loss further contributes to skin fibrosis and injury., Summary: Potentially, halting or reversing the transdifferentiation of adipocytes to myofibroblasts along with improving survival of reparative adipose-derived stromal cells (ADSCs) and expression of antifibrotic cytokines may be effective therapeutic avenues.

Objectives: Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc., Methods: The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-β-receptor I., Results: Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis., Conclusions: These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Localized scleroderma (LS) (morphea) is a chronic, inflammatory skin disease with unknown cause that progresses with sclerosis in the skin and/or subcutaneous tissues. Its pathogenesis is not completely understood. Oxidative stress is suggested to have a role in the pathogenesis of localized scleroderma. We have aimed to determine the relationship of morphea lesions with oxidative stress. The total oxidant capacity (TOC), total antioxidant capacity (TAC), paroxonase (PON) and arylesterase (ARES) activity parameters of PON 1 enzyme levels in the serum were investigated in 13 LS patients (generalized and plaque type) and 13 healthy controls. TOC values of the patient group were found higher than the TOC values of the control group (p < 0.01). ARES values of the patient group was found to be higher than the control group (p < 0.0001). OSI was significantly higher in the patient group when compared to the control (p < 0.005). Oxidative stress seems to be effective in the pathogenesis. ARES levels have increased in morphea patients regarding to the oxidative stress and its reduction. Further controlled studies are required in wider series.

Objectives: Interleukin (IL) 22 mRNA in systemic sclerosis (SSc) skin and Th22 cells in SSc peripheral blood are increased, but the role of IL-22 in fibrosis development remains poorly understood., Methods: Biopsies were obtained from the involved skin of 15 SSc, 4 morphea and 8 healthy donors (HD). The presence of IL-22+ cells in the skin was determined by immunostaining. The in vitro response of HD and SSc fibroblasts to IL-22, IL-22 in conjunction with tumour necrosis factor (TNF) or keratinocyte conditioned medium was assessed by ELISA, radioimmunoassay (RIA), real-time PCR and western blot. The in vivo response in mice was assessed by histomorphometry., Results: IL-22+ cells were over-represented in the dermis and epidermis of morphea and in the epidermis of SSc compared with HD. The majority of dermal IL-22+ cells were T cells. Dermal fibroblasts expressed both IL-22 receptor subunits IL-10RB and IL-22RA, expression of which was enhanced by TNF and reduced by transforming growth factor (TGF)-β. IL-22 induced rapid phosphorylation of p38 and ERK1/2 in fibroblasts, but failed to induce the synthesis of chemokines and extracellular matrix components. However, IL-22 enhanced the production of monocyte chemotactic protein 1, IL-8 and matrix metalloproteinase 1 induced by TNF. Fibroblast responses were maximal in the presence of conditioned medium from keratinocytes activated by IL-22 in conjunction with TNF. Dermal thickness was maximal in mice injected simultaneously with IL-22 and TNF., Conclusions: IL-22 capacitates fibroblast responses to TNF and promotes a proinflammatory fibroblast phenotype by favouring TNF-induced keratinocyte activation. These results define a novel role for keratinocyte-fibroblast interactions in the context of skin fibrosis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Localized scleroderma (LS) is a disease characterized by fibrotic changes in the dermis. Connective tissue growth factor and transforming growth factor β2 are the main mediators of fibrogenesis; this, along with excessive connective tissue production, affects epidermal keratinocytes, and thereby contributes to the changed quality of skin barrier. The objective of this article was to study the objective measurement of the skin barrier quality in LS with transepidermal water loss (TEWL) meter. The measurements of TEWL were performed on LS plaques in all three stages of various body locations. Control measurements were made on the contralateral side of healthy skin. The difference between TEWL in LS area and the contralateral side of the healthy skin was evaluated. A higher average TEWL 7.86 g/m(2) /h (SD 5.29) was observed on LS plaques compared with the control measurements on healthy skin 6.39 g/m(2) /h (SD 2.77). TEWL average values decreased from the inflammatory stage, through the sclerotic and to the atrophic stage. The mean difference 1.301 g/m(2) /h (SD 5.16) was found between TEWL on LS plaques and on the contralateral healthy skin in 82 measurements, i.e., a higher TEWL was observed in LS. The difference was statistically significant with p = 0.0250. Although fibrogenesis in scleroderma is localized in dermis, the skin barrier changes can be detected., (© 2016 Wiley Periodicals, Inc.)

