Clinical, Histologic, and Transcriptomic Evaluation of Sequential Fat Grafting for Morphea: A Nonrandomized Controlled Trial.

Importance: Morphea is a rare disease of unknown etiology without satisfactory treatment for skin sclerosis and soft tissue atrophy. Objective: To provide clinical, histologic, and transcriptome evidence of the antisclerotic and regenerative effects of sequential fat grafting with fresh fat and cryopreserved stromal vascular fraction gel (SVF gel) for morphea. Design, Setting, and Participants: This single-center, nonrandomized controlled trial was conducted between January 2022 and March 2023 in the Department of Plastic and Reconstructive Surgery of Nanfang Hospital, Southern Medical University and included adult participants with early-onset or late-onset morphea who presented with varying degrees of skin sclerosis and soft tissue defect. Interventions: Group 1 received sequential grafting of fresh fat and cryopreserved SVF gel (at 1 and 2 months postoperation). Group 2 received single autologous fat grafting. All patients were included in a 12-month follow-up. Main Outcome and Measures: The primary outcome included changes in the modified Localized Scleroderma Skin Severity Index (mLoSSI) and Localized Scleroderma Skin Damage Index (LoSDI) scores as evaluated by 2 independent blinded dermatologists. The histologic and transcriptome changes of morphea skin lesions were also evaluated. Results: Of 44 patients (median [IQR] age, 26 [23-33] years; 36 women [81.8%]) enrolled, 24 (54.5%) were assigned to group 1 and 20 (45.5%) to group 2. No serious adverse events were noted. The mean (SD) mLoSSI scores at 12 months showed a 1.6 (1.50) decrease in group 1 and 0.9 (1.46) in group 2 (P = .13), whereas the mean (SD) LoSDI scores at 12 months showed a 4.3 (1.34) decrease in group 1 and 2.1 (1.07) in group 2 (P < .001), indicating that group 1 had more significant improvement in morphea skin damage but not disease activity compared with group 2. Histologic analysis showed improved skin regeneration and reduced skin sclerosis in group 1, whereas skin biopsy specimens of group 2 patients did not show significant change. Transcriptome analysis of skin biopsy specimens from group 1 patients suggested that tumor necrosis factor α signaling via NFκB might contribute to the immunosuppressive and antifibrotic effect of sequential fat grafting. A total of 15 hub genes were captured, among which many associated with morphea pathogenesis were downregulated and validated by immunohistochemistry, such as EDN1, PAI-1, and CTGF. Conclusions and Relevance: The results of this nonrandomized trial suggest that sequential fat grafting with fresh fat and cryopreserved SVF gel was safe and its therapeutic effect was superior to that of single autologous fat grafting with improved mLoSSI and LoSDI scores. Histological and transcriptomic changes further support the effectiveness after treatment. Trial Registration: Chinese Clinical Trial Registry identifier: ChiCTR2200058003.

Skin biopsy analysis of concurrent keloidal morphoea and systemic sclerosis confirms overlapping pathogenic pathways.

OBJECTIVES: Although localised forms of scleroderma (morphoea) have very different clinical features and outcomes from systemic sclerosis the two conditions can occur together in some patients. In this study we have explored skin gene expression in a series of patients with keloidal morphoea, a distinct clinical variant, concurrently with systemic sclerosis. METHODS: We compared skin gene expression from the keloidal lesions with that from skin elsewhere. We also examined a series of patients with diffuse or limited cutaneous SSc without morphoea and some healthy control skin biopsies. RESULTS: Keloidal morphoea has a distinct gene expression signature that is mainly driven by differential expression of fibroblast-related genes compared with other cell types. Indeed, the signature reflects a profibrotic pattern seen in diffuse cutaneous SSc but is much more extreme. We propose that keloidal morphoea skin provides unique insight into the profibrotic population of cells driving dcSSc. CONCLUSIONS: Understanding the biology of keloidal morphoea may give valuable insight into the molecular and cellular pathology of systemic sclerosis. The discrete nature of keloidal lesions raises the possibility of haematogenous spread and we suggest that the driving cells could represent blood derived cells derived from circulating progenitors.

Gene Expression Signatures in Inflammatory and Sclerotic Morphea Skin and Sera Distinguish Morphea from Systemic Sclerosis.

