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Rationale: Fibrotic response to lung injury depends on development of a fibrogenic population of myofibroblasts. The importance of resident interstitial fibroblasts and role of transforming growth factor β (TGFβ) in this process is unclear.Objectives: To define the importance of TGFβ signaling in resident lung fibroblasts in the development of experimental pulmonary fibrosis.Methods: A compound genetic strategy in which mice homozygous for a floxed high-affinity type II TGFβ receptor (TβRII) allele were crossed with a transgenic strain harboring a fibroblast-specific transgene encoding ligand-dependent Cre-recombinase was used. TβRII was deleted by postnatal administration of tamoxifen over 5 days to compound mutant mice with appropriate littermate controls. Illumina microarray gene profiling and quantitative reverse transcriptase-polymerase chain reaction were used to confirm anergy to TGFβ in explanted lung fibroblasts. Bleomycin lung injury was used to induce lung fibrosis, which was analyzed by histology and biochemical methods. Immunofluorescence was used to define cell populations after lung injury.Measurements and Main Results: There was significant attenuation of fibrosis in mice after deletion of TβRII in resident fibroblasts. At 7 days after injury the number of fibrocytes and myofibroblasts was substantially reduced. Potential regulators of fibrosis were suggested by gene expression profiles that identified key candidate profibrotic genes, including connective tissue growth factor and endothelin-1 expressed by wild-type but not mutant lung fibroblasts.Conclusions: Intact TGFβ signaling in resident pulmonary fibroblasts is essential for pulmonary fibrosis to develop. Our data support a key regulatory role of these cells in determining fibrocyte recruitment and myofibroblast differentiation. [ABSTRACT FROM AUTHOR]