Background: Lipodermatosclerosis (LDS) is a chronic fibrosing panniculitis associated with venous insufficiency. Although LDS is often a clinical diagnosis, it can be confused with other panniculitides. Microscopic examination is therefore essential to support the diagnosis in this condition. Histopathologic changes, however, have not been extensively defined. The purpose of this study was to characterize the histopathologic spectrum of this condition correlated with clinical manifestation., Methods: A total of 25 cases were collected retrospectively, and the clinical information and histopathologic findings were reviewed., Results: Of 25 patients, the female to male ratio was 4 : 1. The mean age was 54 years (range, 31-74 years). Clinical features were acute in eight (32%), subacute in 12 (48%), and chronic in five (20%). The microscopic study mostly demonstrated vascular stasis changes of varying degrees depending on the age of the lesion. Adipocyte necrosis with thickened septa, extravasation of erythrocytes, and lymphocytic infiltration were major findings in the early lesions. In the chronic lesion, lipomembranous fat necrosis with microcyst formation, vascular stasis changes in subcutaneous tissue, and septal fibrosis were predominant features. Iron deposition or hemosiderin extending to the subcutaneous layer was always seen in all specimens at the subacute and chronic stages., Conclusion: The diagnosis of LDS still needs clinicopathologic correlation. The constellation of findings including septal fibrosis, lipomembranous fat necrosis, prominent vascular changes of stasis, and erythrocytic extravasation can be used to define LDS histopathologically. Interestingly, iron deposition in the subcutaneous tissue is a useful finding for this chronic condition., (© 2015 The International Society of Dermatology.)

Scleroderma is a group of rare diseases associated with early and transient inflammation and vascular injury, followed by fibrosis affecting the skin and multiple internal organs. Fibroblast activation is the hallmark of scleroderma, and disrupting the intracellular TGFβ signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, both PPARγ and CB2 receptors represent attractive targets for the development of cannabinoid-based therapies. We have developed a non-thiophilic and chemically stable derivative of the CBD quinol (VCE-004.8) that behaves as a dual agonist of PPARγ and CB2 receptors, VCE-004.8 inhibited TGFβ-induced Col1A2 gene transcription and collagen synthesis. Moreover, VCE-004.8 inhibited TGFβ-mediated myofibroblast differentiation and impaired wound-healing activity. The anti-fibrotic efficacy in vivo was investigated in a murine model of dermal fibrosis induced by bleomycin. VCE-004.8 reduced dermal thickness, blood vessels collagen accumulation and prevented mast cell degranulation and macrophage infiltration in the skin. These effects were impaired by the PPARγ antagonist T0070907 and the CB2 antagonist AM630. In addition, VCE-004.8 downregulated the expression of several key genes associated with fibrosis, qualifying this semi-synthetic cannabinoid as a novel compound for the management of scleroderma and, potentially, other fibrotic diseases.

Systemic sclerosis (SSc) is a chronic autoimmune disease with a high morbidity and mortality. Skin and organ fibrosis are key manifestations of SSc, for which no generally accepted therapy is available. Thus, there is a high unmet need for novel anti-fibrotic therapeutic strategies in SSc. At the same time, important progress has been made in the identification and characterization of potential molecular targets in fibrotic diseases over the recent years. In this review, we have selected four targeted therapies, which are tested in clinical trials in SSc, for in depths discussion of their preclinical characterization. Soluble guanylate cyclase (sGC) stimulators such as riociguat might target both vascular remodeling and tissue fibrosis. Blockade of interleukin-6 might be particularly promising for early inflammatory stages of SSc. Inhibition of serotonin receptor 2b signaling links platelet activation to tissue fibrosis. Targeting simultaneously multiple key molecules with the multityrosine kinase-inhibitor nintedanib might be a promising approach in complex fibrotic diseases such as SSc, in which many partially independent pathways are activated. Herein, we also give a state of the art overview of the current classification, clinical presentation, diagnostic approach, and treatment options of localized scleroderma. Finally, we discuss whether the novel targeted therapies currently tested in SSc could be used for localized scleroderma.

Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-β induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Radiation-induced morphea is a rare complication of radiation therapy. The affected areas are generally restricted to the radiation field or to the nearby surrounding area., Case Report: A 67-year-old Japanese woman with a history of right breast cancer followed by adjuvant radiotherapy was referred our hospital because of 7-year history of symmetrical indurated erythematous plaques on her trunk. Three months after completion of irradiation, erythematous plaques developed on her right chest and gradually spread accompanied tenderness. She did not have a history of trauma to her right chest. Laboratory testing was positive for antinuclear antibody test at 1: 640 but negative for anti-SS-A/B, anti-U1-RNP, anti-DNA, anti-Sm, anticentromere, anti-topoisomerase I antibodies, and Borrelia and cytomegalovirus infection. She had no Raynaud's phenomenon, sclerodactyly, or nail-fold bleeding. She did not have interstitial lung disease or other internal organ involvement. A biopsy specimen revealed reticular dermal fibrosis with thickened collagen bundles with superficial and deep perivascular infiltration of mononuclear cells. These findings were consistent with morphea. Furthermore, mucin deposition was present in the papillary dermis upon Alcian blue staining, which has been reported to be observed in generalized morphea. Consequently, a diagnosis of generalized morphea induced by radiotherapy was made. She had been treated with oral hydroxychloroquine sulfate, resulting in the resolution of tenderness but the erythematous plaques remained., Conclusions: To the best of our knowledge, this is the first report of radiation-induced generalized morphea with prominent mucin deposition. Hydroxychloroquine sulfate may be efficacious for radiation-induced morphea-associated tenderness.

Anabolic steroids have been used to treat lower extremity ulcerations, including venous and cryofibrinogenemic ulcers and lipodermatosclerosis (LDS). Yet there have been no studies to determine the severity and reversibility of side effects of anabolic steroids on liver enzymes and lipid profiles in elderly patients. We therefore evaluated, in a prospective, randomized, double-blinded, placebo-controlled trial, the extent and reversibility of abnormal liver enzymes and lipid profiles in patients with LDS and venous leg ulcers treated with stanozolol at 2 mg twice daily for up to 6 months. Follow-up laboratory testing was done for 2 months after cessation of treatment. A total of 44 patients with LDS and venous ulcers were enrolled and treated with either leg compression alone (placebo) or leg compression plus oral stanozolol 2 mg twice daily (active). Baseline and follow-up laboratory testing of liver enzymes and lipid profiles were obtained. A total of 21 active and 23 placebo patients were treated and evaluated. We measured liver enzymes (aspartate aminotransferase [AST/SGOT], alanine aminotransferase [ALT/SGPT], γ-glutamyl transferase [GGT]) and lipid profile components (high-density lipoprotein [HDL], low-density lipoprotein [LDL], total cholesterol) before, during, and after the treatment period. We found that AST/SGOT and ALT/SGPT became significantly elevated in 29% (P = .0415 at 2 months) and 33% (P = .0182 at 1 month) of patients treated with stanozolol or placebo, respectively, with return to baseline in the posttreatment period. Unexpectedly, 91% of patients on stanozolol developed a significant (P < .0001) decrease in HDL levels, by as much as 37 U/L. All patients remained asymptomatic and levels returned to baseline after discontinuation of the drug. We conclude that low-dose stanozolol, 2 mg twice daily, produces asymptomatic and temporary elevation of liver transaminases and depression of the HDL level in a significant proportion of patients., (© The Author(s) 2015.)

Background: High interleukin (IL)-17A levels are characteristically found in the skin of systemic sclerosis (SSc) individuals. Our aim was to investigate whether the dermal expression of IL-17A and related IL-17 family members (i.e. IL-17C, IL-17E and IL-17F) could distinguish fibrotic from healthy skin and could show similarities in SSc and morphea, two disorders with presumed distinct pathogenesis, but characterized by skin fibrosis., Methods: Biopsies were obtained from the involved skin of 14 SSc, 5 morphea and 8 healthy donors (HD) undergoing plastic surgery. Immunohistochemistry/immunofluorescence techniques were coupled to a semi-automated imaging quantification approach to determine the presence of the IL-17 family members in the skin. The in vitro effects induced by the IL-17 family members on fibroblasts from normal and SSc individuals were assessed by ELISA and RIA., Results: Positive cells for each of the IL-17 isoforms investigated were present in the dermis of all the individuals tested, though with variable frequencies. SSc individuals had increased frequency of IL-17A+ (p = 0.0237) and decreased frequency of IL-17F+ (p = 0.0127) and IL-17C+ cells (p = 0.0008) when compared to HD. Similarly, morphea individuals had less frequent IL-17C+ cells (p = 0.0186) in their skin but showed similar number of IL-17A+ and IL-17F+ cells when compared to HD. Finally, IL-17E+ cells were more numerous in morphea (p = 0.0109) and tended to be more frequent in SSc than in HD. Fibroblast production of IL-6, MMP-1 and MCP-1 was enhanced in a dose-dependent manner in the presence of IL-17E and IL-17F, but not in the presence of IL-17C. None of the cytokine tested had significant effect on type I collagen production. Of interest, in SSc the frequency of both IL-17A and IL-17F positive cells increased with disease duration., Conclusions: The frequency of IL-17A and IL-17F distinguish SSc to morphea individuals while dermal expression of IL-17C (low) and IL-17E (high) identifies a fibrosis-specific motif. The specific IL-17C/IL-17E cytokine combination may thus play a role in the development of fibrosis.