Morphea is an inflammatory fibrotic disorder of the skin that has been likened to systemic sclerosis (SSc). We sought to examine the molecular landscape of morphea by examining lesional skin gene expression and blood biomarkers and comparing the gene expression profiles with those from site-matched nonlesional and SSc lesional skin. We found the morphea transcriptome is dominated by IFN-γ-mediated T helper 1 immune dysregulation, with a relative paucity of fibrosis pathways. Specifically, expression profiles of morphea skin clustered with the SSc inflammatory subset and were distinct from the those of SSc fibroproliferative subset. Unaffected morphea skin also differed from unaffected SSc skin because it did not exhibit pathological gene expression signatures. Examination of downstream IFN-γ-mediated chemokines, CXCL9 and CXCL10, revealed increased transcription in the skin but not in circulation. In contrast to transcriptional activity, CXCL9 was elevated in serum and was associated with active, widespread cutaneous involvement. Taken together, these results indicate that morphea is a skin-directed process characterized by T helper 1 immune-mediated dysregulation, which contrasts with fibrotic signatures and systemic transcriptional changes associated with SSc. The similarity between morphea and the inflammatory subset of SSc on transcriptional profiling indicates that therapies under development for this subset of SSc are also promising for treatment of morphea.

Serum vitamin D levels and vitamin D receptor gene ApaI and TaqI polymorphisms in patients with morphea: a case-control study.

A uncommon inflammatory condition called morphea causes fibrosis in the skin and subcutaneous tissue. The key stages in the pathophysiology are vascular damage, immunological response, and fibrosis. Numerous research have examined the relationships between the immune system, fibrosis, and vitamin D, but the exact pathogenetic pathways of morphea remain poorly understood. The purpose of this study was to investigate serum 25(OH)D levels and the ApaI (rs7975232) and TaqI (rs731236) polymorphisms of the vitamin D receptor (VDR) in morphea patients. There were 48 age- and sex-matched controls and 41 morphea patients total. VDR polymorphisms were found using PCR tests and gel electrophoresis, and serum 25(OH)D levels were determined using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The patient group consisted of 37 females (90.2%) and 4 males (9.8%). The patients' mean age was 38.68 ± 17.54 years. In terms of VDR ApaI and TaqI polymorphisms, there was no discernible difference between the patient and control groups. TaqI polymorphism heterozygosity was discovered in all patients with progressive disease, and this finding was statistically significant (p = 0.012). Patients' mean serum 25(OH)D levels were 16.98 ± 11.55 ng/mL, while those in the control group were 18.02 ± 14.30 ng/mL. VDR polymorphisms, vitamin D levels, disease subtype, age of onset, and responsiveness to treatment did not significantly correlate. In our research, we discovered that TaqI polymorphism may be related to the severity of the disease and that the polymorphisms of the VDR ApaI and TaqI were not associated with morphea susceptibility.

Genetic Association of VDR gene Apa1 and Taq1 Variants with Scleroderma in an Iranian Northeast Population.

BACKGROUND: Despite vigorous research efforts, the etiology of scleroderma (systemic sclerosis (SSc)) remains still unclear and both genetic and environmental factors clearly contribute to the pathogenesis of scleroderma. Reports of aberrant vitamin D status in scleroderma patients suggest a need for considering the genotype and allele frequencies of VDR gene polymorphisms. This case-control study aimed to investigate the possible association of two common polymorphisms of the VDR gene (ApaI, and TaqI) with susceptibility to scleroderma in an Iranian population. METHODS: Using polymerase chain reaction and restriction fragment length polymorphism (PCRRFLP), ApaI and TaqI polymorphisms in the VDR region were genotyped in 51 patients with scleroderma and 50 healthy controls. Logistic regression analysis was performed to calculate the genotypes odds ratios (ORs) as a measure of association with the presence of scleroderma. Haplotype and linkage disequilibrium analyses were also performed on the detected genotypes. RESULTS: No significant differences were found for the allelic and genotype distributions of ApaI and TaqI polymorphisms between patients with scleroderma and healthy controls (p>0.05). In haplotype analysis, three haplotypes TA, CA, and TC, with a frequency greater than 1% were identified. However, none of them was associated with the risk of scleroderma. CONCLUSION: Our preliminary study showed no evidence of an association between ApaI and TaqI polymorphisms and scleroderma. As the association between VDR polymorphisms and autoimmune diseases varies across the different ethnic populations, further large cohort studies are necessary to confirm the results.

Identification of lncRNA expression profiles in pediatric localized scleroderma.

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.

Clinical and laboratory characterization of patients with localized scleroderma and response to UVA-1 phototherapy: In vivo and in vitro skin models.

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.

The Immunogenetics of Morphea and Lichen Sclerosus.