OBJECTIVE: There is increasing evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in controlling cell differentiation, and that PPARgamma ligands can modify inflammatory and fibrotic responses. The aim of the present study was to examine the role of PPARgamma in a mouse model of skin scleroderma, in which mice bearing a fibroblast-specific deletion of PPARgamma were used. METHODS: Cutaneous sclerosis was induced by subcutaneous injection of bleomycin, while untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast-specific deletion of PPARgamma were investigated for changes in dermal thickness, inflammation, collagen content, and the number of alpha-smooth muscle actin-positive cells. The quantity of the collagen-specific amino acid hydroxyproline was also measured. In addition, the effect of PPARgamma deletion on transforming growth factor beta1 (TGFbeta1) signaling in the fibroblasts was investigated. RESULTS: Bleomycin treatment induced marked cutaneous thickening and fibrosis in all treated mice. Deletion of PPARgamma resulted in enhanced susceptibility to bleomycin-induced skin fibrosis, as indicated by increases in all measures of skin fibrosis and enhanced sensitivity of fibroblasts to TGFbeta1 in PPAR-deficient mice. CONCLUSION: These results indicate that PPARgamma suppresses fibrogenesis. Specific agonists of PPARgamma may therefore alleviate the extent of the development of cutaneous sclerosis. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Activated adhesive signaling is a hallmark of fibroblasts isolated from the scars of scleroderma (systemic sclerosis) lesions. Beta-1 integrin plays a key role in adhesive signaling. The aim of the present study was to examine the role of beta1 integrin in a mouse model of skin scleroderma using mice bearing a fibroblast-specific deletion of beta1 integrin. METHODS: Cutaneous sclerosis was induced by subcutaneous injection of bleomycin. Control groups were treated with phosphate buffered saline. Mice bearing a fibroblast-specific deletion of beta1 integrin and control mice were investigated. Dermal thickness, collagen production, and the number of alpha-smooth muscle actin-positive cells were determined. The quantity of the collagen-specific amino acid hydroxyproline was also measured. RESULTS: Bleomycin treatment induced marked cutaneous thickening and fibrosis in control mice. Conversely, the deletion of beta1 integrin resulted in resistance to bleomycin-induced fibrosis. CONCLUSION: Expression of beta1 integrin by fibroblasts is required for fibrogenesis. Inhibition of beta1 integrin may be a viable method to alleviate the development of cutaneous sclerosis. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor beta (TGFbeta) and is a mediator of some profibrotic effects of TGFbeta in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I alpha2) in this mouse model and in human pulmonary fibroblasts. METHODS: Transgenic mice that were carrying luciferase and beta-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts. RESULTS: In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by approximately 25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling. CONCLUSION: Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Fibrosis is believed to occur through normal tissue remodeling failing to terminate. Tissue repair intimately involves the ability of fibroblasts to contract extracellular matrix (ECM), and enhanced ECM contraction is a hallmark of fibrotic cells in various conditions, including scleroderma. Some fibrogenic transcriptional responses to transforming growth factor beta (TGFbeta), including alpha-smooth muscle actin (alpha-SMA) expression and ECM contraction, require focal adhesion kinase/Src (FAK/Src). The present study was undertaken to assess whether TGFbeta-activated kinase 1 (TAK1) acts downstream of FAK/Src to mediate fibrogenic responses in fibroblasts. METHODS: We used microarray, real-time polymerase chain reaction, Western blot, and collagen gel contraction assays to assess the ability of wild-type and TAK1-knockout fibroblasts to respond to TGFbeta1. RESULTS: The ability of TGF to induce TAK1 was blocked by the FAK/Src inhibitor PP2. JNK phosphorylation in response to TGFbeta1 was impaired in the absence of TAK1. TGFbeta could not induce matrix contraction or expression of a group of fibrotic genes, including alpha-SMA, in the absence of TAK1. CONCLUSION: These results suggest that TAK1 operates downstream of FAK/Src in mediating fibrogenic responses and that targeting of TAK1 may be a viable antifibrotic strategy in the treatment of certain disorders, including scleroderma. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To explore increased susceptibility to fibrosis following experimental injury to alveolar epithelial cells (AECs) in a novel transgenic mouse model of scleroderma with fibroblast-specific perturbation of transforming growth factor beta (TGFbeta) signaling (TbetaRIIDeltak-fib mice). METHODS: Wild-type (WT) and transgenic mice were injured with intratracheally administered saline or bleomycin, and the lungs were harvested for biochemical, histologic, and electron microscopic analysis. RESULTS: Electron microscopy revealed AEC abnormalities in the lungs of untreated transgenic mice and bleomycin-treated WT mice; the lungs of transgenic mice treated with bleomycin showed severe epithelial damage. Compared with lungs from bleomycin-treated WT mice, lungs from bleomycin-treated transgenic mice demonstrated increased fibroproliferation, myofibroblast persistence, and impaired hyperplasia and increased apoptosis of type II AECs. The lungs from saline-treated transgenic mice and those from bleomycin-treated WT mice had phenotypic similarities, suggesting enhanced susceptibility to minor epithelial injury in the transgenic strain. The level of collagen was increased in the lungs from transgenic mice compared with that in the lungs from WT mice after treatment with either bleomycin or saline. Persistent fibrosis in bleomycin-treated transgenic mice was independent of ongoing neutrophil inflammation but was associated with impaired alveolar epithelial repair. CONCLUSION: These results suggest that in the context of fibroblast-specific perturbation of TGFbeta signaling, even minor epithelial injury induces significant fibrosis. The model supports a central role for TGFbeta in determining fibrosis and demonstrates that lung fibroblasts may regulate the response of AECs to injury. Our findings provide insight into likely pathogenic mechanisms in scleroderma-associated pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Fibrosis is excessive scarring caused by the accumulation and contraction of extracellular matrix proteins and is a common end pathway in many chronic diseases, including scleroderma (systemic sclerosis [SSc]). Indeed, pulmonary fibrosis is a major cause of death in SSc. Transforming growth factor beta (TGFbeta) induces endothelin 1 (ET-1) in human lung fibroblasts by a Smad-independent, JNK-dependent mechanism. The goal of this study was to assess whether ET-1 is a downstream mediator of the profibrotic effects of TGFbeta in lung fibroblasts. METHODS: We used a specific endothelin receptor antagonist to determine whether ET-1 is a downstream mediator of TGFbeta responses in lung fibroblasts, using microarray technology, real-time polymerase chain reaction, and Western blot analyses. RESULTS: The ability of TGFbeta to induce the expression of a cohort of profibrotic genes, including type I collagen, fibronectin, and CCN2, and to contract a collagen gel matrix, depends on ET-1. CONCLUSION: ET-1 contributes to the ability of TGFbeta to promote a profibrotic phenotype in human lung fibroblasts, consistent with the notion that endothelin receptor antagonism may be beneficial in controlling fibrogenic responses in lung fibroblasts. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To use a specific transforming growth factor beta receptor type I (TGFbetaRI; activin receptor-like kinase 5 [ALK-5]) kinase inhibitor (SD208) to determine the role of activation of the TGFbetaRI kinase (ALK-5) in maintaining the profibrotic phenotype of dermal fibroblasts in systemic sclerosis (SSc). METHODS: The effect of SD208 on the expression of key biochemical markers of the fibrotic phenotype was compared in fibroblasts cultured from clinically involved (lesional) and clinically uninvolved skin of patients with diffuse cutaneous SSc (dcSSc) and in fibroblasts from healthy controls matched for age, sex, and anatomic site. Protein expression was compared together with the ability of fibroblasts to adhere to the extracellular matrix and to remodel and contract a free-floating fibroblast-populated type I collagen lattice. RESULTS: Inhibiting TGFbetaRI kinase reduced the expression of a cohort of fibrotic markers by dermal fibroblasts from patients with dcSSc, including type I collagen and beta1 integrin. Moreover, inhibition also attenuated the elevated adhesive and contractile abilities of dcSSc fibroblasts. CONCLUSION: Our data suggest that some of the key profibrotic features of lesional SSc fibroblasts are dependent upon ALK-5 activity. Thus, TGFbetaRI kinase-mediated signaling may contribute to dermal fibrosis in dcSSc. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Endothelin 1 (ET-1) has been implicated in the pathogenesis of fibrotic and inflammatory diseases, including scleroderma. In addition to modulating vascular tone and extracellular matrix turnover, ET-1 up-regulates cell surface adhesion molecules including intercellular adhesion molecule 1 (ICAM-1), which is key to cell-cell and cell-matrix adhesion and leukocyte infiltration. This study was undertaken to delineate the signal transduction pathways utilized by ET-1 and compare them with those adopted by proinflammatory cytokine interleukin-1beta (IL-1beta) in normal and scleroderma dermal fibroblasts. METHODS: Protein expression induced by ET-1 and IL-1beta on normal dermal fibroblasts, with or without signaling inhibitors, was detected by enzyme-linked immunosorbent assay, while messenger RNA (mRNA) levels were analyzed by LightCycler polymerase chain reaction. Expression of protein kinase Cdelta (PKCdelta) and PKCepsilon protein in normal dermal fibroblasts and scleroderma dermal fibroblasts was determined by Western blotting, and PKCepsilon involvement in ET-1 signaling was confirmed through transfection of an ICAM-1 promoter construct into murine PKCepsilon-/- fibroblasts. NF-kappaB activation was confirmed via electrophoretic mobility supershift assay, and analysis of the ICAM-1 promoter region was achieved via transfection of deletion constructs into human dermal fibroblasts. RESULTS: In normal dermal fibroblasts, ET-1 induced ICAM-1 mRNA and surface protein expression in a dose- and time-dependent manner via both receptor subtypes, ET(A) and ET(B); antagonism of both abolished the ET-1 response. MEK was involved in the signaling cascade, but phosphatidylinositol 3-kinase and p38 MAPK were not. Key to the cascade was activation of NF-kappaB, achieved by ligation of either receptor subtype. PKCepsilon activation led to downstream activation of MEK and, in part, NF-kappaB. IL-1beta signaling required NF-kappaB and MEK activation, along with activation of PKCdelta. ET-1 and IL-1beta each utilized the same ICAM-1 promoter region and the same NF-kappaB site at -157 bp. Responses to ET-1 and IL-1beta differed in scleroderma dermal fibroblasts, with ET-1 sensitivity decreasing and IL-1beta responses remaining intact. Expression of PKCepsilon and PKCdelta in scleroderma dermal fibroblasts was also altered. CONCLUSION: The findings of this study indicate that differences in sensitivity to ET-1 and IL-1beta in scleroderma dermal fibroblasts may be explained by altered expression of the PKC isoforms and cytokine receptors. [ABSTRACT FROM AUTHOR]

