Development Of Skin Fibrosis Research Articles

Let‑7f‑5p Regulated by Hsa_circ_0000437 Ameliorates Bleomycin-Induced Skin Fibrosis.

The current treatment of skin fibrosis is limited in its effectiveness due to a lack of understanding of the underlying mechanisms. Previous research has shown a connection between microRNAs (miRNAs) and the development of skin fibrosis. Therefore, investigating miRNA for the treatment of skin fibrotic diseases is highly important and merits further exploration. In this study, we have discovered that let-7f-5p could suppress the proliferation, migration, and expression of collagen type I alpha 1 (COL1A1) in human dermal fibroblasts (HDFs). It was further determined that let-7f-5p could target thrombospondin-1 (THBS1), thereby inhibiting the TGF-β2/Smad3 signaling pathway and exerting its biological effects. Additionally, let-7f-5p is regulated by Hsa_circ_0000437, which acts as a sponge molecule for let-7f-5p and consequently regulates the biological function of HDFs. Furthermore, our findings indicate that in vivo overexpression of let-7f-5p leads to a reduction in dermal thickness and COL1A1 expression, effectively inhibiting the progression of bleomycin (BLM)-induced skin fibrosis in mice. Hence, our research enhances the comprehension of the Hsa_circ_0000437/let-7f-5p/THBS1/TGF-β2/Smad3 regulatory network, highlighting the potential of let-7f-5p as a therapeutic approach for the treatment of skin fibrosis.

Single-cell transcriptional analysis of irradiated skin reveals changes in fibroblast subpopulations and variability in caveolin expression

BackgroundRadiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury.MethodsPaired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing.ResultsComparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0–4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses.ConclusionIn response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.

Lymphatic Endothelial-to-Myofibroblast Transition: A Potential New Mechanism Underlying Skin Fibrosis in Systemic Sclerosis.

At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-β1 (TGFβ1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFβ1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development.

Immunohistochemical and histochemical analysis of the rat skin after local electron irradiation.

Skin cancer is the most frequently diagnosed type of cancer among all malignant neoplasms. The decrease in mitotic activity and the death of intact keratinocytes arise due to the constantly renewing epithelium being highly sensitive to ionizing radiation. The aim of the study is immunohistochemical evaluation of the proliferative-apoptotic balance of keratinocytes, the fibrous component of the skin, and the expression of pro-inflammatory and anti-inflammatory cytokines after single or fractional local electron irradiation. Wistar rats (n = 80) were taken from the ITM&B Vivarium (Sechenov University) and divided into groups: I-control, which were injected with saline; and experimental groups, local electron irradiation at doses: II-8 Gy (single), III-40 Gy (single), IV-summary dose 78 Gy (fractional; 13 Gy per day for 6 days). We performed histological analysis, histochemical analysis using Masson, safranine, and picrosirius red staining, immunohistochemical (Ki-67, caspase-3, p53, types I and III collagens, IL-1, IL-6, IL-4, and IL-10) and morphometric analysis of skin fragments of the outer surface of the thigh, irradiated in accordance with the design of the experiment. The early and delayed effects of local electron irradiation at different doses were studied. After local electron irradiation, dose-dependent morphological changes in the skin of the experimental groups were observed: violation of the histoarchitectonics of the skin confirmed by morphological and morphometric analysis, the proliferation of connective tissue according to the results of histochemical and immunohistochemical studies with signs of the radiation-induced skin fibrosis development, an increase in the levels of pro- and anti-inflammatory cytokines. We observed the most pronounced signs of radiation-induced skin damage in the group of fractional irradiation after 3 months. 8 Gy and 40 Gy single local electron irradiation leads to a shift in the proliferative-apoptotic balance of keratinocytes toward their apoptosis, which activity is directly correlated with the dose of ionizing radiation, and 78 Gy in fractions leads to partial desquamation of the epithelium and inflammatory infiltration. In addition, after 3 months a significant increase in the expression of type I and III collagen fibers and the development of radiation-induced skin fibrosis takes place against the background of 78 Gy fractional local electron irradiation.