Morphea and lichen sclerosis et atrophicus (LSA) are two distinct immune-mediated diseases with a dominant presentation of dermal fibrosis and sclerosis. The two diseases have many similar clinical and histological features and tend to co-occur. Both diseases are thought to result from a derailment of the normal response to environmental triggers. Positive family history is more common in LSA than morphea but individuals with morphea have a higher frequency of concomitant and familial autoimmunity. These findings hint at the involvement of inheritance in susceptibility to LSA and morphea and thus provide a rationale for exploring the disease genetics. This chapter contains a comprehensive review of the pathogenesis of the two diseases and their known genetic associations including HLA class I and II genes.

RNA sequencing of chronic GVHD skin lesions defines shared and unique inflammatory pathways characterizing lichen planus and morphea.

Cutaneous involvement of chronic graft-versus-host disease (cGVHD) has a wide range of manifestations including a lichenoid form with a currently assumed mixed Th1/Th17 signature and a sclerotic form with Th1 signature. Despite substantial heterogeneity of innate and adaptive immune cells recruited to the skin and of the different clinical manifestations, treatment depends mainly on the severity of the skin involvement and relies on systemic, high-dose glucocorticoids alone or in combination with a calcineurin inhibitor. We performed the first study using RNA sequencing to profile and compare the transcriptome of lichen planus cGVHD (n = 8), morphea cGVHD (n = 5), and healthy controls (n = 6). Our findings revealed shared and unique inflammatory pathways to each cGVHD subtype that are both pathogenic and targetable. In particular, the deregulation of IFN signaling pathway was strongly associated with cutaneous cGVHD, whereas the triggering receptor expressed on myeloid cells 1 pathway was found to be specific of lichen planus and likely contributes to its pathogenesis. The results were confirmed at a protein level by performing immunohistochemistry staining and at a transcriptomic level using real-time quantitative polymerase chain reaction.

Transcriptomic Evaluation of Juvenile Localized Scleroderma Skin With Histologic and Clinical Correlation.

OBJECTIVE: Juvenile localized scleroderma (LS) is an autoimmune disease of the skin whose pathogenesis is not well understood due to the rarity of the disease. This study was undertaken to determine the skin transcriptome in skin biopsy tissue from children with juvenile LS compared to pediatric healthy controls, with identification of significant molecular targets using RNA sequencing (RNA-Seq). In this study, differentially expressed genes (DEGs) were assessed for correlations with histopathologic and clinical features in children with juvenile LS, and were used to group the children into distinct genetic clusters based on immunophenotype. METHODS: RNA-Seq was performed on sections of paraffin-embedded skin tissue obtained from 28 children with juvenile LS and 10 pediatric healthy controls. RNA-Seq was carried out using an Illumina HTS TruSeq RNA Access library prep kit, with data aligned using STAR and data analysis using a DESeq2 platform. A standardized histologic scoring system was used to score skin sections for the severity of inflammation and levels of collagen deposition. Histologic scoring was completed by 2 pathologists who were blinded with regard to the status of each sample. Spearman's rank correlation coefficients were used to assess significant correlations between DEG expression profiles and skin histologic findings in patients with juvenile LS. RESULTS: We identified 589 significant DEGs in children with juvenile LS as compared to healthy controls. Hierarchical clustering was used to demonstrate 3 distinct juvenile LS immunophenotype clusters. The histologic scores of skin inflammation (based on numbers and categories of inflammatory cell infiltrates) were significantly correlated with the expression levels of HLA-DPB1, HLA-DQA2, HLA-DRA, and STAT1 genes (rs > 0.5, P < 0.01). Collagen thickness correlated with the expression levels of collagen organization genes as well as with genes found to be correlated with the severity of inflammation, including genes for major histocompatibility complex (MHC) class I, MHC class II, and interferon-γ signaling. CONCLUSION: Among children with juvenile LS, 3 distinct genetic signatures, or clusters, were identified. In one cluster, inflammation-related pathways were up-regulated, corresponding to the histologic skin inflammation score. In the second cluster, fibrosis-related pathways were up-regulated. In the third cluster, gene expression in the skin corresponded to the patterns seen in healthy controls. Up-regulation of HLA class II genes was observed within the first cluster (characterized by predominant inflammation), a feature that has also been observed in the peripheral blood of patients with morphea and in the skin of patients with systemic sclerosis.

Circulating miRNA-181b-5p, miRNA-223-3p, miRNA-210-3p, let 7i-5p, miRNA-21-5p and miRNA-29a-3p in patients with localized scleroderma as potential biomarkers.