Background: Localized scleroderma (LSc) exhibits fibrosis of the skin and subcutaneous tissue. LSc shows an excessive deposition of type 1 collagen., Objectives: To elucidate the mechanism of type 1 collagen overexpression in LSc, we investigated the epigenetics, focusing on microRNA (miRNA)., Materials & Methods: miRNA expression profile was determined by PCR array analysis. The expression of microRNA-196a (miR-196a) in the skin tissue was examined by in situ hybridization or real-time PCR. The serum levels of miR-196a were measured by real-time PCR., Results: PCR array analysis demonstrated that the miR-196a level was markedly decreased in LSc skin tissue in vivo. The transfection of specific inhibitor for miR-196a into normal cultured human dermal fibroblasts led to the up-regulation of type 1 collagen protein in vitro. Furthermore, the serum levels of miR-196a were significantly decreased in LSc patients., Conclusion: Down-regulation of miR-196a and subsequent overexpression of type 1 collagen in dermal fibroblasts may play a key role in the pathogenesis of LSc. The serum levels of miR-196a may be useful as a diagnostic marker of LSc.

Skin fibrosis is characterized by activated fibroblasts and an altered architecture of the extracellular matrix. Excessive deposition of extracellular matrix proteins and altered cytokine levels in the dermal collagen matrix are common to several pathological situations such as localized scleroderma and systemic sclerosis, keloids, dermatosclerosis associated with venous ulcers and the fibroproliferative tissue surrounding invasively growing tumors. Which factors contribute to altered organization of dermal collagen matrix in skin fibrosis is not well understood. We recently demonstrated that cartilage oligomeric matrix protein (COMP) functions as organizer of the dermal collagen I network in healthy human skin (Agarwal et al., 2012). Here we show that COMP deposition is enhanced in the dermis in various fibrotic conditions. COMP levels were significantly increased in fibrotic lesions derived from patients with localized scleroderma, in wound tissue and exudates of patients with venous leg ulcers and in the fibrotic stroma of biopsies from patients with basal cell carcinoma. We postulate enhanced deposition of COMP as one of the common factors altering the supramolecular architecture of collagen matrix in fibrotic skin pathologies. Interestingly, COMP remained nearly undetectable in normally healing wounds where myofibroblasts transiently accumulate in the granulation tissue. We conclude that COMP expression is restricted to a fibroblast differentiation state not identical to myofibroblasts which is induced by TGFβ and biomechanical forces., (© Elsevier B.V. All rights reserved.)

Background: Several lines of evidence suggest that the generation of reactive oxygen species (ROS) is of major importance in the pathogenesis of scleroderma, and thus antioxidant therapy may be useful for patients with an impaired oxidative defence mechanism., Aim: To examine the effect of N-acetylcysteine (NAC) on skin fibrosis and oxidative stress in a bleomycin (BLM)-induced mouse model of scleroderma., Methods: We used this mouse model to evaluate the effect of NAC on skin fibrosis and oxidative stress. Skin fibrosis was evaluated by histopathological examination and hydroxyproline content. To measure lipid peroxidation, we used a thiobarbituric acid-reactive species, malondialdehyde (MDA). Oxidative protein damage (carbonyl content) and the activities of catalase (CAT) and superoxide dismutase (SOD) were determined to evaluate oxidative stress in the skin tissue., Results: Treatment with NAC attenuated the skin fibrosis induced by BLM, significantly reducing the MDA and protein carbonyl content in these mice. SOD activity in BLM-only mice and BLM plus NAC-treated mice was increased compared with control mice. However, there was no significant difference in skin SOD activity of mice treated with both BLM and NAC compared with those treated with BLM only. In addition, CAT activity was not altered in the BLM plus NAC mice., Conclusions: NAC treatment attenuates skin fibrosis in a BLM-induced mouse model of scleroderma, and this is associated with diminished oxidative stress. The results suggest that NAC may be a potential therapeutic agent for patients with scleroderma., (© The Author(s) CED © 2013 British Association of Dermatologists.)