Dersimelagon, a novel oral melanocortin 1 receptor agonist, demonstrates disease-modifying effects in preclinical models of systemic sclerosis

BackgroundActivation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc).MethodsThe effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed.ResultsProphylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117.ConclusionsMT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.

Profibrotic Role of Inducible Heat Shock Protein 90α Isoform in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease that affects skin and multiple internal organs. TGF-β, a central trigger of cutaneous fibrosis, activates fibroblasts with the involvement of the stress-inducible chaperone heat shock protein 90 isoform α (Hsp90α). Available evidence supports overexpression and secretion of Hsp90α as a feature in profibrotic pathological conditions. The aim of this work is to investigate the expression and function of Hsp90α in experimental models of skin fibrosis such as human fibroblasts, C57BL/6 mice, and in human SSc. For this purpose, we generated a new experimental model based on doxorubicin administration with improved characteristics with respect to the bleomycin model. We visualized disease progression in vivo by fluorescence imaging. In this work, we obtained Hsp90α mRNA overexpression in human skin fibroblasts, in bleomycin- and doxorubicin-induced mouse fibrotic skin, and in lungs of bleomycin- and doxorubicin-treated mice. Hsp90α-deficient mice showed significantly decreased skin thickness compared with wild-type mice in both animal models. In SSc patients, serum Hsp90α levels were increased in patients with lung involvement and in patients with the diffuse form of SSc (dSSc) compared with patients with the limited form of SSc. The serum Hsp90α levels of patients dSSc were correlated with the Rodnan score and the forced vital capacity variable. These results provide new supportive evidence of the contribution of the Hsp90α isoform in the development of skin fibrosis. In SSc, these results indicated that higher serum levels were associated with dSSc and lung fibrosis.

Interleukin-1β-Activated Microvascular Endothelial Cells Promote DC-SIGN-Positive Alternatively Activated Macrophages as a Mechanism of Skin Fibrosis in Systemic Sclerosis.

To characterize the role of interleukin-1β (IL-1β) and microvascular endothelial cells (MVECs) in the generation of alternatively activated macrophages in the skin, and to explore their role in the development of skin fibrosis in patients with systemic sclerosis (SSc; scleroderma). Conditioned medium prepared with MVECs purified from the skin of healthy donors and the skin of SSc patients was used to generate monocyte-derived macrophages. Flow cytometry, multiplex protein assessment, real-time quantitative polymerase chain reaction, and tissue immunofluorescence were used to characterize MVEC-induced polarization of alternatively activated macrophages. Coculture experiments were conducted to assess the role of MVEC-induced alternatively activated macrophages in fibroblast activation. Alternatively activated macrophages were characterized in the skin of healthy donors and SSc patients using multiparametric immunofluorescence and multiplex immunostaining for gene expression. Based on our in vitro data, we defined a supervised macrophage gene signature score to assess correlation between the macrophage score and clinical features in patients with SSc, using the Spearman's test. IL-1β-activated MVECs from SSc patients induced monocytes to differentiate into DC-SIGN+ alternatively activated macrophages producing high levels of CCL18, CCL2, and CXCL8 but low levels of IL-10. DC-SIGN+ alternatively activated macrophages showed significant enhancing effects in promoting the production of proinflammatory fibroblasts and were found to be enriched in perivascular regions of the skin of SSc patients who had a high fibrosis severity score. A novel skin transcriptomic macrophage signature, defined from our in vitro findings, correlated with the extent of skin fibrosis (Spearman's r = 0.6, P = 0.0018) and was associated with early disease manifestations and lung involvement in patients with SSc. Our findings shed new light on the vicious circle implicating unabated IL-1β secretion, MVEC activation, and the generation of DC-SIGN+ alternatively activated macrophages in the development of skin fibrosis in patients with SSc.

