Scleroderma Fibroblasts Research Articles

Transplantation of in vitro prefabricated adipose organoids attenuates skin fibrosis by restoring subcutaneous fat and inducing dermal adipogenesis.

Localized scleroderma is a complex autoimmune disease characterized by dermal fibrosis and loss of cutaneous fat. While cytotherapy offers a promising treatment option, stem cell transplantation results in low survival rates and fails in target cell differentiation. In this study, we aimed to prefabricate syngeneic adipose organoids (ad-organoids) using microvascular fragments (MVFs) via three-dimensional (3D) culturing and transplant them beneath the fibrotic skin to restore subcutaneous fat and reverse the pathological manifestation of localized scleroderma. We employed 3D culturing of syngeneic MVFs with stepwise angiogenic and adipogenic induction to produce ad-organoids and evaluated their microstructure and paracrine function in vitro. C57/BL6 mice with induced skin scleroderma were treated with adipose-derived stem cells (ASCs), adipocytes, ad-organoids, and Matrigel, and the therapeutic effect was assessed histologically. Our results showed that ad-organoids derived from MVF contained mature adipocytes and a well-established vessel network, secreted multiple adipokines, promoted adipogenic differentiation of ASCs, and suppressed proliferation and migration of scleroderma fibroblasts. Subcutaneous transplantation of ad-organoids reconstructed the subcutaneous fat layer and stimulated dermal adipocyte regeneration in bleomycin-induced scleroderma skin. It reduced collagen deposition and dermal thickness, attenuating dermal fibrosis. Moreover, ad-organoids suppressed macrophage infiltration and promoted angiogenesis in the skin lesion. In conclusion, 3D culturing of MVFs with stepwise angiogenic and adipogenic induction is an effective strategy for the fabrication of ad-organoids, and the transplantation of prefabricated ad-organoids can improve skin sclerosis by restoring cutaneous fat and attenuating skin fibrosis. These findings offer a promising therapeutic approach for the treatment of localized scleroderma.

Fat transplantation induces dermal adipose regeneration and reverses skin fibrosis through dedifferentiation and redifferentiation of adipocytes

BackgroundLocalized scleroderma causes cosmetic disfigurement, joint contractures, and other functional impairment, but no currently available medications can reverse the resulting skin lesions. Fat grafting is beneficial for reversing skin fibrosis; however, the mechanism by which adipose tissue transplantation contributes to lesion improvement has not been fully clarified. The purpose of our study was to verify the therapeutic effect of fat grafts in reversing skin fibrosis.MethodsInguinal fat pads from AdipoqCreER+;mT/mG mice, which were treated with tamoxifen, were transplanted to the skin lesion in bleomycin-treated wild-type C57 mice. Tdtomato transgenic mice-derived adipocytes, adipose-derived stem cells (ASCs), dedifferentiated adipocytes (DAs) were embedded in matrigel and transplanted beneath the skin lesion of bleomycin-treated wild-type C57 mice. A transwell co‐culture system was used to verify the effect of ASCs, adipocytes or DAs on scleroderma fibroblasts or monocytes.ResultsAdipocytes from the fat grafts could undergo dedifferentiation and redifferentiation for dermal adipose tissue re-accumulation within the skin lesion. Moreover, compared with ASCs and adipocytes, DAs show greater potency of inducing adipogenesis. ASCs and DAs showed comparable effect on inducing angiogenesis and suppressing macrophage infiltration in fibrotic skin. Co-culture assay showed that DAs and ASCs were able to reduce fibrosis-related genes in human scleroderma fibroblasts and drive M2 macrophage polarization.ConclusionOur results indicated that adipocytes would transform into a more functional and dedifferentiated state and reverse dermal fibrosis, by promoting dermal adipose tissue regeneration, improving angiogenesis, suppressing macrophage-mediated inflammation and myofibroblast accumulation.

Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-β signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1’s association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.

Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-β/Wnt3a Is Mediated by GLI2 Upregulation.