Objectives: Overexpression of vascular endothelial growth factor (VEGF) in scleroderma (SSc) skin may play a role in the pathogenesis of the disease. Our study was undertaken to evaluate whether dermal fibroblasts function as one of the sources of the increased VEGF in SSc, and to clarify its mechanism., Methods: Protein and mRNA levels of VEGF were analyzed using immunoblotting, enzyme-linked immunosorbent assay, and real-time PCR. The DNA-binding ability of Smad3 was evaluated by DNA affinity precipitation., Results: VEGF mRNA expression in vivo was increased in SSc skin compared to skin with other collagen diseases. Expression of VEGF protein and mRNA in cultured SSc dermal fibroblasts was constitutively and significantly upregulated. Ectopic TGF-β stimulation induced VEGF synthesis in normal fibroblasts, and TGF-β knockdown normalized the upregulated VEGF levels in SSc fibroblasts. Furthermore, Smad3 overexpression induced VEGF levels. We found that bp -532 to -521 on the VEGF promoter is a putative binding site for Smads, and that the binding activity of Smad3 to VEGF promoter was constitutively increased in SSc fibroblasts as well as in normal fibroblasts treated with exogenous TGF-β1., Conclusions: We demonstrated that VEGF were overexpressed due to autocrine TGF-β/Smad signaling in SSc. TGF-β signaling may contribute to the pathogenesis of angiopathy as well as tissue fibrosis.

Scleroderma is a skin disorder characterized by persistent fibrosis. Macrophage properties influencing cutaneous fibrogenesis remain to be fully elucidated. In this rat (F344 rats) model of scleroderma, at 1, 2, 3, and 4 weeks after initiation of daily subcutaneous injections of bleomycin (BLM; 100 μl of 1 mg/ml daily), skin samples were collected for histological and immunohistochemical evaluations. Immunohistochemically, the numbers of cells reacting to ED1 (anti-CD68; phagocytic activity) and ED2 (anti-CD163; inflammatory factor production) began to increase at week 1, peaked at week 2, and decreased thereafter. In contrast, the increased number of cells reacting to OX6 (anti-MHC class II molecules) was seen from week 2 and remained elevated until week 4. α-Smooth muscle actin-positive myofibroblasts were increased for 4 weeks. Double labeling revealed that galectin-3, a regulator of fibrogenic factor TGF-β1, was expressed in CD68+, CD163+, and MHC class II+ macrophages and myofibroblasts. mRNA expression of TGF-β1, as well as MCP-1 and CSF-1 (both macrophage function modulators), were significantly elevated at weeks 1 to 4. This study shows that the increased number of macrophages with heterogeneous immunophenotypes, which might be induced by MCP-1 and CSF-1, could participate in the sclerotic lesion formation, presumably through increased fibrogenic factors such as galectin-3 and TGF-β1; the data may provide useful information to understand the pathogenesis of the human scleroderma condition.

Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.

Introduction: The purpose of this study was to evaluate the presence and levels of interferon-gamma inducible protein-10 (IP-10) in the plasma and skin of pediatric localized scleroderma (LS) patients compared to those of healthy pediatric controls and to determine if IP-10 levels correlate to clinical disease activity measures., Methods: The presence of IP-10 in the plasma was analyzed using a Luminex panel in 69 pediatric patients with LS and compared to 71 healthy pediatric controls. Of these patients, five had available skin biopsy specimens with concurrent clinical and serological data during the active disease phase, which were used to analyze the presence and location of IP-10 in the skin by immunohistochemistry (IHC)., Results: IP-10 levels were significantly elevated in the plasma of LS patients compared to that of healthy controls and correlated to clinical disease activity measures in LS. Immunohistochemistry staining of IP-10 was present in the dermal infiltrate of LS patients and was similar to that found in psoriasis skin specimens, the positive disease control., Conclusions: Elevation of IP-10 levels in the plasma compared to those of healthy controls and the presence of IP-10 staining in the affected skin of LS patients indicates that IP-10 is a potential biomarker in LS. Furthermore, significant elevation of IP-10 in LS patients with active versus inactive disease and correlations between IP-10 levels and standardized disease outcome measures of activity in LS strongly suggest that IP-10 may be a biomarker for disease activity in LS.

Introduction: Adenosine, acting through the A(2A) receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A(2A) receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A(2A) receptor produces its fibrogenic effects., Methods: The effects of A(2A) receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A(2A) receptor stimulation on CTGF and TGF-β1 expression are also examined., Results: A(2A) receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A(2A) receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A(2A) receptor-mediated enhancement of collagen type I production. A(2A)R activation, however, resulted in a decrease in TGF-β1 protein release., Conclusions: Our results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A(2A) receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.

Background: A noncanonical pathway of transforming growth factor-β signalling, the c-Abl/protein kinase C-δ (PKC-δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts., Objectives: To investigate the significance of the c-Abl/PKC-δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc)., Methods: The activation status of the c-Abl/PKC-δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet-derived growth factor receptor inhibitor AG1296 and gene silencing of c-Abl on the expression levels of type I collagen were evaluated by immunoblotting., Results: The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c-Abl were elevated compared with cultured normal fibroblasts and PKC-δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c-Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts., Conclusions: Constitutive activation of the c-Abl/PKC-δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc., (© 2012 The Authors. BJD © 2012 British Association of Dermatologists.)

Background: Localized scleroderma (LS) is a disfiguring inflammatory autoimmune disease of the skin and underlying tissue. As in systemic sclerosis, a key feature is the presence of T cells in inflammatory lesions., Aim: To evaluate the effect of polymerized type I collagen vs. methylprednisolone (MP) in LS, and to determine the influence of this polymerized collagen (PC) on CD4+ peripheral T cells expressing interleukin (IL)-4, IL-17A, interferon-γ and Forkhead box protein (Foxp)3, and on cells expressing transforming growth factor (TGF)-β1, IL-17A, IL-22 and Foxp3 in the skin., Methods: In total, 16 patients with LS were treated for 3 months with monthly subcutaneous intralesional injections of 0.1 mL MP (giving a total dose of 20 mg/mL each month) and 15 patients were treated, with weekly subcutaneous intralesional injections of PC, ranging from 0.2 mL (equivalent to 1.66 mg collagen) for a lesion of 50 mm in size, up to a maximum of 1.0 mL (8.3 mg collagen) for a lesion > 100 mm in size, and followed up for a further 6 months. Skin biopsies were obtained from lesions at baseline (before treatment) and 9 months later (6 months after treatment end). Tissue sections were evaluated by histology and immunohistochemistry (IL-17A, IL-22, TGF-β1 and Foxp3). CD4+ T-cell subsets were determined in peripheral blood by flow cytometry., Results: Abnormal tissue architecture was seen in the biopsies taken from patients treated with MP, whereas the PC treatment restored normal skin architecture. PC downregulated pro-inflammatory/profibrotic cytokine expression in peripheral cells, and upregulated the number of regulatory T cells (Tregs) in skin. PC was safe and well tolerated., Conclusions: PC is not only an antifibrotic/fibrolytic agent but also an immunomodulator biodrug that restores the balance between T helper (Th)1, Th2, Th17 and Tregs, downregulates production of pro-inflammatory or profibrogenic cytokines (IL-17A, IL-22 and TGF-β1), and renews skin architecture, without adverse effects., (© The Author(s). CED © 2012 British Association of Dermatologists.)