Localized scleroderma (LoSc) is a rare disease manifested by an inflammation and sclerosis of the skin. The latest studies focused on glycoprotein Krebs von den Lungen-6, surfactant protein-D, chemokine ligand 18 and dipeptidylpeptidase 4 as potential biomarkers of skin fibrosis in systemic scleroderma. Our study aimed to identify 6 miRNAs with elevated or decreased levels in 38 LoSc patients (31 females, 7 males) compared to healthy volunteers (HVs) and to correlate the selected miRNAs' serum levels with the severity and the clinical symptoms of LoSc and some laboratory parameters with the selected miRNAs' serum levels. The serum levels of miRNAs, i.e. miRNA-181b-5p, miRNA-223-3p, miRNA-21-5p, let 7i-5p, miRNA-29a-3p and miRNA-210-3p were significantly increased in the LoSc patients compared to the HVs. The level of let-7i increase in the female LoSc patients correlated negatively with BSA (r = - 0.355, p = 0.049) and mLoSSI (r = - 0.432, p = 0.015). Moreover, the female patients with inactive LoSc had significantly higher level of let-7i (2.68-fold on average) in comparison to those with active disease (p = 0.045). The exact role of those molecules has not been revealed in LoSc and a long-term longitudinal research is pivotal to confirm their prognostic value.

Regulation of skin fibrosis by RALDH1-producing dermal dendritic cells via retinoic acid-mediated regulatory T cell induction: A role in scleroderma.

BACKGROUND: Skin fibrosis of systemic sclerosis (SSc) is believed to be driven by complex processes including immune abnormalities, but the underlying immune response remains enigmatic. In particular, the role of dermal dendritic cells (DCs) is totally unknown. OBJECTIVE: We investigated the impact of CD103 loss on bleomycin-induced skin fibrosis because CD103 is a critical molecule determining DC phenotypes. METHODS: Bleomycin-induced skin fibrosis was generated with Cd103-/- mice. The alterations of tissue fibrosis and related inflammation were investigated by histologic examination, hydroxyproline assay, quantitative reverse transcription PCR and flow cytometry. SSc skin samples were evaluated by immunofluorescence. RESULTS: CD103 loss decreased bleomycin-induced dermal thickness and collagen contents, along with TGF-ß1 and CTGF suppression. Treg proportion was increased, while Th1/Th2/Th17 cell proportions were decreased in the skin of bleomycin-treated Cd103-/- mice. Bleomycin injection enhanced CD11b-CD103- DC proportion in wild-type mice, which was further augmented in Cd103-/- mice. Importantly, RALDH1/ALDH1A1 enzyme oxidizing retinaldehyde to retinoic acid, an inducer of Tregs, was preferentially expressed by CD11b-CD103- DCs and its expression levels were elevated in bleomycin-injected skin lesions, to a greater extent in Cd103-/- mice than in wild-type mice. Importantly, the number of RALDH1-positive DCs was decreased in the lesional skin of SSc patients and tended to inversely correlate with skin fibrosis severity. CONCLUSION: This study identified a critical role of dermal DCs as a regulator of Treg development through RALDH1 in bleomycin-treated mice and possibly in human SSc. This finding sheds new light on dermal DCs as a new therapeutic target of SSc.

MicroRNA in localized scleroderma: a review of literature.

Localized scleroderma (LoSc) is rare connective tissue disease that manifests with inflammation and fibrosis of the skin. Depending on the LoSc subtype, adjacent structures such as subcutaneous tissue, fascia, muscles, bones may be affected. The hallmark of fibrosis is tissue remodelling with excess deposition of extracellular matrix proteins (ECM), principally collagens. MicroRNAs (miRNAs) are small, noncoding RNA molecules that consist of 19-24 nucleotides and act as negative regulators of gene expression at the posttranscriptional level. Based on the current articles, approximately 40 microRNAs have been linked to fibrosis in different organs and diseases. The majority of these molecules promote or inhibit fibrosis by targeting connective tissue growth factor (CTGF), extracellular matrix proteins, TGF-ß pathway and MAPK (mitogen-activated protein kinase) pathway. Further, particular microRNAs regulate fibrogenesis by altering epithelial-to-mesenchymal transition (EMT) or activating proliferation of myofibroblasts. MiRNAs are relatively stable, detectable in tissues and body fluids (serum, plasma) which suggest that they may serve as beneficial biomarkers to monitor the course of the disease and response to treatment. Herein, we report the present state of knowledge on microRNA expression in localized scleroderma.

Immunopathogenesis of Pediatric Localized Scleroderma.

