Mouse Model Of Systemic Sclerosis Research Articles

Neutrophil-Driven M2-Like Macrophages Are Critical for Skin Fibrosis in a Systemic Sclerosis Model

Systemic sclerosis (SSc) is a challenging autoimmune disease characterized by progressive fibrosis affecting the skin and internal organs. Despite the known infiltration of macrophages and neutrophils, their precise contributions to SSc pathogenesis remain elusive. In this study, we elucidated that CD206hiMHCIIlo M2-like macrophages constitute the predominant pathogenic immune cell population in the fibrotic skin of a bleomycin (BLM)-induced SSc mouse model. These cells emerged as pivotal contributors to the profibrotic response by orchestrating the production of TGF-β1 through a MerTK signaling-dependent manner. Notably, we observed that neutrophil infiltration was a prerequisite for the accumulation of M2-like macrophages. Strategies such as neutrophil depletion or inhibition of CXCR1/2 were proven effective in reducing M2-like macrophages, subsequently mitigating SSc progression. Detailed investigations revealed that in fibrotic skin, neutrophil-released neutrophil extracellular traps (NETs) were responsible for the differentiation of M2-like macrophages. Our findings illuminate the significant involvement of the neutrophil-macrophage-fibrosis axis in SSc pathogenesis, offering critical information for the development of potential therapeutic strategies.

The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud's phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer's disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects.

SARS-CoV-2 spike protein accelerates systemic sclerosis by increasing inflammatory cytokines, Th17 cells, and fibrosis

BackgroundCoronavirus disease 2019 (COVID-19) induces a dysfunctional immune response, inflammation, autoantibody production, and coagulopathy, which are symptoms that bear resemblance to those of autoimmune diseases, including systemic sclerosis (SSc).MethodsWhile there is a single case report suggesting an association between COVID-19 and SSc, the effects of COVID-19 on SSc are not yet fully understood. Human embryonic kidney 293 (HEK293) cells were transfected with the SARS-CoV-2 spike protein gene, in the presence of TGF-β. The expression levels of fibrosis-related proteins were measured via Western blotting. A bleomycin (BLM)-induced SSc mouse model was employed, wherein mice were injected with the gene encoding the SARS-CoV-2 spike protein and the ACE2 receptor. The levels of fibrosis, autoantibodies, thrombotic factors, and inflammatory cytokines in tissues and serum were analyzed.ResultsIn vitro, the expression levels of fibrosis marker proteins were elevated in the spike protein group compared to the control group. In vivo, the skin thickness of SSc mice increased following exposure to the SARS-CoV-2 spike protein. Furthermore, the levels of autoantibodies and thrombotic factors, such as anti-phospholipid antibodies (APLA), were significantly increased in the presence of the protein. Flow cytometry analysis revealed increased expression of the proinflammatory cytokine IL-17 in the skin, lungs, and blood. Moreover, tissue fibrosis and levels of inflammatory cytokines in skin and lung tissues were markedly escalated in SSc mice subjected to the protein.ConclusionCOVID-19 may accelerate the development and progression of SSc by intensifying fibrosis through the upregulation of inflammation, autoantibody production, and thrombosis.

Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

Tanshinone IIA ameliorates the development of dermal fibrosis in systemic sclerosis.

We previously revealed the role of tanshinone IIA (TAN IIA) on endothelial cells and the impact of TAN IIA on the endothelial-to-mesenchymal transition in systemic sclerosis (SSc). In this study, we sought to further determine whether TAN IIA can directly act on the skin fibroblasts of scleroderma and look into its underlying anti-fibrotic mechanisms. Bleomycin was used to establish the SSc mouse model. After TAN IIA treatment, dermal thickness, type I collagen and hydroxyproline content were measured. Primary fibroblasts were acquired from SSc patients and cultured in vitro, and the effects of TAN IIA on proliferation, apoptosis and the cell cycle of fibroblasts were detected. In a bleomycin-induced SSc model, we discovered that TAN IIA significantly improved skin thickness and collagen deposition, demonstrating a potent anti-fibrotic action. TAN IIA inhibits the proliferation of skin fibroblasts derived from SSc patients by causing G2/M cell cycle arrest and promoting apoptosis. Additionally, TAN IIA downregulated extracellular matrix gene transcription and collagen protein expression in skin fibroblasts in a dose-gradient-dependent manner. Furthermore, we showed how TAN IIA can reduce the activation of the transforming growth factor-β (TGF-β)/Smad and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are important factors in SSc. In summary, these data suggest that TAN IIA can reduce SSc-related skin fibrosis by modulating the TGF-β/Smad and MAPK/ERK signalling pathways. More importantly, our results imply that TAN IIA can directly act on the skin fibroblasts of SSc, therefore, inhibiting fibrosis.

Antifibrotic effect of apremilast in systemic sclerosis dermal fibroblasts and bleomycin-induced mouse model

Phosphodiesterase (PDE) 4 inhibitors have been reported to suppress the progression of dermal fibrosis in patients with systemic sclerosis (SSc); however, the precise mechanisms remain to be elucidated. Therefore, we conducted experiments focusing on the antifibrotic and anti-inflammatory effects of apremilast using dermal fibroblasts derived from patients with SSc and an SSc mouse model. Dermal fibroblasts derived from healthy controls and patients with SSc were incubated with apremilast in the presence or absence of 10 ng/ml transforming growth factor (TGF)-β1 for the measurement of intracellular cAMP levels and evaluation of mRNA and protein expression. A bleomycin-induced dermal fibrosis mouse model was used to evaluate the inhibitory effects of apremilast on the progression of dermal fibrosis. Intracellular cAMP levels were significantly reduced in dermal fibroblasts derived from patients with SSc compared with those derived from healthy controls. Apremilast reduced the mRNA expression of profibrotic markers and the protein expression of type I collagen and Cellular Communication Network Factor 2 (CCN2) in dermal fibroblasts. Additionally, apremilast inhibited the progression of dermal fibrosis in mice, partly by acting on T cells. These results suggest that apremilast may be a potential candidate for treating dermal fibrosis in SSc.

Abstract 14651: Noncoding RNA Drug, TY1, Attenuates Cardiac Fibrosis in Two Mouse Models of Systemic Sclerosis

Introduction: Systemic sclerosis (SSc) is a connective tissue disease characterized by inflammation and fibrosis of the skin and vital organs. The disease has no specific treatment options and a high mortality (~30% at 3 years). We have previously demonstrated anti-fibrotic and anti-inflammatory properties of EV-YF1, a small Y RNA abundant in extracellular vesicles secreted by cardiosphere-derived cells (CDCs). Here we describe the therapeutic bioactivity of a shorter, engineered derivative, TY1. Methods: 5-7-week-old female C57BL/6J wild type mice (n=100 total) were injected with phosphate-buffered saline (PBS; WT) or 100 μL bleomycin (1 mg/ml) subcutaneously every other day for 3 weeks. Animals then received either vehicle (formulation only, in PBS), TY1, or scrambled TY1 (as an RNA control) via oral gavage. Endpoints include exercise capacity, lung weight, systolic and diastolic function by echocardiography, and histologic quantification of fibrosis in heart and skin. To confirm the antifibrotic findings in an inflammation-independent model, 4-6-week-old Tight-skin (tsk-1) mice (n=18 total) received oral TY1, scramble or vehicle for 4 weeks. Results: Diastolic dysfunction was evident in bleomycin mice, but was normalized by TY1 ingestion (E/E’ ratios in WT: 18.91, vehicle: 32.29, scramble: 36.19, TY1: 20.65 p <0.0001). Likewise, lung congestion and exercise intolerance were ameliorated by TY1 ingestion (p<0.0001). In both models (bleomycin-induced and tsk-1), skin and cardiac fibrosis were greater in vehicle or scramble groups than in WT, but were normalized by TY1 after 4 weeks (p <0.001). Conclusions: These results demonstrate the therapeutic bioactivity of a bioinspired new chemical entity in two complementary mouse models of SSc. Thus, TY1 merits investigation as a potential novel therapeutic for SSc.