The Contribution of LIGHT (TNFSF14) to the Development of Systemic Sclerosis by Modulating IL-6 and T Helper Type 1 Chemokine Expression in Dermal Fibroblasts

Systemic sclerosis (SSc) is an autoimmune and vascular disease resulting in multiple organ fibrosis, in which IL-6 and T helper (Th)2/Th17 cytokines serve as critical disease drivers. LIGHT is a proinflammatory cytokine promoting IL-6 production in lung fibroblasts and Th1 chemokine expression in dermal fibroblasts (DFs) stimulated with IFN-γ. In this study, we investigated the potential contribution of LIGHT to SSc development using clinical samples and animal models. In SSc-involved skin, LIGHT was upregulated in inflammatory cells, whereas herpesvirus entry mediator (HVEM), a receptor of LIGHT, was downregulated in DFs. Similar expression profiles of LIGHT and HVEM were reproduced in bleomycin-treated mice. Transcription factor FLI1 bound to the HVEM promoter, and FLI1 small interfering RNA suppressed HVEM expression in normal DFs. In SSc DFs, LIGHT significantly increased IL-6 production, whereas IFN-γ/LIGHT-dependent Th1 chemokine induction was decreased compared with that in normal DFs. Importantly, LIGHT small interfering RNA significantly attenuated bleomycin-induced skin fibrosis, and serum LIGHT levels were elevated in patients with diffuse cutaneous SSc and positively correlated with clinical parameters reflecting skin and pulmonary fibrosis. Taken together, these results suggest that altered response of DFs to LIGHT, namely increased IL-6 production and decreased Th1 chemokine expression, contributes to the development of skin fibrosis in SSc.

The role of Wnt signaling in skin fibrosis.

Skin fibrosis is the excessive deposition of extracellular matrix in the dermis. Cutaneous fibrosis can occur following tissue injury, including burns, trauma, and surgery, resulting in scars that are disfiguring, limit movement and cause significant psychological distress for patients. Many molecular pathways have been implicated in the development of skin fibrosis, yet effective treatments to prevent or reverse scarring are unknown. The Wnt signaling pathways are known to play an important role in skin homeostasis, skin injury, and in the development of fibrotic skin diseases. This review provides a detailed overview of the role of the canonical Wnt signaling pathways in regulating skin scarring. We also discuss how Wnt signaling interacts with other known fibrotic molecular pathways to cause skin fibrosis. We further provide a summary of the different Wnt inhibitor types available for treating skin scarring. Understanding the role of the Wnt pathway in cutaneous fibrosis will accelerate the development of effective Wnt modulators for the treatment of skin fibrosis.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model.

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-β1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-β1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-β1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 μg/100 μL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 μg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

TGFβ promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis

ObjectiveInnate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin...

Exosomes derived from miR-16-5p-overexpressing keratinocytes attenuates bleomycin-induced skin fibrosis

microRNAs have been shown to be associated with the development of skin fibrosis. Therefore, miRNA modulators play an important role in the management of cutaneous fibrotic diseases and are worthy of investigation. However, a major obstacle of miRNAs therapy is to deliver miRNAs to target cell types, tissues or organs. The study reported here investigated the effects of miR-16-5p delivery by keratinocytes-derived exosomes on skin fibrosis in the bleomycin (BLM)-treated mice. In results, miR-16-5p-overexpressing keratinocytes-derived exosomes significantly suppressed the enhancing effects of TGF-β1 on proliferation, migration and COL1A1 expression of fibroblasts. Moreover, we found that miR-16-5p-overexpressing keratinocytes-derived exosomes inhibited the endogenous Smad3 expression. In vivo, subcutaneously injected of miR-16-5p-overexpressing keratinocytes-derived exosomes significantly enhanced miR-16-5p expression in the skin compared with the control group, while suppressing BLM-induced skin fibrosis with reduced dermal thickening and lower COL1A1 expression. In conclusion, our results suggest that the localized delivery of miR-16-5p by keratinocytes-derived exosomes may have potential for efficient clinical treatment of skin fibrosis.

