Bleomycin-induced Mouse Model Of Scleroderma Research Articles

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31−/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

The Therapeutic Effects of Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Scleroderma

Background:Scleroderma is a multisystem disease in which tissue fibrosis is caused by inflammation and vascular damage. The mortality of scleroderma has remained high due to a lack of effective treatments. However, exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs)-Ex have been regarded as potential treatments for various autoimmune diseases, and may also act as candidates for treating scleroderma.Methods:Mice with scleroderma received a single 50 μg HUMSCs-Ex. HUMSCs-Ex was characterized using transmission electron microscopy, nanoparticle tracking analysis and nanoflow cytometry. The therapeutic efficacy was assessed using histopathology, immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and western blot.Results:HUMSCs-Ex ameliorated the deposition of extracellular matrix and suppressed the epithelial-mesenchymal transition process, and the effects lasted at least three weeks. In addition, HUMSCs-Ex promoted M1 macrophage polarization and inhibited M2 macrophage polarization, leading to the restoration of the balance of M1/M2 macrophages.Conclusion:We investigated the potential antifibrotic and anti-inflammatory effects of HUMSCs-Ex in a bleomycin-induced mouse model of scleroderma. So HUMSCs-Ex could be considered as a candidate therapy for scleroderma.

Immunomodulating role of the JAKs inhibitor tofacitinib in a mouse model of bleomycin-induced scleroderma

BackgroundJanus kinase (JAK)-signal transducer and activator of transcription (STAT) was hyperactivated in biopsies from patients with systemic sclerosis (SSc) and in several autoimmune disease models. Tofacitinib, a pan-JAK inhibitor, blocks the downstream signaling of multiple cytokines and has exhibited therapeutic efficacy in various autoimmune diseases, although its immunomodulating property in scleroderma is unclear. ObjectiveTo evaluate the effect of tofacitinib on the modulation of cytokine-producing T and B cells, and proinflammatory cells in a mouse model of SSc. MethodsBleomycin (BLM)-induced SSc was generated by intradermal injection of BLM or PBS for control. Mice received intraperitoneal tofacitinib (20 mg/kg) or vehicle 3 times per week from day 0–28. Mice were sacrificed at day 28 after the last BLM/PBS injection. ResultsTofacitinib administration significantly alleviated fibrosis of the skin and lungs in scleroderma mouse model. Furthermore, tofacitinib suppressed adaptive and innate immune responses by reducing splenocytes, total lymphocytes, CD4+ T helper cells (especially Th2 and Th17 subtypes), IL-6-producing effector B cells, PDCA-1+ dendritic cells in the spleen, and infiltration of F4/80+, CD206+ and CD163+ macrophages in the skin and lungs. Conversely, tofacitinib increased the proportions of splenic regulatory T and B cells. The mRNA expression of extracellular matrix proteins and fibrogenic cytokines was downregulated by tofacitinib in both the skin and lungs. ConclusionThese observations suggest JAK inhibition as a therapeutic approach for the treatment of inflammatory and fibrotic diseases, and highlight the potential of tofacitinib as a promising candidate for treating patients with scleroderma.

The safety and efficacy of light emitting diodes-based ultraviolet A1 phototherapy in bleomycin-induced scleroderma in mice

PurposeTo define the efficacy and safety of narrowband ultraviolet A1 (UVA1) for the treatment of dermal fibrosis in bleomycin-induced mouse model of scleroderma. Materials and methods42 DBA/2 strain mice were included in the study: healthy mice and mice with established scleroderma, treated with high or medium dose of UVA1. Non-treated groups served as control. The equipment emitting 365±5nm UVA1 radiation was used in the study. The average cumulative doses were 1200J/cm2 for high and 600J/cm2 for medium dose course. Histological analysis was performed for the evaluation of the dermal thickness and mast cells density. The expressions of p53 and Ki-67 proteins were assessed by immunohistochemical analyses. ResultsSkin thickness of mice with scleroderma, treated with high and medium dose of UVA1, were lower (272.9±113.2μm and 394±125.9μm, respectively) in comparison to the dermal thickness of non-treated animals (599±55.7μm). The dermal mast cells count in mice with scleroderma was reduced after high and medium dose treatment to 11±1.7 and 13±2.2, respectively, as compared to that in non-treated mice (23±3.0). No significant upregulation of p53 nor Ki-67 proteins was observed in the skin of healthy mice and mice with scleroderma after high- and medium-dose of UVA1. ConclusionsThe results of this study indicate that 365nm UVA1 with the cumulative doses of 1200J/cm2 and 600J/cm2 is safe and effective for the dermal fibrosis treatment.