Coadministration of 3′5-dimaleamylbenzoic acid and quercetin decrease pulmonary fibrosis in a systemic sclerosis model

Systemic sclerosis (SSc) is an autoimmune disease characterized by microvascular compromise and fibrosis. Pulmonary fibrosis, a prominent pulmonary complication in SSc, results in impaired lung function due to excessive accumulation of extracellular matrix components. This study aimed to investigate the effects of coadministration of 3′5-dimaleamylbenzoic acid (AD) and quercetin (Q) on key events in the development and maintenance of pulmonary fibrosis in a bleomycin (BLM)-induced SSc mouse model. The model was induced in CD1 mice through BLM administration using osmotic mini pumps. Subsequently, mice were treated with AD (6 mg/kg) plus Q (10 mg/kg) and sacrificed at 21 and 28 days post BLM administration. Histopathological analysis was performed by hematoxylin and eosin staining and Masson's trichrome staining. Immunohistochemistry was used to determine the expression of proliferation, proinflammatory, profibrotic and oxidative stress markers. The coadministration of AD and Q during the fibrotic phase of the BLM-induced SSc model led to attenuated histological alterations and pulmonary fibrosis, reflected in the recovery of alveolar spaces (30 %, p < 0.01) and decreased collagen deposits (50 %, p < 0.001). This effect was achieved by decreasing the expression of the proliferative markers cyclin D1 (87 %, p < 0.0001) and PCNA (43 %, p < 0.0001), inflammatory markers COX-2 (71 %, p < 0.0001) and iNOS (84 %, p < 0.0001), profibrotic markers α-SMA (80 %, p < 0.0001) and TGF-β (81 %, p < 0.0001) and the lipid peroxidation marker 4-HNE (43 %, p < 0.01). The antifibrotic effect of this combined therapy is associated with the regulation of proliferation, inflammation and oxidative stress, mechanisms involved in the development and progression of the fibrotic process. Our novel therapeutic strategy is the first approach to propose the use of the combination of prooxidant and antioxidant compounds as a potential strategy for SSc-associated pulmonary fibrosis.

039 Inhibitory effect on skin fibrosis by regulating Th17 and regulatory T cell imbalance in a systemic sclerosis mouse model and the involvement of alteration in the intestinal microbiota

039 Inhibitory effect on skin fibrosis by regulating Th17 and regulatory T cell imbalance in a systemic sclerosis mouse model and the involvement of alteration in the intestinal microbiota

Bioinformatics-integrated screening of systemic sclerosis-specific expressed markers to identify therapeutic targets.

Systemic sclerosis (SSc) is a rare autoimmune disease characterized by extensive skin fibrosis. There are no effective treatments due to the severity, multiorgan presentation, and variable outcomes of the disease. Here, integrated bioinformatics was employed to discover tissue-specific expressed hub genes associated with SSc, determine potential competing endogenous RNAs (ceRNA) regulatory networks, and identify potential targeted drugs. In this study, four datasets of SSc were acquired. To identify the genes specific to tissues or organs, the BioGPS web database was used. For differentially expressed genes (DEGs), functional and enrichment analyses were carried out, and hub genes were screened and shown in a network of protein-protein interactions (PPI). The potential lncRNA-miRNA-mRNA ceRNA network was constructed using the online databases. The specifically expressed hub genes and ceRNA network were validated in the SSc mouse and in normal mice. We also used the receiver operating characteristic (ROC) curve to determine the diagnostic values of effective biomarkers in SSc. Finally, the Drug-Gene Interaction Database (DGIdb) identified specific medicines linked to hub genes. The pooled datasets identified a total of 254 DEGs. The tissue/organ-specifically expressed genes involved in this analysis are commonly found in the hematologic/immune system and bone/muscle tissue. The enrichment analysis of DEGs revealed the significant terms such as regulation of actin cytoskeleton, immune-related processes, the VEGF signaling pathway, and metabolism. Cytoscape identified six gene cluster modules and 23 hub genes. And 4 hub genes were identified, including Serpine1, CCL2, IL6, and ISG15. Consistently, the expression of Serpine1, CCL2, IL6, and ISG15 was significantly higher in the SSc mouse model than in normal mice. Eventually, we found that MALAT1-miR-206-CCL2, let-7a-5p-IL6, and miR-196a-5p-SERPINE1 may be promising RNA regulatory pathways in SSc. Besides, ten potential therapeutic drugs associated with the hub gene were identified. This study revealed tissue-specific expressed genes, SERPINE1, CCL2, IL6, and ISG15, as effective biomarkers and provided new insight into the mechanisms of SSc. Potential RNA regulatory pathways, including MALAT1-miR-206-CCL2, let-7a-5p-IL6, and miR-196a-5p-SERPINE1, contribute to our knowledge of SSc. Furthermore, the analysis of drug-hub gene interactions predicted TIPLASININ, CARLUMAB and BINDARIT as candidate drugs for SSc.

