Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Myofibroblast Specific Gene Expression Signature in Systemic Sclerosis, More Than TGF-beta Induced Activation.
Abignano2, Giuseppina, Hermes, Heidi, Gillespie, Justin, Jimenez, Sergio A., Emery, Paul, Del Galdo1, Francesco
The key cellular elements in the pathogenesis of tissue fibrosis are myofibroblasts. It is widely accepted that the number of myofibroblasts is increased in SSc and it correlates with the severity of tissue fibrosis. The mechanisms underlying their increased number and their heterogeneity 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. The purpose of this study was to unravel the specific transcriptome of myofibroblasts derived from SSc skin biopsies.
4 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 and qRT-PCR validation. 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.
qRT-PCR for a-SMA showed in average 3.7 fold increased expression in a-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 a-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 and comprised tropomyosin, reticulocalbin 1, caldesmon 1, and 6 members of Neuroblastome Breakpoint Family (NBPF). IF studies followed by CLSM confirmed the expression, never shown before, of NBPF in dermal fibroblasts. Functional studies on normal dermal fibroblalsts indicated that NBPF was not inducible by 24 or 48 h stimulation with 10 ng/ml TGF-beta neither at mRNA or protein level.
IF followed by CLSM of alpha-SMA positive vs negative cells showed a specific expression profile for pro-collagen-1, caveolin-1, beta-catenin and phospho-RB whereas SMAD3, SMAD1, SMAD5, and NF-kB did not differ between alpha-SMA positive or negative cells.
Myofibroblast secretome displayed, besides predictable genes involved in the increased ECM production and TGF-beta pathway activation, genes involved in several pathways not known to be specific of myofibroblasts or inducible by TGF-beta. The specific expression of these genes may reflect either a specific metabolic status or a specific differentiation lineage of myofibroblasts
To cite this abstract, please use the following information:
Abignano, Giuseppina, Hermes, Heidi, Gillespie, Justin, Jimenez, Sergio A., Emery, Paul, Del Galdo, Francesco; Myofibroblast Specific Gene Expression Signature in Systemic Sclerosis, More Than TGF-beta Induced Activation. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :673