Simultaneous inhibition of c-Abl and Src kinases abrogates the exaggerated expression of profibrotic genes in cultured systemic sclerosis dermal fibroblasts.

OBJECTIVES: To examine the effects of simultaneous inhibition of c-Abl and Src kinases on the gene expression and in vitro production of profibrotic molecules by dermal fibroblasts from patients with diffuse systemic sclerosis (SSc) of recent onset. METHODS: Dermal fibroblasts from normal individuals or from patients with diffuse cutaneous SSc fulfilling the American College of Rheumatology/EULAR SSc classification criteria were cultured in media containing increasing concentrations of the dual c-Abl and Src kinase inhibitor Bosutinib for 24 h. Total soluble collagen in cell culture supernatants was quantified. Western blots were performed for quantitative assessment of type I collagen, fibronectin, and α-smooth muscle actin (α-SMA) production. Quantitative PCR was performed to examine the effects of Bosutinib on the expression of profibrotic and TGF-ß-responsive genes in cultured SSc dermal fibroblasts. RESULTS: Simultaneous inhibition of c-Abl and Src kinases with Bosutinib reduced the expression of numerous fibrosis-associated genes including COL1A1, COL1A3, FN, and TGFß and the production of the corresponding proteins by SSc dermal fibroblasts. Bosutinib also decreased the transition of normal dermal fibroblasts into activated myofibroblasts induced by TGF-ß as evidenced by reduction of α-SMA in cell extracts from normal and SSc dermal fibroblasts. CONCLUSIONS: Simultaneous inhibition of c-Abl and Src kinases with Bosutinib abrogates the exaggerated expression of genes encoding fibrillar collagens, fibronectin, and TGF-ß-responsive genes and reduces type I collagen, fibronectin and α-SMA production by SSc dermal fibroblasts in vitro. Bosutinib also abrogates TGF- ß-induced transition of normal fibroblasts to activated myofibroblasts. These results indicate that inhibition of c-Abl and Src kinases activity may be an effective disease modifying antifibrotic therapeutic intervention for SSc.

Patterns and predictors of skin score change in early diffuse systemic sclerosis from the European Scleroderma Observational Study.

OBJECTIVES: Our aim was to use the opportunity provided by the European Scleroderma Observational Study to (1) identify and describe those patients with early diffuse cutaneous systemic sclerosis (dcSSc) with progressive skin thickness, and (2) derive prediction models for progression over 12 months, to inform future randomised controlled trials (RCTs). METHODS: The modified Rodnan skin score (mRSS) was recorded every 3 months in 326 patients. 'Progressors' were defined as those experiencing a 5-unit and 25% increase in mRSS score over 12 months (±3 months). Logistic models were fitted to predict progression and, using receiver operating characteristic (ROC) curves, were compared on the basis of the area under curve (AUC), accuracy and positive predictive value (PPV). RESULTS: 66 patients (22.5%) progressed, 227 (77.5%) did not (33 could not have their status assessed due to insufficient data). Progressors had shorter disease duration (median 8.1 vs 12.6 months, P=0.001) and lower mRSS (median 19 vs 21 units, P=0.030) than non-progressors. Skin score was highest, and peaked earliest, in the anti-RNA polymerase III (Pol3+) subgroup (n=50). A first predictive model (including mRSS, duration of skin thickening and their interaction) had an accuracy of 60.9%, AUC of 0.666 and PPV of 33.8%. By adding a variable for Pol3 positivity, the model reached an accuracy of 71%, AUC of 0.711 and PPV of 41%. CONCLUSIONS: Two prediction models for progressive skin thickening were derived, for use both in clinical practice and for cohort enrichment in RCTs. These models will inform recruitment into the many clinical trials of dcSSc projected for the coming years. TRIAL REGISTRATION NUMBER: NCT02339441.

Serum levels of soluble carbonic anhydrase IX are decreased in patients with diffuse cutaneous systemic sclerosis compared to those with limited cutaneous systemic sclerosis.

