Skin Fibrosis In Scleroderma Research Articles

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p < 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p < 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p < 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Thy-1 plays a pathogenic role and is a potential biomarker for skin fibrosis in scleroderma.

Thy-1 (CD90) is a well-known marker of fibroblasts implicated in organ fibrosis, but its contribution to skin fibrosis remains unknown. We examined Thy-1 expression in scleroderma skin and its potential role as a biomarker and pathogenic factor in animal models of skin fibrosis. Skin from patients with systemic sclerosis demonstrated markedly elevated Thy-1 expression compared with controls, colocalized with fibroblast activator protein in the deep dermis, and correlated with the severity of skin involvement (modified Rodnan skin score). Serial imaging of skin from Thy-1 yellow fluorescent protein reporter mice by IVIS showed an increase in Thy-1 expression that correlated with onset and progression of fibrosis. In contrast to lung fibrosis, Thy-1-KO mice had attenuated skin fibrosis in both bleomycin and tight skin-1 murine models. Moreover, Thy-1 regulated key pathogenic pathways involved in fibrosis, including inflammation, myofibroblast differentiation, apoptosis, and multiple additional canonical fibrotic pathways. Therefore, although Thy-1 deficiency leads to exacerbated lung fibrosis, in skin it is protective. Moreover, Thy-1 may serve as a longitudinal marker to assess skin fibrosis.

Mouse Models of Skin Fibrosis.

Systemic sclerosis (SSc) is a rare systemic autoimmune disease associated with a high mortality. The first histopathological hallmarks are vasculopathy and inflammation, followed by fibrosis of the skin and internal organs. The molecular and cellular mechanisms are incompletely understood. Rodent models provide important insights into the pathogenesis of SSc and are a mainstay for the development of novel targeted therapies. Here we describe the mechanistic insights of inducible and genetic models, and also discuss in detail the limitations and pitfalls of the most frequently used SSc mouse models. We also describe protocols for running the established bleomycin-induced scleroderma skin fibrosis model.

Pharmacological activation of PPAR-γ: a potential therapy for skin fibrosis.

Skin fibrosis caused by excessive collagen synthesis and deposition in the dermis affects the quality of daily life of hundreds of thousands of people around the world. The skin quality, including its smoothness in young age and wrinkly during the aging process, depends largely on the levels of extracellular matrix proteins such as collagen in skin. As physiological levels of collagen are desirable for skin homeostasis, beauty, and its flexibility, too much collagen deposition in the skin is associated with tight hard skin, loss of adipose layer, and flexibility, the pathological manifestations of skin fibrosis in fibrotic diseases such as scleroderma. To understand the molecular basis of skin fibrosis and in search of its therapy, different cellular, molecular, epigenetic, and preclinical studies have been undertaken to control abnormal excessive synthesis and accumulation of matrix protein collagen. Over the last two decades, numerous phase 1 through 3 clinical trials have been conducted to test the safety and efficacy of a wide variety of compounds in amelioration of skin fibrosis and other pathologies in scleroderma, yet, no effective therapy for skin fibrosis is available. This article solely focuses on the role of a nuclear receptor and transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR-γ), as an anti-skin fibrotic driving force and the potential therapeutic efficacies of PPAR-γ-specific ligands/agonists including antidiabetic drugs and other natural or semi-synthetic compounds derived from cannabis in amelioration of skin fibrosis in scleroderma. The underlying molecular basis of agonist-activated PPAR-γ-mediated suppression of profibrogenic signaling and skin fibrogenesis is also highlighted.

Adipose-derived mesenchymal stromal/stem cells in systemic sclerosis: Alterations in function and beneficial effect on lung fibrosis are regulated by caveolin-1.

The potential value of mesenchymal stromal/stem cell therapy in treating skin fibrosis in scleroderma (systemic sclerosis) and of the caveolin-1 scaffolding domain peptide in treating lung, skin, and heart fibrosis is known. To understand how these observations may relate to differences between mesenchymal stromal/stem cells from healthy subjects and subjects with fibrosis, we have characterized the fibrogenic and adipogenic potential of adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients, from mice with fibrotic lung and skin disease induced by systemic bleomycin treatment, and from healthy controls. Early passage systemic sclerosis adipose-derived mesenchymal stromal/stem cells have a profibrotic/anti-adipogenic phenotype compared to healthy adipose-derived mesenchymal stromal/stem cells (low caveolin-1, high α-smooth muscle actin, high HSP47, low pAKT, low capacity for adipogenic differentiation). This phenotype is mimicked by treating healthy adipose-derived mesenchymal stromal/stem cells with transforming growth factor beta or caveolin-1 small interfering RNA and is reversed in systemic sclerosis adipose-derived mesenchymal stromal/stem cells by treatment with caveolin-1 scaffolding domain peptide, but not scrambled caveolin-1 scaffolding domain peptide. Similar results were obtained with adipose-derived mesenchymal stromal/stem cells from systemic sclerosis patients and from bleomycin-treated mice, indicating the central role of caveolin-1 in mesenchymal stromal/stem cell differentiation in fibrotic disease.