Background: Systemic sclerosis (scleroderma) is a life-threatening autoimmune disease that is characterized by the presence of specific autoantibodies and fibrosis of the skin and major internal organs.Methods: We genotyped a polymorphism (G-945C) in the promoter of the connective-tissue growth factor (CTGF) gene in 1000 subjects in two groups: group 1, consisting of 200 patients with systemic sclerosis and 188 control subjects; and group 2, consisting of 300 patients with systemic sclerosis and 312 control subjects. The combined groups represented an estimated 10% of patients with systemic sclerosis in the United Kingdom. We tested the effect of the polymorphism on the transcription of CTGF.Results: The GG genotype was significantly more common in patients with systemic sclerosis than in control subjects in both groups, with an odds ratio for the combined group of 2.2 (95% confidence interval [CI], 1.5 to 3.2; P<0.001 for trend). Analysis of the combined group of patients with systemic sclerosis showed a significant association between homozygosity for the G allele and the presence of anti-topoisomerase I antibodies (odds ratio, 3.3; 95% CI, 2.0 to 5.6; P<0.001) and fibrosing alveolitis (odds ratio, 3.1; 95% CI, 1.9 to 5.0; P<0.001). We observed that the substitution of cytosine for guanine created a binding site of the transcriptional regulators Sp1 and Sp3. The C allele has high affinity for Sp3 and is associated with severely reduced transcriptional activity. A chromatin immunoprecipitation assay showed a marked shift in the ratio of Sp1 to Sp3 binding at this region, demonstrating functional relevance in vivo.Conclusions: The G-945C substitution represses CTGF transcription, and the -945G allele is significantly associated with susceptibility to systemic sclerosis. [ABSTRACT FROM AUTHOR]