Hypoxia may play an important role in the pathogenesis of systemic sclerosis (SSc). Carbonic anhydrase IX (CA IX) is one of the hypoxia markers and its extracellular domain can be released into the serum. However, the clinical significance of serum CA IX levels in SSc is still unknown. The aim of this study is to evaluate the possibility that serum CA IX levels can be a specific disease marker of SSc. Serum samples were obtained from SSc patients and healthy controls. Patients diagnosed as scleroderma spectrum disorder (SSD), who did not fulfill the ACR criteria of SSc but were thought that they might develop SSc in the future, were also included in this study. Serum CA IX levels were measured with specific enzyme-linked immunosorbent assays. SSD patients had significantly lower CA IX levels than diffuse cutaneous SSc (dcSSc), limited cuntaneous SSc (lcSSc) and healthy control groups. Also, we found a significant decrease in the values in dcSSc patients compared to those of lcSSc patients. Serum levels of CA IX may be useful for the differentiation of lcSSc from SSD. Decreased serum CA IX levels in spite of the presence of hypoxia in SSc may indicate an impaired response to hypoxia, which leads to the persistent hypoxic condition. Our results suggest that the abnormal response to hypoxia may already exist in SSD patients, and may be involved in its pathogenesis.

Serum levels of matrix metalloproteinase-13 in patients with eosinophilic fasciitis.

Matrix metalloproteinase-13 (MMP-13), a member of the collagenase family, has been implicated in the pathogenesis of connective tissue diseases characterized by extracellular matrix remodeling. Since serum MMP-13 levels reflect disease severity of systemic sclerosis and localized scleroderma, we evaluated the clinical significance of serum MMP-13 levels in eosinophilic fasciitis (EF). All the EF patients had serum MMP-13 levels lower than the mean - 2SD of healthy controls. Serum MMP-13 levels were also significantly decreased in EF patients compared with diffuse cutaneous systemic sclerosis, limited cutaneous systemic sclerosis, and generalized morphea patients. Although serum MMP-13 levels did not reflect any clinical and serological features of EF, these results indicate that MMP-13 may be involved in the development of this disease.

Elevated MMP-7 levels in patients with systemic sclerosis: correlation with pulmonary involvement.

BACKGROUND: Fibrosis is characterized by an excessive accumulation of connective tissue because of an imbalance between synthesis and degradation of extracellular matrix proteins. Systemic sclerosis (SSc) is a prototypic chronic inflammatory disease leading to a severe fibrosis of the skin and many internal organs. QUESTIONS ADDRESSED: We investigated whether serum MMP-7 levels reflect the activity of the fibrotic reaction in systemic sclerosis. EXPERIMENTAL DESIGN: Serum samples were obtained from 123 patients with systemic sclerosis. MMP-serum levels of all patients with SSc were compared with age-matched healthy controls. RESULTS: Significantly increased median serum MMP-7 levels were found in patients with SSc when compared with controls. The median MMP-7 serum level of patients with lung fibrosis (LF) was significantly higher compared with those without LF. Accordingly, patients with dyspnea and DLCO (diffusion capacity of the lung for carbon monoxide) levels below 60% showed significantly higher median MMP-7 levels. CONCLUSIONS: Elevated MMP-7 levels are associated with an advanced stage of SSc and LF. These data suggest that in SSc MMP-7 is involved in the process of fibrotic tissue remodelling.

Decreased secretion of MMP by non-lesional late-stage scleroderma fibroblasts after selection via activation of the apoptotic Fas-pathway.

