Rosiglitazone Attenuates Activation Of Human Tenons Fibroblasts Induced By TGF-?1

BACKGROUNDThe first irreversible blind leading disease in the world is glaucoma. In our country, glaucoma filtering surgery (GFS) is still one of the principal means for treating advanced glaucoma. A main problem threatening long-term success rate of GFS is postoperative scarring process. Though antimetabolite drugs could effectively inhibit the formation of scarring, they also related with serious complications such as wound leakage, infection, hypotony maculopathy, even endophthalmitis which limit their application. Antifibrotic therapeutic methods with safe and effective effects are desirable.The activation of Human Tenons fibroblasts (HTFs) is essential for postoperative repairing, however continuous presence of this active state is related to increased deposition of extra cellular matrix (ECM) proteins which lead to postoperative scarring. This process is regulated by various inflammatory factors, among which transforming growth factor (TGF)-?has been recognized as a key cytokine. However besides inducing HTFs activation TGF-?till has other biological activity such as regulating cell differentiation, promoting osteogenesis and hematopoiesis, and inducing angiogenesis. For its complicated biological effects completely blocking TGF-?may potentially suppress immune and block wound healing. So we committed to find a even more specific and safe method to regulate fibrosis after GFS. TGF-?exerts its multiple biologic actions by activating several intracellular signal transduction systems, and Smads family of proteins has been recently identified as a predominant signal transducer of TGF-?.Rosiglitazone one of the specific thiazolidinediones (TZDs) is a synthetic high-affinity ligands for peroxisome proliferator-activated receptor (PPAR)-?a member of the nuclear hormone receptor super family of ligand-activated transcription factors. In clinic it widely used for treating of non-insulin-dependent diabetes mellitus. Besides it effects on metabolism, current studies have begun to indicate additional important roles for PPAR-?agonists in the regulation of inflammation, connective tissue remodeling, and organ fibrosis.In the present study we provided novel data demonstrating the ability of rosiglitazone to block TGF-?mediated profibrotic effects in HTFs. These activities support the concept that rosiglitazone may have exciting potential method for counteract postoperative scarring after GFS.PURPOSETo culture human Tenon's fibroblasts. To investigate the inhibition role of the synthetic peroxisome proliferators-activated receptor (PPAR)-?agonist rosiglitazone on TGF-?induced human Tenon's fibroblasts activation, and to assess the possible mechanism of it.METHODS1. Tenon's biopsy samples were obtained during standard Glaucoma Filtering Surgery and cultured in vitro.2. HTFs were treated with various concentration of TGF-?. The optimization concentration was assessed by Cell count kit-8. The morphologic changes of HTFs were observed. The expression of Alpha smooth muscle action (a-SMA), connective tissue growth factor (CTGF), type?Collagen (Col?), were detected with RT-PCR, Western-Blot,?-SMA was also examined with immunofluorescence. Smad2/3, was assessed by Western-Blot.3. HTFs were pretreated with different concentrations of rosiglitazone (0.1umol/1-3?umol/1) and then activated with 5ng/mL TGF-?1. Cell count kit-8 was accessed for cell viability and proliferation, wound closure assay was performed to assess cell migration. The expression of Alpha smooth muscle action (a-SMA), connective tissue growth factor (CTGF), type?Collagen (Col?), were detected with RT-PCR, Western-Blot, a-SMA was also examined with immunofluorescence. PPAR-y, P-Smad2/3 were assessed by Western-Blot.RESULTS1. HTFs were successfully cultured in vitro. They had normal morphosis and stable growth characteristic, before and after freezing and resusxitation.2. After induced by TGF-?1 the mRNA and protein expression of a-SMA were up-regulation, with a peak at 24 and 48 hours. The mRNA and protein expression of CTGF were up-regulation, with a peak at 12 and 24 hours. The expression of Col?and P-Smad2/3 were also up-regulated.3. Rosiglitazone could attenuate TGF-?1 induced up-regulation of a-SMA, CTGF and Col?. It could availably increase the expression of PPAR-?, and effectively attenuated the phosphorylation of Smad2/3.CONCLUSIONSRosiglitazone can effectively attenuate TGF-?1 induced activation of HTFs, without obvious toxic effect. The possible mechanism might be rosiglitazone interfering the TGF-?/Smad pathway.