The impact of high-dose narrowband ultraviolet A1 on dermal thickness, collagen and matrix-metalloproteinases in animal model of scleroderma

ObjectiveThe main purpose of the present study was to define the impact of high-dose of 365±5nm ultraviolet A1 (UVA1) on dermal fibrosis in the pre-established, bleomycin-induced mouse model of scleroderma. MethodsDBA/2 strain mice with the pre-established, bleomycin-induced scleroderma were irradiated with cumulative UVA1 dose of 1200J/cm2 and in parallel were challenged with prolonged administration of bleomycin. Non-treated groups served as the control. Light source emitting a narrow band UVA1 light of 365±5nm and 21mW/cm2 power density was used in the study. Histological analysis was performed for the evaluation of dermal thickness. The expressions of matrix-metalloproteinase-1 (MMP-1), matrix-metalloproteinase-3 (MMP-3), collagen types I and III were evaluated by immunohistochemical analyses. The Mann - Whitney U test was used for statistical analysis. ResultsDermal thickness in mice injected with bleomycin during all the experiment (8weeks) and irradiated with UVA1 for the last 5weeks was significantly lower than that in mice challenged only with bleomycin for 8weeks (253.96±31.83μm and 497.43±57.83μm, respectively; P=0.002). The dermal thickness after phototherapy was lower as compared with the pre-existing fibrotic changes observed after 3weeks of bleomycin injections (253.96±31.83μm and 443.87±41.76μm, respectively; P=0.002). High-dose of UVA1 induced the 5.8- and 5.2-fold increase in MMP-1 and MMP-3 expressions, respectively, and the 1.2- and 1.4-fold decrease in collagen type I and collagen type III expressions in the pre-established, bleomycin-induced scleroderma model as compared to that in the control non-irradiated mice (P=0.002). ConclusionsOur study has demonstrated that a cumulative 365±5nm UVA1 radiation dosage of 1200J/cm2 not only prevents the progression of dermal fibrosis, but also induces a regression of pre-existing fibrotic changes.

Activation of TGF-β1 by AQP3-Mediated H2O2 Transport into Fibroblasts of a Bleomycin-Induced Mouse Model of Scleroderma

Hydrogen peroxide (H2O2), the most important reactive oxygen species, mediates intracellular signal transmission and is transported into cells by aquaporin 3 (AQP3). However, it remains unclear whether AQP3 is involved in the pathogenesis of scleroderma. In this study, we examined the role of AQP3 in a bleomycin-induced mouse model of scleroderma. We observed that H2O2 and AQP3 levels in mouse skin increased with the bleomycin injection period relative to phosphate-buffered saline-injected control mice. AQP3 mRNA and protein levels were higher in bleomycin mice fibroblasts than in phosphate-buffered saline mice fibroblasts, and AQP3 immunofluorescence signals in the cytoplasm and cell membrane of bleomycin mice fibroblasts were much stronger than those in phosphate-buffered saline mice fibroblasts. Bleomycin-induced increases in H2O2, transforming growth factor-β1, collagen type I, and collagen type III levels in bleomycin mice fibroblasts were blocked by silencing AQP3. In addition, silencing AQP3 decreased H2O2 levels, transforming growth factor-β1 expression, and fibrosis in the scleroderma mouse model. These results demonstrate that AQP3-mediated transport of H2O2 into bleomycin mice fibroblasts activated transforming growth factor-β1, and silencing AQP3 is a potential approach for treating scleroderma.

A convenient method for producing the bleomycin-induced mouse model of scleroderma by weekly injections using a methylcellulose gel

