–SMA and CTGF mRNA Expression of CD90+ and CD90– Orbital Fibroblasts Subsets Induced by TGFß 1

Abstract

Purpose: : Orbital fibroblasts play an important role on the pathogenesis of Thyroid–associated ophthalmopathy(TAO). Fibroblasts may exhibited functional heterogeneity results from phenotypic differences . In this report , orbital fibroblasts were devided into CD90⁺ and CD90^–subsets respect to surface CD90 expression , then determined the effect of recombinant human TGFß1,an known inducer of the myofibroblastic phenotype,on gene expression of α–SMA and CTGF in the two subsets .α–SMA was a characteristic marker of myofibroblasts. CTGF may be the downstream mediator responsible for mediating some of the cellular effects of TGFß1.

Methods: : Fibroblasts subset separation into CD90⁺ and CD90^–subsets was accomplished by three to four rounds of magnetic bead selection, then treated with recombinant human TGFß1. The concentration of TGFß1 was 5ng/ml. The total RNA was extracted at 0.5 hours to 48 hours after adding TGFß1. The expression of α–SMA,CTGF mRNA levels were measured by real–time quantitative PCR.

Results: : The total RNA from each groups was detected by agarose 1% gel electrophoresis. No unexpected band is detected which indicates the purity of the RNA was high. According to each standard curve, the level of α–SMA and CTGF was examined. Orbital fibroblasts expressed α–SMA,CTGF gene at a low level without adding TGFß1. TGFß1 upregulated CTGF mRNA expression of CD90⁺ orbital fibroblasts up to 13 times against control, whereas CD90^– orbital fibroblasts only 3 times against control(p<0.01). CD90⁺ orbital fibroblasts were induced more α–SMA mRNA than CD90^– cells followed CTGF mRNA expression induced by TGF–ß1, the quantity of α–SMA mRNA copies was 35 and 4 times of controls respectively(p<0.01).

Conclusions: : These data suggests that obital fibroblasts are heterogeneous with respect to surface CD90 expression, TGF–ß1 induced more α–SMA mRNA expression in CD90⁺ orbital fibroblasts than in CD90^– cells. Myofibroblast formation stimulated by TGF–ß1 is mediated by CTGF. CD90⁺ orbital fibroblasts maybe have a potential ability about myofibroblast differentiation which are related to the extraocular muscles fibrosis and GAG accumulation in the orbit.

Keywords: orbit • gene/expression • cytokines/chemokines