Effects of Ruxolitinib on fibrosis in preclinical models of systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune fibrotic disorder notably characterized by the production of antinuclear autoantibodies, which have been linked to an excess of apoptotic cells, normally eliminated by a macrophagic efferocytosis. As interferon (IFN) signature and phosphorylation of JAK-STAT proteins are hallmarks of SSc tissues, we tested the hypothesis that a JAK inhibitor, ruxolitinib, targeting the IFN signaling, could improve efferocytosis of IFN-exposed human macrophages in vitro as well as skin and lung fibrosis.In vivo, BLM- and HOCl-induced skin thickness and fibrosis is associated with an increase of caspase-3 positive dermal cells and a significant increase of IFN-stimulated genes expression. In BLM-SSc model, ruxolitinib prevented dermal thickness, fibrosis and significantly decreased the number of cleaved caspase-3 cells in the dermis. Ruxolitinib also improved lung architecture and fibrosis although IFN signature was not entirely decreased by ruxolitinib. In vitro, ruxolitinib improves efferocytosis capacity of human monocyte-differentiated macrophages exposed to IFN-γ or IFN-β. In human fibroblasts derived from lung (HLF) biopsies isolated from patients with idiopathic pulmonary fibrosis, the reduced mRNA expression of typical TGF-β-activated markers by ruxolitinib was associated with a decrease of the phosphorylation of SMAD2 /3 and STAT3.Our finding supports the anti-fibrotic properties of ruxolitinib in a systemic SSc mouse model and in vitro in human lung fibroblasts.

Traditional Chinese Medicine Integrated Responsive Microneedles for Systemic Sclerosis Treatment

Traditional Chinese medicine, such as Tripterygium wilfordii and Paeonia lactiflora, has potential values in treating systemic sclerosis (SSc) and other autoimmune diseases, while their toxic side effect elimination and precise tropical drug delivery are still challenges. Here, we present multiple traditional Chinese medicine integrated photoresponsive black phosphorus (BP) microneedles (MNs) with the desired features for the SSc treatment. By employing a template-assisted layer-by-layer curing method, such MNs with triptolide (TP)/paeoniflorin (Pae) needle tips and BP-hydrogel needle bottoms could be well generated. The combined administration of TP and Pae can not only provide anti-inflammatory, detoxification, and immunomodulatory effects to treat skin lesions in the early stage of SSc but also remarkably reduce the toxicity of single drug delivery. Besides, the additive BPs possess good biocompatibility and near-infrared (NIR) responsiveness, imparting the MN photothermal-controlled drug release capability. Based on these features, we have demonstrated that the traditional Chinese medicine integrated responsive MNs could effectively improve skin fibrosis and telangiectasia, reduce collagen deposition, and reduce epidermal thickness in the SSc mouse models. These results indicated that the proposed Chinese medicine integrated responsive MNs had enormous potential in clinical therapy of SSc and other diseases.