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-β signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-β/SMAD3 co-operates with Wnt3a/β-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of β-catenin and GLI expression or activity abolishes TGF-β/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-β, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors

Sildenafil Counteracts the In Vitro Activation of CXCL-9, CXCL-10 and CXCL-11/CXCR3 Axis Induced by Reactive Oxygen Species in Scleroderma Fibroblasts.

Simple SummarySystemic sclerosis (SSc) is a complex autoimmune disease characterized by different clinical features and a high risk of mortality due to multi-organ fibrosis. Oxidative stress plays a key role in SSc pathogenesis, and an altered redox state could be responsible for abnormal inflammatory condition, tissue damage and fibrosis. Different pro-inflammatory mediators (cytokines and chemokines) occur from the earlier to the last stage of this disease, but their relationship with clinical findings is still unclear. To this purpose, studies are still required which work to find biomarkers mirroring the disease progression and potential pharmacological targets improving treatment response. In this study, we demonstrated that the vasoactive drug sildenafil, a phosphodiesterase type 5 inhibitor commonly used to treat pulmonary hypertension, reduces the expression and secretion of pro-inflammatory chemokines in dermal fibroblasts isolated from SSc patients exposed to reactive oxygen species. We demonstrated that this effect relies on the ability of sildenafil to interfere with the pro-inflammatory pathways involved in the expression of these molecules and its capacity to counteract the plasma membrane translocation of their specific receptor. These results sustain clinical studies to consider the use of sildenafil in preventing tissue damage and fibrosis in systemic sclerosis by targeting essential biomarkers of disease progression and reducing the oxidative-stress-induced inflammation.Oxidative stress plays a key role in systemic sclerosis (SSc) pathogenesis, and an altered redox homeostasis might be responsible for abnormal inflammatory status, fibrosis and tissue damage extension. In this study, we explored the effect of the phosphodiesterase type 5 inhibitor sildenafil in modulating the activation of the CXCL-9, -10, -11/CXCR3 axis, which is fundamental in the perpetuation of inflammation in different autoimmune diseases, in the cell culture of SSc human dermal fibroblasts exposed to a pro-oxidant environment. We observed that sildenafil significantly reduced gene expression and release of CXCL-9, -10 and -11, inhibited the CXCR3 action and suppressed the activation of STAT1-, JNK- and p38MAPK pathways. This in vitro study on dermal fibroblasts supports clinical studies to consider the efficacy of sildenafil in preventing tissue damage and fibrosis in SSc by targeting central biomarkers of disease progression, vascular injuries and fibrosis and reducing the pro-inflammatory activation induced by oxidative stress.

The hard problem: Mechanotransduction perpetuates the myofibroblast phenotype in scleroderma fibrosis.

The effector cells ultimately responsible for fibrosis are myofibroblasts. In the fibrotic autoimmune connective tissue disease scleroderma, myofibroblasts are autonomously activated, and retain their phenotype upon culturing. Since the 1990s, researchers have exploited this fact to use scleroderma fibroblasts as a model system to uncover the fundamental mechanisms underlying myofibroblast persistence in fibrotic conditions. These studies have suggested that an autocrine transforming growth factor (TGF)beta signaling loop is insufficient to explain the persistent myofibroblast phenotype but instead support the hypothesis that fibrotic myofibroblasts possess an intrinsically activated pro-adhesive signaling pathway, and that this contributes to the perpetuation of pathological fibrosis. This review focuses on these observations.

CD47 prevents the elimination of diseased fibroblasts in scleroderma

Scleroderma is a devastating fibrotic autoimmune disease. Current treatments are partly effective in preventing disease progression but do not remove fibrotic tissue. Here, we evaluated whether scleroderma fibroblasts take advantage of the “don’t-eat-me-signal” CD47 and whether blocking CD47 enables the body’s immune system to get rid of diseased fibroblasts. To test this approach, we used a Jun-inducible scleroderma model. We first demonstrated in patient samples that scleroderma upregulated transcription factor JUN and increased promoter accessibilities of both JUN and CD47. Next, we established our scleroderma model, demonstrating that Jun mediated skin fibrosis through the hedgehog-dependent expansion of CD26+Sca1– fibroblasts in mice. In a niche-independent adaptive transfer model, JUN steered graft survival and conferred increased self-renewal to fibroblasts. In vivo, JUN enhanced the expression of CD47, and inhibiting CD47 eliminated an ectopic fibroblast graft and increased in vitro phagocytosis. In the syngeneic mouse, depleting macrophages ameliorated skin fibrosis. Therapeutically, combined CD47 and IL-6 blockade reversed skin fibrosis in mice and led to the rapid elimination of ectopically transplanted scleroderma cells. Altogether, our study demonstrates the efficiency of combining different immunotherapies in treating scleroderma and provides a rationale for combining CD47 and IL-6 inhibition in clinical trials.