Objective: Skin autofluorescence noninvasively assesses expression of advanced glycation endproducts and therefore potentially the presence of oxidative stress that is implicated in the pathogenesis of systemic sclerosis (SSc). We investigated whether autofluorescence was increased in patients with SSc, primary Raynaud's phenomenon (RP), and morphea as compared to healthy controls., Methods: Measurements of autofluorescence were made at 5 upper limb sites in 16 healthy controls, 16 patients with diffuse cutaneous SSc (dcSSc), 15 with limited cutaneous SSc (lcSSc), 15 with primary RP, and 13 with morphea. For patients with morphea, additional measurements were made at the affected and an adjacent unaffected site., Results: Autofluorescence was significantly increased in patients with dcSSc but not lcSSc as compared to controls at the proximal phalanx [dcSSc median 0.15, interquartile range (IQR) 0.10-0.24, vs control 0.10, IQR 0.07-0.13; p = 0.014], dorsum of the hand (dcSSc 0.17, IQR 0.11-0.36, vs control 0.12, IQR 0.09-0.17; p = 0.031), the wrist (dcSSc 0.22, IQR 0.13-0.33, vs control 0.13, IQR 0.09-0.18; p = 0.005), and forearm (dcSSc 0.19, IQR 0.12-0.47, vs control 0.14, IQR 0.10-0.16; p = 0.022). There was a trend for autofluorescence to be increased in patients with lcSSc and at morphea sites, compared to noninvolved skin., Conclusion: Autofluorescence is increased in patients with dcSSc compared to primary RP and to healthy controls. This suggests increased oxidative stress and the potential for autofluorescence as a biomarker.

Morphea is an autoimmune sclerotic skin disease of unknown pathogenesis. As type 1 interferons (IFN) have been implicated in the pathogenesis of systemic sclerosis, we proposed that type 1 IFN promote localized inflammation and fibrosis in morphea. To investigate the expression of the type 1 IFN-inducible protein myxovirus A (MxA) and the presence of plasmacytoid dendritic cells (pDC) in lesions of morphea, lesional skin of 10 patients with morphea was examined by immunohistochemistry for the presence of the type 1 IFN-inducible protein, myxovirus A (MxA), and the pDC markers, CD123 and BDCA-2, and was compared with lesional skin of cutaneous lupus erythematosus, lichen planus and keloid. Lesional and non-lesional morphea skin was compared. MxA was expressed in the epidermis as well as the reticular dermis and subcutis in morphea. pDCs were abundant around vessels and between fibrous bundles. Non-lesional biopsies demonstrated little or no expression of MxA and pDC markers. Keloid showed minimal expression of MxA and pDC markers. We demonstrate the expression of type 1 IFN-related protein MxA and plasmacytoid DCs in lesional but not in non-lesional biopsies of morphea. These findings suggest a potential role for type 1 interferons in the pathogenesis of morphea., (© 2012 John Wiley & Sons A/S.)

Introduction: The aim of this study was to explore the presence and localization of myocardial citrullination in samples from rheumatoid arthritis (RA) patients compared to rheumatic and non-rheumatic disease control groups., Methods: Archived myocardial samples obtained during autopsy from 1995 to 2009 were assembled into four groups: RA; scleroderma; fatal myocarditis; and non-rheumatic disease controls. Samples were examined by immunohistochemistry (IHC) for the presence and localization of citrullination and peptidyl arginine deiminase enzymes (PADs) by a single cardiovascular pathologist blinded to disease group and clinical characteristics., Results: Myocardial samples from seventeen RA patients were compared with those from fourteen controls, five fatal myocarditis patients, and ten scleroderma patients. Strong citrullination staining was detected exclusively in the myocardial interstitium in each of the groups. However, average and peak anti-citrulline staining was 59% and 44% higher, respectively, for the RA group compared to the combined non-RA groups (P < 0.05 for both comparisons). Myocardial fibrosis did not differ between the groups. In contrast to citrullination, PADs 1 to 3 and 6 were detected in cardiomyocytes (primarily PADs 1 and 3), resident inflammatory cells (primarily PADs 2 and 4), and, to a smaller extent, in endothelial cells and vascular smooth muscle cells. PAD staining did not co-localize with anti-citrulline staining in the interstitium and did not vary by disease state., Conclusions: Staining for citrullination was higher in the myocardial interstitium of RA compared to other disease states, a finding that could link autoimmunity to the known increase in myocardial dysfunction and heart failure in RA.