Mindin (SPON2) Is Essential for Cutaneous Fibrogenesis in a Mouse Model of Systemic Sclerosis

Systemic sclerosis is a fibrotic disease that initiates in the skin and progresses to internal organs, leading to a poor prognosis. Unraveling the etiology of a chronic, multifactorial disease such as systemic sclerosis has been aided by various animal models that recapitulate certain aspects of the human pathology. We found that the transcription factor SNAI1 is overexpressed in the epidermis of patients with systemic sclerosis, and a transgenic mouse recapitulating this expression pattern is sufficient to induce many clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin (SPON2) in fibrogenesis. Mindin is produced by SNAI1 transgenic skin keratinocytes and aids fibrogenesis by inducing early inflammatory cytokine production and collagen secretion in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Cathepsin B/NLRP3/GSDMD axis-mediated macrophage pyroptosis induces inflammation and fibrosis in systemic sclerosis

BackgroundPyroptosis is a newly discovered type of programmed cell death associated with inflammatory and fibrotic diseases. Macrophages play an important role in inducing early immune inflammation in systemic sclerosis (SSc). ObjectiveTo investigate the effect of macrophages pyroptosis on fibrosis of SSc. MethodsPyroptosis/inflammatory markers in serum and skin of SSc patients were detected. Bleomycin (BLM) was subcutaneously injected to establish SSc mouse model. The levels of pyroptosis markers, dermal thickness and collagen deposition in skin were assessed before and after the administration of pyroptosis inhibitors, including MCC950, Disulfiram and necrosulfonamide (NSA). Human-derived monocyte-macrophage cell line (THP-1) or mouse bone marrow-derived macrophages (BMDMs) were primed with lipopolysaccharide (LPS) and stimulated by silicon dioxide (SiO2) to induce cell pyroptosis. Fibroblasts from patients with SSc were co-cultured with pyroptotic THP-1 cells, and the collagen production was assessed. ResultsPyroptotic/inflammatory proteins, including NLRP3, cleaved-Caspase (CASP)1, GSDMD-N terminal and IL-18 were increased in the serum, and ASC aggregation and GSDMD were elevated in macrophages in the skin of SSc patients. SSc mice showed increased pyroptosis markers, dermal thickness and collagen deposition in skins, which were alleviated by MCC950, Disulfiram and NSA. Pyroptosis of THP-1 cells and BMDMs was induced by LPS/SiO2, and it was reduced by the inhibitors of Cathepsin B, NLRP3, CASP1 and GSDMD. Co-culture with pyroptotic THP-1 cells increased the fibrotic proteins in fibroblasts, which were alleviated by pyroptosis inhibitors. ConclusionsSSc patients and BLM-induced mouse model presented increased pyroptosis. LPS/SiO2-induced macrophage pyroptosis promoted fibrosis of SSc through Cathepsin B/NLRP3/GSDMD pathway.

Single-cell sequencing reveals the antifibrotic effects of YAP/TAZ in systemic sclerosis

Systemic sclerosis (SSc) is a heterogeneous disease with skin fibrosis. Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) is associated with fibrotic response. This work attempted to determine the precise mechanism of YAP/TAZ in SSc. Single-cell sequencing (scRNA-seq) was used to analyse the differential gene expression between SSc patients and healthy volunteers, showing that the YAP/TAZ signalling pathway was enriched in the fibroblasts of SSc patients. Subsequently, enzyme-linked immunosorbent assay and immunohistochemical analyses were conducted to examine the levels of YAP and TAZ in mild and severe SSc patients. YAP and TAZ were highly expressed in the serum and skin tissues of mild and severe SSc patients, especially severe SSc patients. Additionally, an SSc mouse model was induced by bleomycin, and the impacts of YAP/TAZ knockdown on the pathological changes in skin and lung tissues were detected by haematoxylin and eosin staining and Masson staining. Knockdown of YAP and TAZ inhibited α-SMA mRNA and protein expression in skin and lung tissues of SSc mice. Inhibition of YAP and TAZ reduced skin inflammation and thickness and repressed lung inflammation and fibrosis in SSc mice. Importantly, knockdown of YAP and TAZ synergistically inhibited inflammation and fibrosis in skin and lung tissues in SSc mice. In conclusion, this work demonstrated that knockdown of YAP and TAZ exerted a synergistic effect on alleviating SSc in mice. Thus, this work suggests that YAP/TAZ is a potential target for SSc treatment.