Galectin-1 and Galectin-3 Expression in Lesional Skin of Patients With Systemic Sclerosis-Association With Disease Severity.

The aim of this study was to evaluate Gal-1 and Gal-3 expression in the lesional skin of patients with scleroderma regarding disease severity and organ involvement. A cross-sectional study was conducted on patients diagnosed as systemic sclerosis (SSc), after informed consent. Clinical and serological profiles were reviewed from medical records. Lesional skin biopsies were taken by losange incision from patients. Samples were analyzed by immunohistochemistry and compared with normal skin of a healthy patient. Parametric statistical analysis was done with Student t test and Pearson coefficient. Significance was established as p ≤ 0.05 with a 95% confidence interval. Biopsies of 10 patients and a healthy control (9 female, 1 male) were analyzed. The mean age was 54.5 years (18-74 years). Four of 10 patients had diffuse, 4 had limited scleroderma, 1 had overlap syndrome, and 1 had sclerodermiform graft-versus-host disease. The mean fibroblasts count per field was 13.2 in scleroderma versus 7.2 in normal skin. The mean expression of Gal-1 in scleroderma fibroblasts was 13% (0%-56%) and 47.5% for Gal-3 (6.5%-95.5%); in normal skin, the mean expression was 91% (90%-95%) for Gal-1 and 97% (89%-100%) for Gal-3. A higher Gal-3 expression in scleroderma (within its lower expression compared with normal skin) was associated with pulmonary artery hypertension (p = 0.004) and to a higher modified Rodnan's skin score (p = 0.0003). In a similar manner, anti-centromere antibodies were associated with a higher Gal-1 expression in SSc skin fibroblasts (p = 0.04). Gal-1 and Gal-3 had a lower expression in scleroderma lesional skin compared with a normal control. We found a significant correlation between a higher Gal-3 expression (within the lower ones compared with normal skin) in fibroblasts from SSc patients and severe disease (pulmonary hypertension and a higher modified Rodnan's skin score) compared with patients with lower expression of this protein. Similarly, the presence of anti-centromere antibodies was associated with a higher expression of Gal-1 within this group of patients.

IL-17A Dissociates Inflammation from Fibrogenesis in Systemic Sclerosis

IL-17A is abundant in scleroderma but its role in fibrogenesis is controversial. We interrogated the role of IL-17A in extracellular matrix deposition and inflammation by investigating its effects on keratinocytes and fibroblasts cross-talk and in organotypic skin cultures. Keratinocyte-conditioned media of resting, IL-17A-, and/or transforming growth factor-β-primed primary keratinocytes were used to stimulate healthy donors and scleroderma fibroblasts. Alternatively, organotypic cultures of full human skin were challenged with these cytokines. Keratinocyte-conditioned media tilted the balance of col-I to matrix metalloproteinase-1 production by fibroblasts in favor of matrix metalloproteinase-1, significantly more so in healthy donors than in scleroderma, resulting in enhanced extracellular matrix turnover, further increased by IL-17A. In organotypic skin, transforming growth factor-β induced an extensive pro-fibrotic gene signature, including the enhanced expression of several collagen genes associated with Wnt signaling. IL-17A strongly promoted the expression of pro-inflammatory genes, with no direct effects on collagen genes, and attenuated Wnt signaling induced by transforming growth factor-β. In this model, at the protein level, IL-17A significantly decreased col-I production. Our data strongly support a pro-inflammatory and antifibrogenic activity of IL-17A in the context of keratinocyte-fibroblast interaction and in full skin. These data help in directing and interpreting targeted therapeutic approaches in scleroderma.