Localized scleroderma (LS) is a complex disease characterized by a mixture of inflammation and fibrosis of the skin that, especially in the pediatric population, also affects extracutaneous tissues ranging from muscle to the central nervous system. Although developmental origins have been hypothesized, evidence points to LS as a systemic autoimmune disorder, as there is a strong correlation to family history of autoimmune disease, the presence of shared HLA types with rheumatoid arthritis, high frequency of auto-antibodies, and elevated circulating chemokines and cytokines associated with T-helper cell, IFNγ, and other inflammatory pathways. This inflammatory phenotype of the peripheral blood is reflected in the skin via microarray, RNA Sequencing and tissue staining. Research is underway to identify the key players in the pathogenesis of LS, but close approximation of inflammatory lymphocytic and macrophage infiltrate with collagen and fibroblasts deposition supports the notion that LS is a disease of inflammatory driven fibrosis. The immune system is dynamic and undergoes changes during childhood, and we speculate on how the unique features of the immune system in childhood could potentially contribute to some of the differences in LS between children and adults. Interestingly, the immune phenotype in pediatric LS resembles to some extent the healthy adult cellular phenotype, possibly supporting accelerated maturation of the immune system in LS. We discuss future directions in better understanding the pathophysiology of and how to better treat pediatric LS.

Withaferin A attenuates bleomycin-induced scleroderma by targeting FoxO3a and NF-κß signaling: Connecting fibrosis and inflammation.

Scleroderma is an inflammatory autoimmune disease which begins with inflammation due to tissue injury and advances to progressive accumulation of extracellular matrix resulting in scarring and hardening of the skin. Inflammation is a salutary response to tissue injury caused by varied factors. While inflammation is required for systematic wound healing, dysregulated chronic inflammation often leads to tissue scarring. Prominent role of inflammation in pathology and physiology makes it a double edge sword. The objective of this study was to investigate the role of Withaferin A (WFA), a steroidal lactone from Withania somnifera in a 28-day murine model of bleomycin-induced experimental scleroderma. Withaferin A was administered at two doses 2 and 4 mg/kg intraperitoneally for 28 days. At the time of study termination, we observed significant reduction in dorsal skin thickness. Our results indicate that WFA was able to sufficiently suppress pro-inflammatory phase of fibrosis, TGF-ß/Smad signaling and also significantly repressed fibroblast conversion to myofibroblasts. Additionally, our study also demonstrated that WFA modulates FoxO3a-Akt-dependent NF-κß/IKK-mediated inflammatory cascade, which is a prime signaling pathway in fibrogenesis. The findings of this study are persuasive of WFA as an antifibrotic agent with promising therapeutic effects in scleroderma. © 2018 BioFactors, 44(6):507-517, 2018.

Juvenile Scleroderma-What has Changed in the Meantime?

BACKGROUND: Juvenile scleroderma is a rarely seen chronic connective tissue disorder characterized by stiffening of the skin. The frequency of the disease was reported as one per million. According to organ involvement, the disease is divided into two main forms: systemic and localized scleroderma. OBJECTIVE: Since it is uncommon in children, many aspects of the disease remain discussable. With this review, we aimed to revise recent findings and new developments in this rare condition. METHOD: A systematic literature research was performed, using the following medical databases: Pubmed/ Medline and the Cochrane Library. We searched for up-to-date randomized controlled studies, case-control studies, and cohort studies and cases reports on juvenile scleroderma (both systemic and localized form). RESULTS: Skin manifestations are most prominent features of the systemic form, followed by musculoskeletal and vascular involvement. Cardiovascular, gastrointestinal and renal disorders are rare in childhood. Combination of disease modifying anti- rheumatic drugs (methotrexate, mycophenolatemofetil, cyclosporine) and steroid reprents the first line therapy. Bosentan is used for cases with pulmonary hypertension and for extensive digital ulcerations. Biological treatment emerges as a useful treatment option in most severe form of the disease. Localized scleroderma is characterized with sclerodermatosis of the skin. Internal organ involvement is not expected. Classification of the local scleroderma is made according to the size and localization of the skin changes. There are few different therapeutical options but there is no specific therapy for the localized scleroderma. CONCLUSION: Many data regarding disease features and treatment options in juvenile scleroderma are based on studies among adults. There is a striking need for multicentric, prospective studies among children with juvenile scleroderma, in order to elucidate some questions of clinical course and disease prognosis. Recent genetic studies have revealed the role of the genetic factors (namely HLA class II) in the pathogenesis of the disease. Emerging biological agents and new treatment options are showing promising results.