Fli1 deficiency suppresses RALDH1 activity of dermal dendritic cells and related induction of regulatory T cells: a possible role in scleroderma

BackgroundAldehyde dehydrogenase 1 family member A1 (RALDH1)-producing dermal dendritic cells (DCs), a conventional DC subset regulating skin fibrosis, are decreased in the involved skin of patients with systemic sclerosis (SSc). In this study, we investigated the contribution of Fli1 deficiency, a potential predisposing factor of SSc, to the phenotypical alteration of RALDH1-producing dermal DCs by using SSc model mice and SSc skin samples.MethodsBleomycin (BLM)-induced skin fibrosis was generated with Fli1+/− and wild-type mice. The proportions of DC and CD4+ T cell subsets were determined by flow cytometry in the dermis of BLM-treated mice. Fli1 expression in dermal DCs was evaluated by immunofluorescence with skin samples of SSc and healthy control subjects.ResultsRALDH activity of dermal DCs was significantly decreased in BLM-treated Fli1+/− mice compared with BLM-treated wild-type mice, whereas the proportion of CD103−CD11b− dermal DCs, a major DC subset producing RALDH1 in response to BLM injection, was comparable between groups. Relevant to this finding, the proportion of regulatory T cells (Tregs) in the dermis was decreased in BLM-treated Fli1+/− mice relative to BLM-treated wild-type mice, while the proportions of Th1, Th2 and Th17 cells were unaltered. In the involved skin of SSc patients, Fli1 was downregulated in CD11c+ cells, including dermal DCs.ConclusionsFli1 deficiency inhibits RALDH1 activity of CD103−CD11b− dermal DCs and related induction of Tregs in BLM-treated mice. Considering Fli1 reduction in SSc dermal DCs, Fli1deficiency may impair the dermal DC-Treg system, contributing to the development of skin fibrosis in SSc.

A critical role of AREG for bleomycin-induced skin fibrosis

We report our discovery of an important player in the development of skin fibrosis, a hallmark of scleroderma. Scleroderma is a fibrotic disease, affecting 70,000 to 150,000 Americans. Fibrosis is a pathological wound healing process that produces an excessive extracellular matrix to interfere with normal organ function. Fibrosis contributes to nearly half of human mortality. Scleroderma has heterogeneous phenotypes, unpredictable outcomes, no validated biomarkers, and no effective treatment. Thus, strategies to slow down scleroderma progression represent an urgent medical need. While a pathological wound healing process like fibrosis leaves scars and weakens organ function, oral mucosa wound healing is a scarless process. After re-analyses of gene expression datasets from oral mucosa wound healing and skin fibrosis, we discovered that several pathways constitutively activated in skin fibrosis are transiently induced during oral mucosa wound healing process, particularly the amphiregulin (Areg) gene. Areg expression is upregulated ~ 10 folds 24hrs after oral mucosa wound but reduced to the basal level 3 days later. During bleomycin-induced skin fibrosis, a commonly used mouse model for skin fibrosis, Areg is up-regulated throughout the fibrogenesis and is associated with elevated cell proliferation in the dermis. To demonstrate the role of Areg for skin fibrosis, we used mice with Areg knockout, and found that Areg deficiency essentially prevents bleomycin-induced skin fibrosis. We further determined that bleomycin-induced cell proliferation in the dermis was not observed in the Areg null mice. Furthermore, we found that inhibiting MEK, a downstream signaling effector of Areg, by selumetinib also effectively blocked bleomycin-based skin fibrosis model. Based on these results, we concluded that the Areg-EGFR-MEK signaling axis is critical for skin fibrosis development. Blocking this signaling axis may be effective in treating scleroderma.

Human umbilical cord mesenchymal stem cells ameliorate skin fibrosis development in a mouse model of bleomycin-induced systemic sclerosis.