Driving Role of Interleukin-2-Related Regulatory CD4+ T Cell Deficiency in the Development of Lung Fibrosis and Vascular Remodeling in a Mouse Model of Systemic Sclerosis.

Systemic sclerosis (SSc) is a debilitating autoimmune disease characterized by severe lung outcomes resulting in reduced life expectancy. Fra-2-transgenic mice offer the opportunity to decipher the relationships between the immune system and lung fibrosis. This study was undertaken to investigate whether the Fra-2-transgenic mouse lung phenotype may result from an imbalance between the effector and regulatory arms in the CD4+ T cell compartment. We first used multicolor flow cytometry to extensively characterize homeostasis and the phenotype of peripheral CD4+ T cells from Fra-2-transgenic mice and control mice. We then tested different treatments for their effectiveness in restoring CD4+ Treg cell homeostasis, including adoptive transfer of Treg cells and treatment with low-dose interleukin-2 (IL-2). Fra-2-transgenic mice demonstrated a marked decrease in the proportion and absolute number of peripheral Treg cells that preceded accumulation of activated, T helper cell type 2-polarized, CD4+ T cells. This defect in Treg cell homeostasis was derived from a combination of mechanisms including impaired generation of these cells in both the thymus and the periphery. The impaired ability of peripheral conventional CD4+ T cells to produce IL-2 may greatly contribute to Treg cell deficiency in Fra-2-transgenic mice. Notably, adoptive transfer of Treg cells, low-dose IL-2 therapy, or combination therapy changed the phenotype of Fra-2-transgenic mice, resulting in a significant reduction in pulmonary parenchymal fibrosis and vascular remodeling in the lungs. Immunotherapies for restoring Treg cell homeostasis could be relevant in SSc. An intervention based on low-dose IL-2 injections, as is already proposed in other autoimmune diseases, could be the most suitable treatment modality for restoring Treg cell homeostasis for future research.

Wenyang Huazhuo Tongluo formula alleviates pulmonary vascular injury and downregulates HIF-1α in bleomycin-induced systemic sclerosis mouse model

BackgroundVascular damage, autoimmune abnormalities, and fibrosis are the three pathological features of systemic sclerosis (SSc).However, pulmonary vascular damage is the main factor affecting the progression and prognosis of SSc. The main purpose of this study was to explore the molecular mechanism of Wenyang Huazhuo Tongluo Formula in alleviating pulmonary vascular injury in bleomycin-induced SSc mouse model.MethodsMasson staining and H&E staining were used to analyze the degree of pulmonary vascular fibrosis and the infiltration of leukocyte cells in lung tissue ofbleomycin-induced SSc mouse models treated with saline (BLM group), Wenyang Huazhuo Tongluo Formula (WYHZTL group) and HIF-1α inhibitor KC7F2 (KC7F2 group). Blood vessel exudation was determined by analyzing the cell number and albumin concentration in bronchoalveolar lavage fluid using a cell counter and ELISA assay, respectively. The degree of vascular injury was assessed by measuring the expression levels of vWF, E-selectin, ICAM-1, VCAM-1, VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells using ELISA and immunofluorescence staining. Finally, the effect of Wenyang Huazhuo Tongluo Formula on the expression of HIF-1α was detected using immunofluorescence staining.ResultsWenyang Huazhuo Tongluo Formula and KC7F2 significantly inhibited bleomycin-induced pulmonary vascular fibrosis and the level of perivascular inflammatory cell infiltration. The number of cells and the concentration of albumin were significantly reduced in the bronchoalveolar lavage fluid of the WYHZTL group and KC7F2 group compared with the BLM group. In addition, treatment with Wenyang Huazhuo Tongluo Formula and KC7F2 significantly downregulated the expression levels of vWF, E-selectin, ICAM-1, VCAM-1 and HIF-1α, but upregulated the expression of VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells, compared with treatment with saline.ConclusionsThis study reveals that Wenyang Huazhuo Tongluo Formula plays a new role in the treatment of SSc by alleviating pulmonary vascular damage. Furthermore, we found that Wenyang Huazhuo Tongluo Formula alleviates pulmonary vascular injury and inhibits HIF-1α expression.