Adipocyte-specific Repression of PPAR-gamma by NCoR Contributes to Scleroderma Skin Fibrosis

BackgroundA pivotal role for adipose tissue homeostasis in systemic sclerosis (SSc) skin fibrosis is increasingly recognized. The nuclear receptor PPAR-γ is the master regulator of adipogenesis. Peroxisome proliferator activated receptor-γ (PPAR-γ) has antifibrotic effects by blocking transforming growth factor-β (TGF-β) and is dysregulated in SSc. To unravel the impact of dysregulated PPAR-γ in SSc, we focused on nuclear corepressor (NCoR), which negatively regulates PPAR-γ activity and suppresses adipogenesis.MethodsAn NCoR-regulated gene signature was measured in the SSc skin transcriptome. Experimental skin fibrosis was examined in mice with adipocyte-specific NCoR ablation.ResultsSSc skin biopsies demonstrated deregulated NCoR signaling. A 43-gene NCoR gene signature showed strong positive correlation with PPAR-γ signaling (R = 0.919, p < 0.0001), whereas negative correlations with TGF-β signaling (R = − 0.796, p < 0.0001) and the modified Rodnan skin score (R = − 0.49, p = 0.004) were found. Mice with adipocyte-specific NCoR ablation demonstrated significant protection from experimental skin fibrosis and inflammation. The protective effects were mediated primarily through endogenous PPAR-γ.ConclusionsOur results implicate, for the first time, to our knowledge, deregulated NCoR/PPAR-γ pathways in SSc, and they support a role of adipocyte modulation of skin fibrosis. Pharmacologic restoration of NCoR/PPAR-γ signaling may represent a novel strategy to control skin fibrosis in SSc.

The roles of dermal white adipose tissue loss in scleroderma skin fibrosis.

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. 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. 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.

Systemic sclerosis in 2016: Dermal white adipose tissue implicated in SSc pathogenesis.

Several strands of new research indicate that skin-specific adipocyte progenitor cells regulate myofibroblasts and skin fibrosis in scleroderma.

Dendritic cells maintain dermal adipose-derived stromal cells in skin fibrosis.

Scleroderma is a group of skin-fibrosing diseases for which there are no effective treatments. A feature of the skin fibrosis typical of scleroderma is atrophy of the dermal white adipose tissue (DWAT). Adipose tissue contains adipose-derived mesenchymal stromal cells (ADSCs) that have regenerative and reparative functions; however, whether DWAT atrophy in fibrosis is accompanied by ADSC loss is poorly understood, as are the mechanisms that might maintain ADSC survival in fibrotic skin. Here, we have shown that DWAT ADSC numbers were reduced, likely because of cell death, in 2 murine models of scleroderma skin fibrosis. The remaining ADSCs showed a partial dependence on dendritic cells (DCs) for survival. Lymphotoxin β (LTβ) expression in DCs maintained ADSC survival in fibrotic skin by activating an LTβ receptor/β1 integrin (LTβR/β1 integrin) pathway on ADSCs. Stimulation of LTβR augmented the engraftment of therapeutically injected ADSCs, which was associated with reductions in skin fibrosis and improved skin function. These findings provide insight into the effects of skin fibrosis on DWAT ADSCs, identify a DC-ADSC survival axis in fibrotic skin, and suggest an approach for improving mesenchymal stromal cell therapy in scleroderma and other diseases.

Bosentan May Reduce Skin Fibrosis in Scleroderma

Bosentan May Reduce Skin Fibrosis in Scleroderma

Potential role of SFRP4 in Tsk mice and scleroderma skin fibrosis

Potential role of SFRP4 in Tsk mice and scleroderma skin fibrosis

Fibroblast expression of the coactivator p300 governs the intensity of profibrotic response to transforming growth factor β