NKX2-5 is a member of the homeobox-containing transcription factors critical in regulating tissue differentiation in development. Here, we report a role for NKX2-5 in vascular smooth muscle cell phenotypic modulation in vitro and in vascular remodeling in vivo. NKX2-5 is upregulated in scleroderma patients with pulmonary arterial hypertension. Suppression of NKX2-5 expression in smooth muscle cells halted vascular smooth muscle proliferation and migration, enhanced contractility, and blocked the expression of extracellular matrix genes. Conversely, overexpression of NKX2-5 suppressed the expression of contractile genes (ACTA2, TAGLN, CNN1) and enhanced the expression of matrix genes (COL1) in vascular smooth muscle cells. In vivo, conditional deletion of NKX2-5 attenuated blood vessel remodeling and halted the progression to hypertension in a mouse chronic hypoxia model. This study revealed that signals related to injury such as serum and low confluence, which induce NKX2-5 expression in cultured cells, is potentiated by TGF-β and further enhanced by hypoxia. The effect of TGF-β was sensitive to ERK5 and PI3K inhibition. Our data suggest a pivotal role for NKX2-5 in the phenotypic modulation of smooth muscle cells during pathological vascular remodeling and provide proof of concept for therapeutic targeting of NKX2-5 in vasculopathies.

Autologous fat transfers show promise in treating fibrotic skin diseases, reversing scarring and stiffness, and improving quality of life. Adipose-derived stem cells (ADSCs) within these grafts are believed to be crucial for this effect, particularly their secreted factors, though the specific mechanisms remain unclear. This study investigates transcriptomic changes in ADSCs after in vitro fibrotic, inflammatory, and hypoxic conditioning. High-throughput gene expression assays were conducted on ADSCs exposed to IL1-β, TGF-β1, and hypoxia and in media with fetal bovine serum (FBS). Flow cytometry characterized the ADSCs. RNA-Seq analysis revealed distinct gene expression patterns between the conditions. FBS upregulated pathways were related to the cell cycle, replication, wound healing, and ossification. IL1-β induced immunomodulatory pathways, including granulocyte chemotaxis and cytokine production. TGF-β1 treatment upregulated wound healing and muscle tissue development pathways. Hypoxia led to the downregulation of mitochondria and cellular activity.