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

Background: T lymphocytes induce the transformation of fibroblasts into myofibroblasts, the main mediators of fibrogenesis. The inosine 5'-monophosphate dehydrogenase inhibitor mycophenolate mofetil (MMF) and the anti-CD25 monoclonal antibody daclizumab (DCZ) have been reported to suppress the proliferation of T lymphocytes., Aim: To evaluate the preventive effects of MMF and DCZ in early stages of bleomycin (BLM)-induced scleroderma., Methods: This study involved five groups of Balb/c mice (n = 10 per group). Mice in four of the groups were injected subcutaneously (SC) with BLM [100 μg/day in 100 μL phosphate-buffered saline (PBS)] for 4 weeks; the remaining (control) group received only 100 μL PBS. Three of the BLM-treated groups also received either intraperitoneal MMF 50 or 150 mg/kg/day, or SC DCZ 100 μg/week. At the end of the fourth week, all mice were killed, and blood and tissue samples were obtained for further analysis., Results: In the BLM-treated group, increases were seen in inflammatory-cell infiltration, α-smooth muscle actin-positive (α-SMA+) fibroblastic cell count, tissue hydroxyproline content, and dermal thickness. Dermal fibrosis was histopathologically prominent. In BLM-treated mice also given MMF or DCZ, inflammatory-cell infiltration, tissue hydroxyproline content and dermal thickness were decreased. In the MMF groups, decreases were also noted in α-SMA+ fibroblastic cell count., Conclusion: In this BLM-induced dermal fibrosis model, MMF and DCZ treatments prevented the development of dermal fibrosis. Further studies are needed to evaluate whether targeting T lymphocytes is effective in resolving pre-existing fibrosis in human scleroderma., (© The Author(s). CED © 2011 British Association of Dermatologists.)

Plaque-type morphea, the most common subtype of localized scleroderma (LS), is a connective tissue disease which is characterized by immunological dysregulation, vascular alterations, and skin fibrosis. We aimed to investigate the expression profiles of different cytokines and chemokines in patients with LS and healthy controls. Twenty patients with plaque-type morphea and 18 healthy controls were investigated. Skin biopsies were performed for real-time RT-PCR studies. Median mRNA of interleukin (IL)-6 was significantly higher expressed in LS than in normal skin. By contrast, median IL-1α mRNA levels were significantly decreased in LS as compared to controls. Median mRNA expression of CCL13, IL-1β, IL-2, and tumor necrosis factor-α did not significantly differ between LS lesions and healthy skin. However, we observed significantly increased median chemokine ligand 5/RANTES (CCL5/RANTES) and median chemokine receptor 7 (CCR7) mRNA expression in LS lesions as compared to healthy controls. CCL5/RANTES and CCR7 mRNA expression significantly correlated in LS lesions. We could confirm data of previous studies indicating that gene expression of IL-6 is up-regulated in LS lesions as compared to healthy skin. Moreover, we have shown for the first time a significant increase of mRNA levels of CCR7 and CCL5/RANTES in LS lesions indicating an important pathogenetic role of chemokines in LS., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Caveolin-1 (Cav-1), the principal structural component of caveolae, participates in the pathogenesis of several fibrotic diseases, including systemic sclerosis (SSc). Interestingly, affected skin and lung samples from patients with SSc show reduced levels of Cav-1, as compared to normal skin. In addition, restoration of Cav-1 function in skin fibroblasts from SSc patients reversed their pro-fibrotic phenotype. Here, we further investigated whether Cav-1 mice are a useful pre-clinical model for studying the pathogenesis of SSc. For this purpose, we performed quantitative transmission electron microscopy, as well as biochemical and immuno-histochemical analysis, of the skin from Cav-1 (-/-) null mice. Using these complementary approaches, we now show that skin from Cav-1 null mice exhibits many of the same characteristics as SSc skin from patients, including a decrease in collagen fiber diameter, increased tensile strength, and stiffness, as well as mononuclear cell infiltration. Furthermore, an increase in autophagy/mitophagy was observed in the stromal cells of the dermis from Cav-1 (-/-) mice. These findings suggest that changes in cellular energy metabolism (e.g., a shift towards aerobic glycolysis) in these stromal cells may be a survival mechanism in this "hostile" or pro-inflammatory microenvironment. Taken together, our results demonstrate that Cav-1 (-/-) null mice are a valuable new pre-clinical model for studying scleroderma. Most importantly, our results suggest that inhibition of autophagy and/or aerobic glycolysis may represent a new promising therapeutic strategy for halting fibrosis in SSc patients. Finally, Cav-1 (-/-) null mice are also a pre-clinical model for a "lethal" tumor micro-environment, possibly explaining the link between fibrosis, tumor progression, and cancer metastasis.