Mesenchymal stem cell (MSC) infusion has become a novel therapeutic strategy for complex autoimmune diseases; however, few detailed studies have been performed to investigate the benefit and mechanism of MSC treatment on systemic sclerosis (SSc). The present study aimed to evaluate the therapeutic effect of human umbilical cord derived-MSCs (UC-MSCs) on bleomycin-induced SSc in mice and explore the potential underlying mechanism. The murine SSc model was established by daily subcutaneous injection of bleomycin for 4 weeks, followed with two UC-MSC infusions every 7 days. Skin fibrosis was assessed by H&E and Masson staining. Flow cytometry was used to determine IL-17A, IFN-γ, tumor necrosis factor-β, IL-10 and IL-12 levels in serum samples and T cell subsets in murine spleen. Additionally, gene expression levels of cytokines and fibrosis markers in skin samples were measured by reverse transcription-quantitative PCR. Immunofluorescence staining was performed to track UC-MSC localization and lymphocyte cell infiltration in vivo. UC-MSC treatment exerted an anti-fibrotic role in bleomycin-induced SSc mice, as confirmed by histological improvement, decreased collagen synthesis, and reduced collagen-1α1, collagen-1α2, fibronectin-1 and α-smooth muscle actin gene expression levels. The results indicated that UC-MSC treatment only had a limited systematic effect on cytokine production in serum samples and T cell activation in the spleen. By contrast, T helper (Th)17 cell infiltration and activation in skin were efficiently inhibited after UC-MSC infusion, as evidenced by the decreased IL-17A and retinoic acid-related orphan receptor γt gene expression as well as IL-17A production. UC-MSC administration significantly ameliorated bleomycin-induced skin fibrosis and collagen formation primarily by eliminating local inflammation and Th17 cell activation in the skin; however, the systemic inhibitory effect of UM-MSCs on cytokines was less profound.

Fibrotic Signaling Pathways of Skin Fibroblasts in Nephrogenic Systemic Fibrosis

The focus of this article is to describe the etiology, clinical presentation, treatment, and molecular signaling mechanisms involved in the dermatological manifestations of nephrogenic systemic fibrosis (NSF). The pathogenesis of NSF involves the recruitment of fibrocytes to tissue where they differentiate into the pro-fibrotic myofibroblast phenotype involved in collagen deposition. This review focuses on the different pro-fibrotic molecular signaling pathways of fibroblasts that mediate skin fibrosis in NSF. This article provides an overview of the clinical presentation and treatment of NSF, with an in depth review of the signaling pathways of fibroblasts associated with the development of skin fibrosis in NSF.

Intervention of Inflammatory Monocyte Activity Limits Dermal Fibrosis

Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen species-induced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6Chigh monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2+ inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6Clow monocytes by a nuclear receptor Nur77-agonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12-deficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.

Fibrosis Development in HOCl-Induced Systemic Sclerosis: A Multistage Process Hampered by Mesenchymal Stem Cells

Objectives: Skin fibrosis is the hallmark of systemic sclerosis (SSc) a rare intractable disease with unmet medical need. We previously reported the anti-fibrotic potential of mesenchymal stem cells (MSCs) in a murine model of SSc. This model, based on daily intra-dermal injections of hypochlorite (HOCl) during 6 weeks, is an inducible model of the disease. Herein, we aimed at characterizing the development of skin fibrosis in HOCl-induced SSc (HOCl-SSc), and evaluating the impact of MSC infusion during the fibrogenesis process.Methods: After HOCl-SSc induction in BALB/c mice, clinical, histological and biological parameters were measured after 3 weeks (d21) and 6 weeks (d42) of HOCl challenge, and 3 weeks after HOCl discontinuation (d63). Treated-mice received infusions of 2.5 × 105 MSCs 3 weeks before sacrifice (d0, d21, d42).Results: HOCl injections induced a two-step process of fibrosis development: first, an ‘early inflammatory phase’, characterized at d21 by highly proliferative infiltrates of myofibroblasts, T-lymphocytes and macrophages. Second, a phase of ‘established matrix fibrosis’, characterized at d42 by less inflammation, but strong collagen deposition and followed by a third phase of ‘spontaneous tissue remodeling’ after HOCl discontinuation. This phase was characterized by partial fibrosis receding, due to enhanced MMP1/TIMP1 balance. MSC treatment reduced skin thickness in the three phases of fibrogenesis, exerting more specialized mechanisms: immunosuppression, abrogation of myofibroblast activation, or further enhancing tissue remodeling, depending on the injection time-point.Conclusion: HOCl-SSc mimics three fibrotic phenotypes of scleroderma, all positively impacted by MSC therapy, demonstrating the great plasticity of MSC, a promising cure for SSc.