Background: Morphoea can have a significant disease burden. Aetiopathogenesis remains poorly understood, with very limited existing genetic studies. Linear morphoea (LM) may follow Blascho's lines of epidermal development, providing potential pathogenic clues., Objective: The first objective of this study was to identify the presence of primary somatic epidermal mosaicism in LM. The second objective was tTo explore differential gene expression in morphoea epidermis and dermis to identify potential pathogenic molecular pathways and tissue layer cross-talk., Methodology: Skin biopsies from paired affected and contralateral unaffected skin were taken from 16 patients with LM. Epidermis and dermis were isolated using a 2-step chemical-physical separation protocol. Whole Genome Sequencing (WGS; n = 4 epidermal) and RNA-seq (n = 5-epidermal, n = 5-dermal) with gene expression analysis via GSEA-MSigDBv6.3 and PANTHER-v14.1 pathway analyses, were performed. RTqPCR and immunohistochemistry were used to replicate key results., Results: Sixteen participants (93.8% female, mean age 27.7 yrs disease-onset) were included. Epidermal WGS identified no single affected gene or SNV. However, many potential disease-relevant pathogenic variants were present, including ADAMTSL1 and ADAMTS16. A highly proliferative, inflammatory and profibrotic epidermis was seen, with significantly-overexpressed TNFα-via-NFkB, TGFβ, IL6/JAKSTAT and IFN-signaling, apoptosis, p53 and KRAS-responses. Upregulated IFI27 and downregulated LAMA4 potentially represent initiating epidermal 'damage' signals and enhanced epidermal-dermal communication. Morphoea dermis exhibited significant profibrotic, B-cell and IFN-signatures, and upregulated morphogenic patterning pathways such as Wnt., Conclusion: This study supports the absence of somatic epidermal mosaicism in LM, and identifies potential disease-driving epidermal mechanisms, epidermal-dermal interactions and disease-specific dermal differential-gene-expression in morphoea. We propose a potential molecular narrative for morphoea aetiopathogenesis which could help guide future targeted studies and therapies., (© 2023. The Author(s).)

Adipose-derived stem cells (ADSCs) as part of autologous fat grafting have anti-fibrotic and anti-inflammatory effects, but the exact mechanisms of action remain unknown. By simulating the interaction of ADSCs with fibroblasts and endothelial cells (EC) from scleroderma (SSc) skin in silico, we aim to unravel these mechanisms. Publicly available single-cell RNA sequencing data from the stromal vascular fraction of 3 lean patients and biopsies from the skin of 10 control and 12 patients with SSc were obtained from the GEO and analysed using R and Seurat. Differentially expressed genes were used to compare the fibroblast and EC transcriptome between controls and SSc. GO and KEGG functional enrichment was performed. Ligand-receptor interactions of ADSCs with fibroblasts and ECs were explored with LIANA. Pro-inflammatory and extracellular matrix (ECM) interacting fibroblasts were identified in SSc. Arterial, capillary, venous and lymphatic ECs showed a pro-fibrotic and pro-inflammatory transcriptome. Most interactions with both cell types were based on ECM proteins. Differential interactions identified included NTN1 , VEGFD , MMP2 , FGF2, and FNDC5 . The ADSC secretome may disrupt vascular and perivascular inflammation hubs in scleroderma by promoting angiogenesis and especially lymphangiogenesis. Key phenomena observed after fat grafting remain unexplained, including modulation of fibroblast behaviour.

Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-β, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Beesley, Goldman, Taher, Denton, Abraham, Mageed and Ong.)

Background: The TβRII∆k-fib transgenic (TG) mouse model of scleroderma replicates key fibrotic and vasculopathic complications of systemic sclerosis through fibroblast-directed upregulation of TGFβ signalling. We have examined peroxisome proliferator-activated receptor (PPAR) pathway perturbation in this model and explored the impact of the pan-PPAR agonist lanifibranor on the cardiorespiratory phenotype., Methods: PPAR pathway gene and protein expression differences from TG and WT sex-matched littermate mice were determined at baseline and following administration of one of two doses of lanifibranor (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. The prevention of bleomycin-induced lung fibrosis and SU5416-induced pulmonary hypertension by lanifibranor was explored., Results: Gene expression data were consistent with the downregulation of the PPAR pathway in the TβRII∆k-fib mouse model. TG mice treated with high-dose lanifibranor demonstrated significant protection from lung fibrosis after bleomycin and from right ventricular hypertrophy following induction of pulmonary hypertension by SU5416, despite no significant change in right ventricular systolic pressure., Conclusions: In the TβRII∆k-fib mouse strain, treatment with 100 mg/kg lanifibranor reduces the development of lung fibrosis and right ventricular hypertrophy induced by bleomycin or SU5416, respectively. Reduced PPAR activity may contribute to the exaggerated fibroproliferative response to tissue injury in this transgenic model of scleroderma and its pulmonary complications., (© 2021. The Author(s).)