Systemic sclerosis (SSc) is a connective tissue disease characterized by vasculopathy, excessive accumulation of extracellular matrix, and fibrosis of the skin and internal organs. An animal model of SSc, the bleomycin-induced mouse model, has been established and used extensively to investigate the pathogenesis of SSc and to seek novel therapeutic agents. We recently developed thermo-reversible combination gels that can be injected subcutaneously and are made in aqueous solution by forming a complex coacervate with the substance of interest and cationic macromolecules, followed by co-formulation with methylcellulose (MC) as a negative thermosensitive polysaccharide. The objective of this study was to demonstrate whether weekly injections of bleomycin using combination gels loaded with bleomycin can induce the skin fibrosis model of SSc in susceptible mouse strains. A low molecular weight MC (4%) gel with 4.5% ammonium sulfate was made in aqueous solution, and mixed with bleomycin. This was injected subcutaneously into female C3H/He mice at weekly intervals. Control mice were injected with the gel made with phosphate-buffered saline. After 4 weeks, histological examination and gene expression assays of cytokines were performed. Examination in vitro showed that more than 80% of the bleomycin was released from the gel by the 4th day. Histological examination showed that dermal thickness increased in the MC-bleomycin-injected group compared with the control, and semi-quantitative analysis indicated that the extent of inflammation did not differ between the groups. In the MC-bleomycin-injected group, dermal fibrosis assessed with the Masson-Trichrome stain and numbers of alpha-smooth muscle actin-positive fibroblastic cells also increased. The procedure for inducing scleroderma in which bleomycin is injected weekly as an easily-made gel system using methylcellulose, can induce dermal fibrosis in susceptible mice without causing inflammation. We believe this system represents a time- saving and convenient procedure that should facilitate research on SSc.

Induction of Animal Model of Scleroderma with Repeated Injection of Bleomycin

Objective: To induce a mouse model of scleroderma with repeated bleomycin injections for research into human scleroderma at our research laboratory. Methods: The protocol of Yamamoto et al. was replicated to establish the bleomycin-induced mouse model of scleroderma. Results: A mouse model of scleroderma was induced by repeated subcutaneous injections of bleomycin. The dermal thickness increased with homogeneous and thickened collagen bundles. Semiquantitative measurements of dermal fibrosis were prominent in bleomycin-treated mice. Conclusion: A mouse model of scleroderma was induced with repeated injections of bleomycin at our laboratory.

Effectiveness of etanercept in bleomycin-induced experimental scleroderma

To evaluate the effects of etanercept and thalidomide in the mouse model of bleomycin-induced scleroderma (BLM-IS). This study involved four groups (n = 8 mice in each group). Dermal sclerosis was induced by repeated subcutaneous injections of BLM (10 microg) for 4 weeks in BALB/c mice. Control group received only phosphate-buffered saline. The second group received only BLM; the third and fourth groups were also given an intraperitoneal injection of 100 microg etanercept or 150 mg/kg thalidomide, respectively. BLM increased serum TGF-beta1, tissue hydroxyproline levels and expression of alpha-smooth muscle actin (alpha-SMA), and dermal fibrosis was histopathologically prominent. Although thalidomide had no significant effect, etanercept caused decreases in levels of serum TGF-beta1, tissue hydroxyproline and number of alpha-SMA-positive cells. Inhibition of TNF-alpha with etanercept in BLM-IS was resulted in a significant reduction of the dermal sclerosis, collagen accumulation and the number of infiltrating myofibroblastic cells. TNF-alpha may play a key role in the progression of BLM-IS and TNF-alpha antagonists may be useful in the management of scleroderma.

Anti-sclerotic effect of transforming growth factor-beta antibody in a mouse model of bleomycin-induced scleroderma.

Recent studies have demonstrated the evidence of the crucial role of transforming growth factor-beta (TGF-beta) in the pathogenesis of tissue fibrosis; however, its precise role has not been fully elucidated. Administration of anti-TGF-beta antibody is shown to be effective for inhibiting lung fibrosis induced by bleomycin in an experimental animal model. We have recently established a mouse model for scleroderma by repeated injections of bleomycin. In this study, we examined whether the suppression of TGF-beta leads to the improvement of dermal sclerotic lesion by using this model. We induced dermal sclerosis in C3H mice by subcutaneous injections of bleomycin (100 microg/ml) for 3 weeks, and separate groups of mice were also injected with bleomycin with either anti-TGF-beta antibody (10 microg/ml) or control normal rabbit serum for 3 weeks. Thus treated skins were harvested and analyzed for histological sclerosis, serum cytokine, and influx of mast cells and eosinophils, both of which are known to release fibrogenic cytokines or several mediators responsible for tissue fibrosis. The result showed that anti-TGF-beta antibody caused a significant reduction in cutaneous sclerosis characterized by histological features and hydroxyproline contents. Examination of tissue sections also revealed a significant suppression of influx of mast cells and eosinophils. Serum interleukin-4 (IL-4) and IL-6 levels determined by enzyme-linked immunosorbent assay exhibited a significant reduction after anti-TGF-beta antibody treatment. Our results suggest that administration of an antibody against TGF-beta is useful in preventing experimental dermal sclerosis induced by bleomycin and raises a possibility of the therapeutic approach of anti-TGF-beta antibody in scleroderma.