TRPV4 channels regulates matrix stiffness and TGFβ1‐induced epithelial‐mesenchymal transition

Epithelial‐mesenchymal transition (EMT) has important functions in cellular processes including development, wound healing, oncogenesis, and fibrosis. Emerging data support a role for both a mechanical signal, and a biochemical signal, e.g., transforming growth factor β1 (TGFβ1), in EMT. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), calcium‐permeable channel and member of TRP superfamily, is the likely mediator of EMT in response to both TGFβ1 and matrix stiffness. Specifically, we found that: i) genetic ablation of TRPV4 blocked TGFβ1‐induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology and alterations of expression of EMT markers including E‐cadherin (ECAD), N‐cadherin (NCAD), and α‐smooth muscle actin (α‐SMA), and ii) TRPV4 deficiency prevented matrix stiffness‐induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α‐SMA, in a bleomycin‐induced murine skin fibrosis model. We found an increased co‐localization of TRPV4 with NCAD, and decreased co‐localization of TRPV4 with epithelial marker ECAD in skin tissues of bleomycin‐treated wild‐type mice compared to saline controls. Mechanistically, we found that: i) TRPV4 was essential for the nuclear translocation of YAP/TAZ (Yes‐associated protein/transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1, ii) TRPV4 deletion inhibited both matrix stiffness‐ and TGFβ1‐induced expression of YAP/TAZ proteins, and iii) TRPV4 deletion abrogated both matrix stiffness‐ and TGFβ1‐induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT, and thus suggest that therapeutic targeting of TRPV4 may provide a selective approach to ameliorate the development of skin fibrosis and oncogenesis.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to SOR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Blockade of TGF-\u03b2/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

BackgroundTransforming growth factor-β (TGF-β)/Smad signaling is well known to play a critical role in the pathogenesis of systemic sclerosis (SSc). We previously developed an artificial molecule, the histidine-pyridine-histidine ligand derivative HPH-15, which may have an antifibrotic effect. The purpose of the present study was to clarify the effects of this drug in human skin fibroblasts and in a preclinical model of SSc.MethodsThe effects of HPH-15 on expression of extracellular matrix components and TGF-β signaling in human dermal fibroblasts were analyzed. The antifibrotic properties of HPH-15 and its mechanisms were also examined in a bleomycin-induced skin fibrosis mouse model.ResultsHPH-15 suppressed the TGF-β-induced phosphorylation of Smad3 and inhibited the expression of collagen I, fibronectin 1, connective tissue growth factor, and α-smooth muscle actin induced by TGF-β in cultured human skin fibroblasts. In the bleomycin-induced skin fibrosis model, oral administration of HPH-15 protected against the development of skin fibrosis and ameliorated established skin fibrosis. Additionally, HPH-15 suppressed the phosphorylation of Smad3 in various cells, including macrophages in the bleomycin-injected skin. Further, in the treated mice, dermal infiltration of proinflammatory macrophages (CD11b+Ly6Chi) and M2 profibrotic macrophages (CD11b+CD204+ or CD11b+CD206+) was significantly decreased during the early and late stages, respectively. HPH-15 treatment resulted in decreased messenger RNA (mRNA) expression of the M2 macrophage markers arginase 1 and Ym-1 in the skin, whereas it inversely augmented expression of Friend leukemia integration 1 and Krüppel-like factor 5 mRNAs, the transcription factors that repress collagen synthesis. No apparent adverse effects of HPH-15 were found during the treatment.ConclusionsHPH-15 may inhibit skin fibrosis by inhibiting the phosphorylation of Smad3 in dermal fibroblasts and possibly in macrophages. Our results demonstrate several positive qualities of HPH-15, including oral bioavailability, a good safety profile, and therapeutic effectiveness. Thus, this TGF-β/Smad inhibitor is a potential candidate therapeutic for SSc clinical trials.