Background and Objective: The course of systemic sclerosis-associated interstitial lung disease (SSc-ILD) is highly variable, and accurate prognostic markers are needed. KL-6 is a mucin-like glycoprotein (MUC1) expressed by type II pneumocytes, while CYFRA 21-1 is expressed by alveolar and bronchiolar epithelial cells. Both are released into the blood from cell injury., Methods: Serum KL-6 and CYFRA 21-1 levels were measured in a retrospective (n = 189) and a prospective (n = 118) cohort of SSc patients. Genotyping of MUC1 rs4072037 was performed. Linear mixed-effect models were used to evaluate the relationship with change in lung function parameters over time, while association with survival was evaluated with Cox proportional hazard analysis., Results: In both cohorts, KL-6 and CYFRA 21-1 were highest in patients with lung involvement, and in patients with extensive rather than limited ILD. KL-6 was higher in patients carrying the MUC1 rs4072037 G allele in both cohorts. In patients with SSc-ILD, serum KL-6, but not CYFRA 21-1, was significantly associated with DL CO decline in both cohorts (P = 0.001 and P = 0.004, respectively), and with FVC decline in the retrospective cohort (P = 0.005), but not the prospective cohort. When combining the cohorts and subgrouping by severity (median CPI = 45.97), KL-6 remained predictive of decline in DL CO in both milder (P = 0.007) and more severe disease (P = 0.02) on multivariable analysis correcting for age, gender, ethnicity, smoking history and MUC1 allele carriage., Conclusion: Our results suggest serum KL-6 predicts decline in lung function in SSc, suggesting its clinical utility in risk stratification for progressive SSc-ILD., (© 2020 The Authors. Respirology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Respirology.)

Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-β-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia/Sugen model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although several genetic associations with scleroderma (SSc) are defined, very little is known on genetic susceptibility to SSc-associated interstitial lung disease (SSc-ILD). A number of common polymorphisms have been associated with SSc-ILD, but most have not been replicated in separate populations. Four SNPs in IRF5, and one in each of STAT4, CD226 and IRAK1, selected as having been previously the most consistently associated with SSc-ILD, were genotyped in 612 SSc patients, of European descent, of whom 394 had ILD. The control population (n = 503) comprised individuals of European descent from the 1000 Genomes Project. After Bonferroni correction, two of the IRF5 SNPs, rs2004640 (OR (95% CI)1.30 (1.10-1.54), p corr  = 0.015) and rs10488631 (OR 1.48 (1.14-1.92), p corr  = 0.022), and the STAT4 SNP rs7574865 (OR 1.43 (1.18-1.73), p corr  = 0.0015) were significantly associated with SSc compared with controls. However, none of the SNPs were significantly different between patients with SSc-ILD and controls. Two SNPs in IRF5, rs10488631 (OR 1.72 (1.24-2.39), p corr  = 0.0098), and rs2004640 (OR 1.39 (1.11-1.75), p corr  = 0.03), showed a significant difference in allele frequency between controls and patients without ILD, as did STAT4 rs7574865 (OR 1.86 (1.45-2.38), p corr  = 6.6 × 10 -6 ). A significant difference between SSc with and without ILD was only observed for STAT4 rs7574865, being less frequent in patients with ILD (OR 0.66 (0.51-0.85), p corr  = 0.0084). In conclusion, IRF5 rs2004640 and rs10488631, and STAT4 rs7574865 were significantly associated with SSc as a whole. Only STAT4 rs7574865 showed a significant difference in allele frequency in SSc-ILD, with the T allele being protective against ILD.Key points• We confirm the associations of the IRF5 SNPs rs2004640 and rs10488631, and the STAT4 SNP rs7574865, with SSc as a whole.• None of the tested SNPs were risk factors for SSc-ILD specifically.• The STAT4 rs7574865 T allele was protective against the development of lung fibrosis in SSc patients.• Further work is required to understand the genetic basis of lung fibrosis in association with scleroderma.