Our hypothesis is that the development of lesional areas of skin in patients with systemic sclerosis (SSc) originates from the selection of profibrotic cell subpopulations within their non-lesional skin areas, due to their greater resistance to apoptosis. Sensitivity to apoptosis of early-stage or late-stage SSc fibroblasts as well as of healthy cells was compared using extrinsic or intrinsic apoptotic pathway-inducers. Subpopulations of non-lesional SSc cells and healthy cells obtained after repeated Fas-induced apoptosis were compared with respect to their fibrotic parameters such as collagen and MMP secretion. Only late-stage lesional SSc cells were more resistant to Fas-induced apoptosis than their non-lesional counterparts isolated from the same patient. This result correlated with an increase in the levels of the anti-apoptotic proteins cFLIPs and cIAP in lesional cells compared to non-lesional cells. Healthy and non-lesional cell populations could be selected to generate a subpopulation that was more resistant to apoptosis. However, only the late-stage non-lesional SSc fibroblast populations showed a significant decrease in MMP secretion, one of parameters of the fibrosis. Our results show that resistance to apoptosis is an important characteristic of the late-stage lesional SSc fibroblast phenotype. We thus hypothesized that a selection of specific fibroblast subpopulations from late-stage non-lesional SSc skin areas could be at the origin of lesional populations. These cells should become independent of any exogenous stimuli and can induce or maintain SSc skin lesions.

Genetic polymorphisms of CYP2D6 oxidation in patients with systemic sclerosis.

BACKGROUND: Involvement of genetic and environmental factors in the pathogenesis of scleroderma has contributed to a number of studies whose aim is to elucidate the way in which xenobiotics exert effects on the occurrence of autoimmune processes resulting in development of systemic sclerosis (SSc). OBJECTIVE: The study dealt with the evaluation of the genetically determined polymorphism of CYP2D6, one of the phase I drug metabolizing isoenzymes, in patients suffering from SSc. Usefulness of the CYP2D6 genotype examination and prevalence of CYP2D6 gene mutation in light of susceptibility to SSc development were assessed. METHODS: Forty-three patients with SSc and 129 healthy volunteers were included in the study. Of the 43 patients with SSc, 17 fulfilled the criteria of diffuse SSc (dSSc) and 26 of limited SSc (lSSc). The determination of the CYP2D6 oxidative polymorphism was performed with the PCR-RFLP method. RESULTS: Relative risk of SSc development for particular genotype carriers expressed by the odds ratio (OR) was statistically significantly higher for subjects with CYP2D6*1/CYP2D6*4 (OR = 4.8; P < 0.001). A statistically significant correlation between the CYP2D6*4 allele prevalence and the risk for developing SSc was found (OR = 2.6; P = 0.0002). CONCLUSION: Higher prevalence of the CYP2D6*4 mutated alleles in patients with SSc and the obtained OR values suggest that this mutation has the effect of increasing SSc morbidity rate.

Poly(ADP-ribose) polymerase activity in systemic lupus erythematosus and systemic sclerosis.

The aim of this study is to investigate the role of poly(ADP-ribose) polymerase (PARP), involved in DNA repair and in autoimmune pathologic conditions such as systemic lupus erythematosus (SLE) and both limited systemic sclerosis (lSSc) and diffuse systemic sclerosis (dSSc), to assess its possible implication in the pathogenetic processes. The relationship between PARP activity and the intracellular concentration of its substrate nicotinamide adenine dinucleotide (NAD) is also investigated. Peripheral mononuclear cells (PMC) from controls and patients with SLE, lSSc, and dSSc were irradiated with ultraviolet light (UV) and PARP activity was assayed by a radiochemical method. Pyridine nucleotide concentrations were assayed by a high-performance liquid chromatography-linked method. PARP activity was detectable in nonirradiated cells and showed similar values in all groups. The activity significantly increased after UV irradiation in control, SLE, and lSSc cells, but not in dSSc cells. Irradiated PMC from both SLE and dSSc showed lower enzyme activity with respect to irradiated controls. Higher intracellular NAD content was found in all of the pathologic conditions in comparison to values in the control; this difference was statistically significant in dSSc. Our data demonstrate a lower PARP activity in response to UV damage in PMC from patients affected by the above pathologic conditions compared with controls. An inverse relationship between PARP activity and NAD content was also observed.