2-Methoxyestradiol inhibits hypoxia-induced scleroderma fibroblast collagen synthesis by phosphatidylinositol 3-kinase/Akt/mTOR signalling.

To investigate the mechanism of 2-methoxyestradiol (2-ME) in inhibiting hypoxia-induced collagen synthesis of fibroblasts in SSc. The expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and connective tissue growth factor (CTGF) in skin specimens derived from SSc patients and healthy volunteers were examined by immunohistochemistry. HIF-1α was knocked down by lentiviral transduction, and SSc dermal fibroblasts cultured under normoxic (21% O2) or hypoxic (1% O2) condition were treated with PI3K inhibitor LY294002, rapamycin or 2-ME (25 μM). The protein levels of HIF-1α, CTGF, collagen I, p-Akt and p-mTOR were examined by western blotting or immunofluorescence. Apoptosis and cell cycle of fibroblasts were assessed by flow cytometry and by measuring caspase 3 activity, and cell proliferation was evaluated by Cell Counting Kit-8. The expressions of HIF-1α and CTGF were increased in skins of SSc patients compared with healthy controls. Hypoxia up-regulated the protein levels of HIF-1α, CTGF and collagen I in SSc fibroblasts. In contrast, 2-ME inhibited PI3K/Akt/mTOR pathway and down-regulated protein levels of HIF-1α, CTGF and collagen I. Knockdown of HIF-1α reduced expressions of CTGF and collagen I, which were further down-regulated by 2-ME intervention. Moreover, 2-ME promoted the apoptosis and inhibited the proliferation of SSc fibroblasts by arresting the cell cycle at the G2/M phase. 2-ME reduced the production of CTGF and collagen I in SSc fibroblasts induced by hypoxia through PI3K/Akt/mTOR/HIF-1α signalling and inhibited the proliferation of fibroblasts. These findings suggested that 2-ME could be employed as a promising antifibrotic therapy for SSc.

Methyl-CpG-binding protein 2 mediates antifibrotic effects in scleroderma fibroblasts

ObjectiveEmerging evidence supports a role for epigenetic regulation in the pathogenesis of scleroderma (SSc). We aimed to assess the role of methyl-CpG-binding protein 2 (MeCP2), a key epigenetic regulator, in...

211 NOX1/NOX4 inhibition blocks the profibrotic activity of scleroderma fibroblasts

211 NOX1/NOX4 inhibition blocks the profibrotic activity of scleroderma fibroblasts

Regulatory mechanisms of collagen expression by interleukin-22 signaling in scleroderma fibroblasts

BackgroundVarious cytokines have been indicated to be involved in the pathogenesis of systemic sclerosis (SSc). IL-22 is one of the member of IL-10 cytokine family, and several studies have implicated IL-22 signaling in the pathogenesis of autoimmune diseases. ObjectivesTo clarify the role of IL-22 in the regulatory mechanism of ECM expression and to determine the contribution of IL-22 to the phenotype of SSc. MethodsThe effect of IL-22 on ECM expression in normal fibroblasts was determined by using PCR array, real-time PCR and immunoblotting. microRNA expression was evaluated by real-time PCR. The expression levels of IL-22 in the skin and sera were determined by using immunohistochemical staining and ELISA. ResultsIL-22 significantly increased the expression of type I collagen protein without changing its mRNA levels in cultured normal human dermal fibroblast. The expression of let-7a, one of the microRNAs which have negative effect on type I collagen expression, was significantly decreased by the treatment with IL-22 in dermal fibroblasts. There was no significant difference in the serum levels of IL-22 between SSc patients and control subjects. However, the expression of IL-22 was detected in the infiltrated lymphocytes in the SSc dermis, but not in normal dermis. IL-22 receptors were expressed in both normal and SSc dermal fibroblasts to the similar extent. ConclusionIL-22 expressed in infiltrated lymphocytes may stimulate the up-regulation of type I collagen protein in dermal fibroblasts via let-7a down-regulation in SSc skin.

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1);...

Antioxidants and NOX1/NOX4 inhibition blocks TGFβ1-induced CCN2 and α-SMA expression in dermal and gingival fibroblasts.

TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents.

Dysregulated interleukin-23 signalling contributes to the increased collagen production in scleroderma fibroblasts via balancing microRNA expression.

The overexpression of IL-12 family cytokines is implicated in the pathogenesis of SSc, but their exact role is still unclear. The aim of this study was to investigate the regulation of extracellular matrix expression by IL-23 and its contribution to the phenotype of SSc. The mRNA expression was determined by PCR array and real-time PCR. The expression levels of proteins were determined by immunoblotting and immunohistochemical staining. The effect of IL-23 on dermal fibrosis in vivo was examined in a mouse model of SSc induced by bleomycin injection. Among the IL-12 family members, IL-23 decreased expression of type I collagen protein in cultured normal dermal fibroblasts. We found that miR-4458 and miR-18a mediated the reduction of collagen expression by IL-23. On the contrary, IL-23 up-regulated type I collagen expression in SSc fibroblasts. The paradoxical effects of IL-23 in SSc fibroblasts were also mediated by the balance between miR-4458 and miR-18a expression. Moreover, we revealed that injection of IL-23 into the mouse skin accelerated skin fibrosis. This is the first study to report that the balance of two miRNAs is involved in the collagen dysregulation in SSc fibroblasts. Clarification of the regulatory mechanism of tissue fibrosis by IL-23 in SSc skin may lead to a better understanding of this disease and new therapeutic approaches.

IL-13 mediates collagen deposition via STAT6 and microRNA-135b: a role for epigenetics.

Systemic sclerosis is an autoimmune connective tissue disease in which T cells play a prominent role. We and others have previously demonstrated a role for T cell-derived IL-13 in mediating the induction of collagen in dermal fibroblasts and that blockade with IL-13 antibodies attenuates this increase. In this study we want to probe the signalling that underpins IL-13 mediated matrix deposition. Isolated dermal fibroblasts were incubated with recombinant IL-13 and gene expression by qRT-PCR was performed for collagen1A1 and TGF-β1. Small interfering RNA (siRNA) was used to knock down STAT6 and a small molecule inhibitor was also used to block this pathway. MiR-135b was transfected into fibroblasts plus and minus IL-13 to see if this miR plays a role. miR-135b was measured in systemic sclerosis fibroblasts isolated from patients and also in serum. Results showed that IL-13 increased collagen expression and that this is independent from TGF-β1. This is dependent on STAT6 as targeting this blocked induction. MiR-135b reduces collagen induction in fibroblasts and scleroderma fibroblasts have lower constitutive levels of the miR. We further demonstrate that miR135b is repressed by methylation and may include MeCP2. In conclusion we show that STAT6 and miR-135b regulate IL-13-mediated collagen production by fibroblasts.

Global DNA hypomethylation and hypoxia-induced expression of the ten eleven translocation (TET) family, TET1, in scleroderma fibroblasts.

The precise mechanisms of tissue fibrosis have not yet been elucidated in systemic sclerosis (SSc). However, studies of the regulation of DNA methylation, the most widely studied epigenetic mechanism, have confirmed the involvement of the TET family proteins, recently identified DNA demethylases, in the pathogenesis of SSc. The mRNA levels of TET family members were compared in normal and SSc fibroblasts. The effects of hypoxia and siRNA specific to HIF-1α on TET expression were also examined. Global methylation status was analysed by LUMA. The presence of 5-hydroxymethylcytosine (5hmC) in SSc was examined by immunohistochemistry. The level of TET1 mRNA in SSc fibroblasts was elevated by 1.68 fold compared with that of normal fibroblasts, but the expression levels of TET2 and TET3 were comparable between both cell types. The expression levels of DNMT1 and DNMT3B mRNA have a tendency to elevate in SSc fibroblasts. Among TET family members, the expression of TET1 was exclusively induced by hypoxia via HIF-1α-independent pathways in SSc fibroblasts, but not in normal fibroblasts. The methylation level was decreased in SSc fibroblasts relative to normal fibroblasts, and 5hmC was present in dermal fibroblasts of skin sections from patients with SSc. TET1 expression in SSc fibroblasts was abnormally regulated in the hypoxic environment and accompanied by global DNA hypomethylation, suggesting the involvement of aberrant DNA methylation in the pathogenesis of SSc.

