Comparison of a Suggested Model of Fibrosis in Human Dermal Fibroblasts by Serum from Systemic Sclerosis Patients with Transforming Growth Factor β Induced in vitro Model.

Abstract

Systemic sclerosis (SSc) is a chronic autoimmune disease, featuring fibrosis in multiple organs. The serum from SSc patients contain inflammatory mediators, contributing to SSc pathogenesis and could be used to develop cell culture models. Here, we compared the fibrotic effects of serum samples from SSc patients with TGFβ1 on human dermal fibroblasts (HDFs). HDF cells were cultured in four different culture media supplementations; 10% SSc serum, 10% healthy human serum, 10% fetal bovine serum or 10% FBS supplemented with 10 ng/Ml human TGFβ. The collagen content in cell layers was measured by spectrophotometric Picro-Sirius red staining. The mRNA expression of α-SMA, COL I and III, TGFβ1, arginase and E-Cadherin genes were determined by real time RT-PCR. TGF-β1 levels in cell culture supernatants were measured using ELISA. Cell layer collagen content was significantly increased following TGF-β1 treatment, compared with FBS group and SSc serum treatment in comparison with healthy controls. Although not statistically significant, the mRNA expression of α-SMA, COLI and III, TGFβ1, and arginase increased upon TGF-β1 treatment in comparison with FBS group, and in SSc serum treatment group in comparison with healthy controls. E-Cadherin decreased following TGF-β1 treatment and SSc serum treatment in comparison with their counterparts. TGF-β1 levels increased in cell culture supernatants of HDF cells exposed to TGF-β1 and SSc serum. An in vitro model of SSc serum-induced fibrosis using human HDF cells was evaluated in comparison to the TGF-β1 fibrosis induced model and data suggested that it may be used in documenting the role of pro-fibrotic factors in serum or plasma from SSc patients.