CTGF Promotes The Role Of Human Tenon’s Capsule Fibroblast In Fibrosis And The Study Of Relative MicroRNAs

Filtering surgery is a commonly performed surgical procedure in treating Glaucoma when medications fail to control intraocular pressure (IOP). Non-functioning filtering bleb and excessive scar formation account for the main cause of surgery failure in most cases, as is revealed by pathological findings. Former studies also suggest that, this postoperative process of scar formation is a pathological consequence of tissue remodeling, consisting of the complex interactions between cells, various kinds of cytokines and extracellular matrix (ECM), and implicating the homeostasis of ECM deposition and degradation.As a response to injury, fibroblasts may transform into another contractile and secretory phenotype, the myofibroblasts (MFs), which behaves like an intermediate form between fibroblast and smooth muscle cell. The pivotal role that MF plays in wound healing and pathological remodeling is well established and amply demonstrated in various organs, with its significant function in ECM synthesis and organization as well as cytokine secretory. It can be identified by the expression of a-smooth muscle actin (a-SMA), which is subsequently organized into stress fibers and furthermore participates in local wound contraction in situ. Among all the various cytokines involved, transforming growth factor β (TGF-β) exerts a critical role in phenotype transition, as is evidenced by numerous publications. It stimulates fibroblast proliferation, extracellular matrix synthesis, and decreases extracellular matrix degradation.Since TGF-β is an upstream and initial factor with a broad spectrum of biotic effects, the notion gained more attention that blocking TGF-β stimuli at the ligand or receptor level may potentially impair some of its beneficial effect such as epithelial healing, and may result in various adverse consequences. Therefore the emerging trend to seek downstream factors involved in the phenotype conversion introduces connective tissue growth factor (CTGF) into current vision field of study. CTGF (also known as CCN2with regard to the CCN protein family) is a cysteine-rich, heparin binding protein. Multiple studies have suggested that CTGF is a critical downstream mediator of TGF-β activity in fibroblasts, involved to mediate key cellular events of fibroblast subsequently in response to TGF-β-induced proliferation, migration, differentiation, contraction, along with extracellular matrix production and accumulation, which are all essential elements of a fibrotic response.Another important issue on its character is that CTGF is largely absent in normal tissues but highly expressed in scars or lesions with fibrotic disease, qualifying it more feasible and effective as a potential therapy target than TGF-β.With this background, we undertook the following study to investigate the effect of CTGF on the proliferation, myofibroblast differentiation, migration and ECM synthesis of HTCF, while comparing with that of TGF-β respectively, aiming to generate new insights into the postoperative scar formation in filtering surgery.Also introduced in our study was the emerging technology of miRNAs as the participation of them were also investigated.Purpose:To investigate the effect of CTGF on the phenotype-transition, proliferation, migration and ECM synthesis of HTCF, while comparing with those of TGF-β respectively, aiming to generate new insights into the postoperative scar formation in filtering surgery. The participation of miRNAs was also observed.Methods:HTCF were obtained from patients with cataract during surgery. They were induced by different concerntration of CTGF for different durations. Western Blot as well as Immunofluorescence were used to observe the expression of Alpha smooth muscle actin (α-SM-actin a marker of myofibroblast) protein and certify the existence of phenotype transition, while Real-time-PCR was used quantify the expression lever of mRNAs.ERK pathway was observed as a possible signal transductive way. MTT was performed to observe the effect of CTGF on the proliferation of HTCF and transwell tests arranged for its migration.Col Ⅰ and FN were recruited as bio-markers of fibrosis in which their expression of both proteins and mRNA were tested after induction of CTGF and TGF-β.Two ways of induction were compared with all the above as well as their difference in the expression of various miRNAsResults:CTGF was capable and sufficient for the phenotype transition of HTCF with the most propriet concerntration at50μ g/L at the peak time of48hours,while still lower than that of TGF-β1(β<0.05)。Immunofluorescence and real-time-PCR also confirmed this process.Erk pathway participated this process activated at the peak time of1h.CTGF could promote the proliferation, migration of HTCF with no obvious difference with TGF-β. Raised Col Ⅰ and FN levels on both protein and mRNAs implied stronge effect of ECM synthesis for CTGF compared to TGF-β (P<0.05).Many miRNAs participated in the process as showed by chip-tese and certified by Real Time PCR. Levels of mir-145and mir-194was reduced in phenotype-induced groups (P<0.05)Conclusions:CTGF could induce the phenotype transition of HTCF and promotes its migration, proliferation and matrix synthesis, implying itself as a more safer and efficient target for suppression therapy. Many miRNAs participated in the process and levels of mir-145and mir-194was reduced.