Background The key cellular elements in the pathogenesis of tissue fibrosis are myofibroblasts. It is widely accepted that the number of myofibroblasts is increased in Scleroderma (SSc) and it correlates with the severity of tissue fibrosis. The mechanisms underlying their increased number in SSc are unknown. The heterogeneity in their frequency may explain the inconsistency of some in vitro studies published on SSc fibroblast biology and may mirror the clinical heterogeneity of SSc patients both in natural history and severity of skin fibrosis.

Objectives The purpose of this study was to unravel the specific transcriptome of myofibroblasts derived from SSc skin biopsies.

Methods Four patients with diffuse rapidly progressive SSc, within 18 months from skin involvement and before any immunosuppression, were enrolled in the study. Skin biopsy on forearm was performed and the fibroblasts subcultured for three passages. 250 acetone fixed alpha-SMA positive cells were isolated by laser capture microdissection (LCM) for mRNA analysis by Affymetrix Gene array. qRT-PCR and in situ hybridization were employed for validation of the results. Pathway analysis was conducted according to David-NIH software. Immunofluorescence (IF) followed by confocal laser scanning microscopy (CLSM) was conducted as well. Normal dermal fibroblasts were utilized to evaluate the effects of TGF-beta stimulation both at mRNA and protein level.

Results qRT-PCR for alpha-SMA showed, in average, 3.7 fold increased expression in alpha-SMA in the LCM captured cells. Microarray analisis identified 269 genes upregulated more than 2 fold in the myofibroblasts. Of these, 24 were clearly reconducible to profibrotic activation, including alpha-SMA, Collagens I, VI and XI, Fibronectin, several Integrin genes, FGF7, CD36, IGF and Rho; 16 were ribosomial genes; 14 were mitochondrial genes involved in oxidative phosphorylation, including COX1,2, 3 and 6 ND1 to 6, CYT-b and F-type ATP-ase; 28 genes were involved in cell to cell adhesion including, JAM2, ERM, and MLC and 7 in antigen processing and presentation including RAB13, B2-microgrobiulin, cathepsin, HSPs, calnexin, and calreticulin. The remaining genes were not classifiable in any specific functional pathway. IF studies followed by CLSM confirmed the specific pattern of protein expression in myofibroblasts vs alpha-SMA negative fibroblasts from the same donor. Long non coding RNA tiling array of the same cells indicated that alpha-SMA positive fibroblasts had a specific up-regulation of HOX-A9 also known as HOTAIR, which is usually expressed mainly in the dermis of the palmo-plantar region. qRT-PCR and in situ hybridization confirmed the increased expression of HOTAIR in SSc skin. Consistently with these results, skin biopsies from the forearms of SSc patients showed an increased level of the palmo-plantar specific Keratin-9.

Conclusions Myofibroblast secretome displayed, besides predictable genes involved in the increased ECM production and TGF-beta pathway activation, a specific gene expression non TGF-inducible and suggestive of a distinct and stable differentiation lineage usually present only in the palmo-plantar region. This distinct differentiation status may account for the dermal and epidermal changes in SSc.

Disclosure of Interest None Declared