Upregulation of the N-formyl Peptide receptors in scleroderma fibroblasts fosters the switch to myofibroblasts.

Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis. N-Formyl peptide (fMLF) receptors (FPRs) are chemotactic receptors involved in inflammation. Three FPRs have been identified: FPR1, FPR2, and FPR3. We have examined, by RT-PCR, Western blot and immunohistochemistry, FPRs expression in skin fibroblasts from 10 normal subjects and 10 SSc patients, showing increased expression in SSc fibroblasts. Several functions of FPRs occur through the interaction with a region of the urokinase-type plasminogen activator receptor (uPAR88-92), able to interact with FPRs and to mediate urokinase (uPA) or fMLF-dependent cell migration. Soluble uPAR84-95 peptide can act as a direct ligand of FPRs. Furthermore, uPA or its aminoterminal fragment (ATF) can promote the exposure of the uPAR88-92 region. The WKYMVm peptide is a FPRs pan-agonist. We investigated the functional effects of these agonists on normal and SSc fibroblasts. ATF, uPAR84-95, and WKYMVm regulated adhesion, migration, and proliferation of normal fibroblasts. Despite FPR overexpression, the response of SSc fibroblasts to the same agonists was greatly reduced, except for the proliferative response to ATF. SSc fibroblasts showed increased α-smooth muscle actin expression and improved capability to induce wound closure. Indeed, they overexpressed a cleaved uPAR form, exposing the uPAR88-92 region, and vitronectin, both involved in fibrosis and in the fibroblast-to-myofibroblast transition. FPR stimulation promoted α-smooth muscle actin expression in normal fibroblasts as well as motility, matrix deposition, αvβ5 integrin expression, and radical oxygen species generation in normal and SSc fibroblasts. This study provides evidence that FPRs may play a role in fibrosis and in the fibroblast-to-myofibroblast transition.

A reactive oxygen species-mediated loop maintains increased expression of NADPH oxidases 2 and 4 in skin fibroblasts from patients with systemic sclerosis.

Reactive oxygen species (ROS) contribute to the pathogenesis of fibrosis in systemic sclerosis (SSc; scleroderma), and NADPH oxidase (NOX) is an important source of ROS. Since the role of single NOX isoforms has not been previously investigated in SSc, this study was undertaken to assess the expression of NOX in SSc fibroblasts compared to normal healthy cells and to analyze their role in cell activation. Expression of NOX isoforms in dermal fibroblasts from patients with SSc and healthy control subjects was analyzed by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. NOX isoforms were silenced using small interfering RNA. Production of ROS was measured by fluorometry and confocal microscopy. Scleroderma fibroblasts showed up-regulation of NOX-2 and NOX-4 protein and messenger RNA (mRNA) expression. Treatment of the cells with diphenyleneiodonium, a nonselective inhibitor of flavin-containing enzymes, and silencing of NOX2 and NOX4 decreased the production of ROS as well as the expression of type I collagen and α-smooth muscle actin in SSc fibroblasts. ROS generated by NOX-2 and NOX-4 were involved in DNA damage and activation of a DNA repair checkpoint. Incubation of healthy control fibroblasts with platelet-derived growth factor (PDGF) or with IgG isolated from SSc patient serum enhanced the expression of NOX2 and NOX4 mRNA, via ROS, in a time-dependent manner. Treatment with actinomycin D, a transcription inhibitor, reversed the effects of PDGF stimulation but not the effects of SSc IgG. Both NOX2 and NOX4 generate ROS in SSc fibroblasts and play a critical role in cell activation and DNA damage. Expression of NOX-2 and NOX-4 in SSc fibroblasts is maintained by a ROS-mediated loop.