Transforming growth factor beta (TGFbeta) induces profibrotic responses in normal fibroblasts, and plays a fundamental role in the pathogenesis of fibrosis in scleroderma (systemic sclerosis [SSc]). The intensity of cellular responses elicited by cytokines is modulated by transcriptional coactivators such as the histone acetylase p300. The objective of these studies was to delineate the physiologic role of p300 in Smad-dependent profibrotic responses elicited by TGFbeta. Ectopic p300 was transiently expressed in normal dermal fibroblasts. Cellular p300 levels were suppressed using p300-specific ribozymes. The regulation of gene expression was examined by transient transfection assays, Northern blotting, and immunoblot analysis. The expression of p300 in normal and scleroderma fibroblasts was evaluated by confocal microscopy and immunoblotting, and p300 levels in skin from mice with experimental scleroderma were assessed by immunohistochemistry. In normal fibroblasts, TGFbeta induced an increase in the levels of p300. Forced expression of ectopic p300 in these cells dramatically enhanced the magnitude of TGFbeta responses, whereas selective depletion of p300 using ribozyme resulted in abrogation of TGFbeta-induced collagen synthesis and promoter activity. Furthermore, TGFbeta lost its ability to induce Smad-dependent transcription in p300-depleted fibroblasts. These responses could be fully rescued with ectopic p300. Abrogation of Smad-mediated TGFbeta signaling was not due to alterations in the levels or the ligand-dependent phosphorylation or intracellular trafficking of endogenous Smads. Immunohistochemical analysis demonstrated substantially increased p300 expression in lesional skin from mice with chronic graft-versus-host disease, an animal model of scleroderma. Furthermore, levels of p300 were 2-3-fold higher in cultured fibroblasts derived from SSc patients than in fibroblasts from matched normal controls. These results establish, for the first time, that the coactivator histone acetylase p300, itself a target of TGFbeta regulation, is an essential component of the cellular TGFbeta signal transduction pathways mediating stimulation of collagen synthesis in fibroblasts. Since the cellular abundance of p300 appears to govern the intensity of profibrotic responses elicited by TGFbeta, elevated p300 expression in lesional tissue may contribute to the progression of skin fibrosis in scleroderma.

Effects of methotrexate on glycosaminoglycan production by scleroderma fibroblasts in culture.

To determine the effects of increasing concentrations of methotrexate on the proliferation and glycosaminoglycan (GAG) synthesis of cultured dermal fibroblasts from patients with scleroderma. Cultured dermal fibroblasts from nine patients with scleroderma and nine normal volunteers were grown for 72 hours in media containing various concentrations of methotrexate. The GAG synthesis in each cell was measured after incubating the fibroblasts with [3H]glucosamine and [35S]sulphate. A negative correlation was found between the concentration of methotrexate and numbers of fibroblasts from patients with scleroderma and normal controls. A positive correlation was found between GAG synthesis in each cell, as measured by [3H]glucosamine and [35S]sulphate incorporation, and increasing methotrexate concentrations in fibroblasts from patients with scleroderma and normal controls. These data indicate increased GAG synthesis in scleroderma and normal fibroblasts with increasing concentrations of methotrexate. Therefore the reported beneficial effect of methotrexate on skin fibrosis in scleroderma is most probably not the result of direct inhibition of GAG synthesis by fibroblasts.

Mast Cells' Contribution to the Fibrosing Alveolitis of the Scleroderma Lung

Fibrosing alveolitis is frequently seen in scleroderma. Although usually causing progressive symptomatology, it may be found in patients reporting no respiratory symptoms. Mast cells may play a role in the pathogenesis of skin fibrosis in scleroderma. We determined the mast cells and other cellular content, as well as measuring the inflammatory mediators in the bronchoalveolar lavage fluid (BALF) in 17 scleroderma patients and nine control subjects to correlate BALF features with fibrosing alveolitis as determined by lung function testing and high-resolution computed tomographic scans. Bronchoalveolar lavage cells were enumerated after May-Giemsa-Grünwald (MGG), alcian blue, and safranine blue staining. Histamine, tryptase, eosinophil cationic protein, hyaluronic acid, and neutrophil-specific myeloperoxidase were measured by radioimmunoassay. In comparison with normal subjects the BALF of scleroderma patients showed an increased percentage of mast cells (p < 0.002, Mann-Whitney U test), and increased levels of histamine (p < 0.005), tryptase (p < 0.02), and hyaluronic acid (p < 0.004). The BALF of the eight scleroderma patients with an abnormal chest X-ray had a significantly greater number of mast cells (p < 0.04, Mann-Whitney U test), and significantly higher levels of histamine (p < 0.03, Mann-Whitney U test) and tryptase (p = 0.02, Mann-Whitney U test) than the nine scleroderma patients with a normal chest X-ray. This study demonstrates the importance of mast cells and mast-cell activation in the pathogenesis of the fibrosing alveolitis of scleroderma.