Oro-facial fibrosis in systemic sclerosis (Scleroderma;SSc) has a major impact on mouth function, facial appearance, and patient quality of life. Lipotransfer is a method of reconstruction that can be used in the treatment of oro-facial fibrosis. The effect of this treatment not only restores oro-facial volume but has also been found to reverse the effects of oro-facial fibrosis. Adipose derived stem cells (ADSCs) within the engrafted adipose tissue have been shown to be anti-fibrotic in SSc and are proposed as the mechanism of the anti-fibrotic effect of lipotransfer. A cohort of 62 SSc patients with oro-facial fibrosis were assessed before and after stem cell enriched lipotransfer treatment. Clinical evaluation included assessment of mouth function using a validated assessment tool (Mouth Handicap in Systemic Sclerosis Scale-MHISS), validated psychological measurements and pre and post-operative volumetric assessment. In addition, to understand the mechanism by which the anti-fibrotic effect of ADSCs occur, SSc derived fibroblasts and ADSCs from this cohort of patients were co-cultured in direct and indirect culture systems and compared to monoculture controls. Cell viability, DNA content, protein secretion of known fibrotic mediators including growth factor- β1 (TGF β-1) and connective tissue growth factor (CTGF) using ELISA analysis and fibrosis gene expression using a fibrosis pathway specific qPCR array were evaluated. Mouth function (MHISS) was significantly improved (6.85±5.07) (p<0.0001) after treatment. All psychological measures were significantly improved: DAS 24 (12.1±9.5) (p<0.0001); HADS-anxiety (2.8±3.2) (p<0.0001), HADS-depression (2.0±3.1) (p<0.0001); BFNE (2.9 ± 4.3) (p<0.0001); VAS (3.56±4.1) (p<0.0001). Multiple treatments further improved mouth function (p<0.05), DAS (p<0.0001) and VAS (p = 0.01) scores. SSc fibroblast viability and proliferation was significantly reduced in co-culture compared to monoculture via a paracrine effect over 14 days (p < 0.0001). Protein secretion of transforming growth factor (TGF-β1) and connective tissue growth factor (CTGF) was significantly reduced in co-culture compared to monoculture (p < 0.0001). Multiple fibrosis associated genes were down regulated in SSc co-culture compared to monoculture after 14 days including Matrix metalloproteinase-8 (MMMP-8), Platelet derived growth factor-β (PDGF-β) and Integrin Subunit Beta 6 (ITG-β6). Autologous stem cell enriched lipotransfer significantly improved the effects of oro-facial fibrosis in SSc in this open cohort study. Lipotransfer may reduce dermal fibrosis through the suppression of fibroblast proliferation and key regulators of fibrogenesis including TG-β1 and CTGF. Our findings warrant further investigation in a randomised controlled trial., Competing Interests: The authors have declared that no competing interests exist.

Background: Angiogenesis and vascular remodeling are complementary, innate responses to ischemic cardiovascular events, including peripheral artery disease and myocardial infarction, which restore tissue blood supply and oxygenation; the endothelium plays a critical function in these intrinsic protective processes. C-type natriuretic peptide (CNP) is a fundamental endothelial signaling species that coordinates vascular homeostasis. Herein, we sought to delineate a central role for CNP in angiogenesis and vascular remodeling in response to ischemia., Methods: The in vitro angiogenic capacity of CNP was examined in pulmonary microvascular endothelial cells and aortic rings isolated from wild-type, endothelium-specific CNP -/- , global natriuretic peptide receptor (NPR)-B -/- and NPR-C -/- animals, and human umbilical vein endothelial cells. These studies were complemented by in vivo investigation of neovascularization and vascular remodeling after ischemia or vessel injury, and CNP/NPR-C expression and localization in tissue from patients with peripheral artery disease., Results: Clinical vascular ischemia is associated with reduced levels of CNP and its cognate NPR-C. Moreover, genetic or pharmacological inhibition of CNP and NPR-C, but not NPR-B, reduces the angiogenic potential of pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, and isolated vessels ex vivo. Angiogenesis and remodeling are impaired in vivo in endothelium-specific CNP -/- and NPR-C -/- , but not NPR-B -/- , mice; the detrimental phenotype caused by genetic deletion of endothelial CNP, but not NPR-C, can be rescued by pharmacological administration of CNP. The proangiogenic effect of CNP/NPR-C is dependent on activation of G i , ERK1/2, and phosphoinositide 3-kinase γ/Akt at a molecular level., Conclusions: These data define a central (patho)physiological role for CNP in angiogenesis and vascular remodeling in response to ischemia and provide the rationale for pharmacological activation of NPR-C as an innovative approach to treating peripheral artery disease and ischemic cardiovascular disorders.