Elevated Alpha 1(I) to Alpha 2(I) Collagen Ratio in Dermal Fibroblasts Possibly Contributes to Fibrosis in Systemic Sclerosis.

Systemic sclerosis (SSc) is characterized by excessive collagen deposition in the skin and internal organs. Activated fibroblasts are the key effector cells for the overproduction of type I collagen, which comprises the α1(I) and α2(I) chains encoded by COL1A1 and COL1A2, respectively. In this study, we examined the expression patterns of α1(I) and α2(I) collagen in SSc fibroblasts, as well as their co-regulation with each other. The relative expression ratio of COL1A1 to COL1A2 in SSc fibroblasts was significantly higher than that in control fibroblasts. The same result was observed for type I collagen protein levels, indicating that α2(I) collagen is more elevated than α2(I) collagen. Inhibition or overexpression of α1(I) collagen in control fibroblasts affected the α2(I) collagen levels, suggesting that α1(I) collagen might act as an upstream regulator of α2(I) collagen. The local injection of COL1A1 small interfering RNA in a bleomycin-induced SSc mouse model was found to attenuate skin fibrosis. Overall, our data indicate that α2(I) collagen is a potent regulator of type I collagen in SSc; further investigations of the overall regulatory mechanisms of type I collagen may help understand the aberrant collagen metabolism in SSc.

Iguratimod inhibits skin fibrosis by regulating TGF-β1/Smad signalling pathway in systemic sclerosis.

Iguratimod (T-614), exerting a powerful anti-inflammatory ability, has therapeutic efficacy in multiple autoimmune diseases. However, the effect of T-614 on systemic sclerosis (SSc) is unclear. Here, we investigate the effect and molecular mechanism of T-614 in experimental SSc models. In vitro, cultured dermal fibroblasts from four SSc patients were subjected to different doses of T-614 in the presence or absence of TGF-β1stimulation. Cell proliferation, apoptosis and migration were determined by CCK-8, flow cytometry and transwell assay, respectively. Fibrosis markers and smad signalling pathway-related proteins were detected by immunoblotting and immunofluorescence. In vivo, a bleomycin-induced SSc mouse model was used to evaluate the effect of T-614 on skin fibrosis. Pathological changes in skin tissues were evaluated by HE, Masson staining and immunohistochemistry. In the study, we found T-614 inhibited TGF-β1-induced cell proliferation, migration and promoted apoptosis in a dose-dependent manner (all p<0.01). T-614 partially reversed TGF-β1-induced upregulation of fibrosis markers and phosphorylation of smad2 and smad3 and blocked p-Smad3 nuclear translocation (all p<0.05), suggesting T-614may inhibit dermal fibroblasts activation by regulating TGF-β1/smad pathway. In vivo experiments, T-614 alleviated skin thickness in bleomycin-induced SSc mice (all p<0.05). The expression of fibrosis markers and the infiltration of macrophages in skin tissue were significantly decreased after T-614 treatment (all p<0.05). Our preliminary data indicated T-614 inhibited dermal fibroblasts activation and skin fibrosis at least partly by regulating TGF-β1/smad pathway in experimental SSc models and may be a promising therapeutic agent for SSc.

α2-Antiplasmin as a Potential Therapeutic Target for Systemic Sclerosis.

Systemic sclerosis is a connective tissue disease of unknown origin that is characterized by immune system abnormalities, vascular damage, and extensive fibrosis of the skin and visceral organs. α2-antiplasmin is known to be the main plasmin inhibitor and has various functions such as cell differentiation and cytokine production, as well as the regulation of the maintenance of the immune system, endothelial homeostasis, and extracellular matrix metabolism. The expression of α2-antiplasmin is elevated in dermal fibroblasts from systemic sclerosis patients, and the blockade of α2-antiplasmin suppresses fibrosis progression and vascular dysfunction in systemic sclerosis model mice. α2-antiplasmin may have promise as a potential therapeutic target for systemic sclerosis. This review considers the role of α2-antiplasmin in the progression of systemic sclerosis.