Plant lectins are carbohydrate-binding proteins, which can interact with cell surfaces to initiate anti-inflammatory pathways, as well as immunomodulatory functions. Here, we have extracted, purified and part-characterized the bioactivity of Jacalin, Frutalin, DAL and PNA, before evaluating their potential for wound healing in cultured human skin fibroblasts. Only Frutalin stimulated fibroblast migration in vitro, prompting further studies which established its low cytotoxicity and interaction with TLR4 receptors. Frutalin also increased p-ERK expression and stimulated IL-6 secretion. The in vivo potential of Frutalin for wound healing was then assessed in hybrid combination with the polysaccharide galactomannan, purified from Caesalpinia pulcherrima seeds, using both hydrogel and membrane scaffolds formulations. Physical-chemical characterization of the hybrid showed that lectin-galactomannan interactions increased the pseudoplastic behaviour of solutions, reducing viscosity and increasing Frutalin's concentration. Furthermore, infrared spectroscopy revealed -OH band displacement, likely caused by interaction of Frutalin with galactose residues present on galactomannan chains, while average membrane porosity was 100 μm, sufficient to ensure water vapor permeability. Accelerated angiogenesis and increased fibroblast and keratinocyte proliferation were observed with the optimal hybrid recovering the lesioned area after 11 days. Our findings indicate Frutalin as a biomolecule with potential for tissue repair, regeneration and chronic wound healing., (Copyright © 2018. Published by Elsevier B.V.)

Background: Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro-inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear., Design: In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL-17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases., Results: The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis., Conclusion: This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases., (© 2018 Stichting European Society for Clinical Investigation Journal Foundation.)

Purpose of Review: The persistence of myofibroblasts is a key feature of fibrosis and in fibrotic diseases including scleroderma. This review evaluates the emerging concepts of the origins and cell populations that contribute to myofibroblasts and the molecular mechanisms that govern phenotypic conversion and that highlight opportunities for new interventional treatments in scleroderma., Recent Findings: Studies have defined heterogeneity in fibroblast-like cells that can develop into myofibroblast in normal wound healing, scarring and fibrosis. Characterizing these distinct cell populations and their behaviour has been a key focus. In addition, the overarching impact of epigenetic regulation of genes associated with inflammatory responses, cell signalling and cell communication and the extracellular matrix (ECM) has provided important insights into the formation of myofibroblast and their function. Important new studies include investigations into the relationship between inflammation and myofibroblast production and further evidence has been gathered that reveal the importance of ECM microenvironment, biomechanical sensing and mechanotransduction., Summary: This review highlights our current understanding and outlines the increasing complexity of the biological processes that leads to the appearance of the myofibroblast in normal functions and in diseased tissues. We also focus on areas of special interest in particular, studies that have therapeutic potential in fibrosis and scleroderma.

Objective: NKX2-5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down-regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2-5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)-associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications., Methods: We explored NKX2-5 expression in biopsy samples from patients with SSc-associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease-associated putative functional single-nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein-DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses., Results: Increased NKX2-5 expression in biopsy samples from patients with SSc-associated PH was localized to remodeled vessels and PASMCs. Meta-analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease-associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA-6, c-Jun, and myocyte-specific enhancer factor 2C. We also characterized an activator/coactivator transcription-enhancer factor domain 1 (TEAD1)/Yes-associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5., Conclusion: NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2-5 activation in cardiovascular disease characterized by vascular remodeling., (© 2018 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)