Effect Of Aptamers Targeting TGF-β Receptor Ⅱ On Transdifferentiation Of Tenon’s Fibroblasts

Background:Filtration surgery is principally used in glaucoma when the intraocular pressure (IOP)is not well controlled by medicines. However, the surgery does not always work owing tothe occurrence of conjunctival scar at the surgical site. Clinically, mitomycin C (MMC) and5-fluorouracil (5-FU) have been found to remarkably improve surgical outcomes, theirapplication was limited because of severe side effects even blindness. Therefore, it isdesirable to search for new strategy more effective and safe in modulating wound healingand preventing scar formation. The cytokine transforming growth factor-β (TGF-β) hasbeen recognized as a pivotal mediator in wound healing and tissue repair. However aninappropriately high level of TGF-β activity at the wound sites is associated with excessivescarring and fibrosis. Researchers have recognized the importance of antagonizing andblocking the TGF-β to modulate wound healing, and these findings suggest that inhibitingthe effects of TGF-β may reduce scar formation. There are three isoforms of TGF-β inhumans: TGF-β1, TGF-β2and TGF-β3. Of which TGF-β2has been found as thepredominant isoform in ocular scarring diseases such as conjunctival scar and proliferativevitreoretinopathy. The binding of a TGF-β2ligand to a type II receptor dimer initializes torecruit a type I receptor dimer to form a hetero-tetrameric complex and to catalyze thephosphorylation of the GS domain of type I receptor, leading to the cascade of downstreamsignaling. Therefore, it may be an effective strategy to impair TGF-β2activity throughtargeting split in the interaction between TGF-β2ligand and type II receptor. Our researchhave screened TβRII-binding aptamers by Systematic Evolution of Ligands by ExponentialEnrichment (SELEX) from a ssDNA library. But the action of aptamers was still unclear,and needed further verification.Objective：This study was designed to assay effect of TβRII-binding aptamers on TGF-β-inducedtransdifferentiation of fibroblasts for evaluating their potential relieving scarring activities. Methods：Human Tenon fibroblasts (HTFs) were cultured and treated with TGF-β2at differentconcentrations. Western blot was performed to determine α-smooth muscle actin (α-SMA).α-SMA subcellular distribution and F-actin with rhodamine-phalloidin staining wereevaluated by confocal immunofluorescence microscopy. TβRII-binding aptamers S58/68were synthesized artificially. HTFs were treated with TGF-β2and aptamer S58/68, oraptamer S58/68. α-SMA and phosphorylation of the signaling proteins Smad2weremeasured by Western blot. α-SMA and pSmad2subcellular distribution and F-actin withrhodamine-phalloidin staining were evaluated by confocal immunofluorescence microscopy.Cell contractility was assessed in collagen gel contraction assays.Results：1. TGF-β2stimulated α-SMA and F-actin expression and α-SMA incorporation intostress fibers with a characteristic concentration-dependent response, with peak activities at2-5ng/ml, that were significantly different from control (P <0.05). TGF-β2also stimulatedHTF-mediated collagen contraction. At concentrations above and below peak activites,HTF activity was reduced, demonstrating biphasic effects of TGF-β2.2. Twenty-one sequences were obtained after eight rounds of selection, and twopreferential sequences, aptamer S58and S68, were isolated and used in the followingexperiments. Aptamer S58significantly inhibited α-SMA expression and its incorporationinto actin stress fibers induced by TGF-β2, and suppressed TGF-β2-induced cell contraction.Furthermore, it also inhibited TGF-β2-induced phosphorylation and nuclear translocation ofSmad2.3. However we failed to find any effect of aptamer S68on TGF-β2activity in vitro.Conclusions：1. TGF-β2induced a response in HTFs that is concentration-dependent, with differentfunctions being maximally stimulated at different concentrations. This response highlightsthe significance of the concentration profile of TGF-β2at the wound site in filtrationsurgery. TGF-β2induced transdifferentiation of fibroblasts to myofibroblasts (MFs) thatexert increased contractile activity associated with the expression of α-SMA.2. Our study revealed that a novel aptamer binding TβRII inhibited TGF-β2-inducedmyofibroblast transdifferentiation in human Tenon fibroblasts. Aptamer S58inhibits TGF-β2-induced α-SMA expression in HTFs. It might be potential to relieve scarring afteroperation, such as glaucoma filter surgery.3. Aptamer S58inhibited TGF-β2-induced phosphorylation and nuclear translocationof Smad2. The result indicates that aptamer S58inhibited TGF-β2activity partially viaSmad signal transduction pathway.4. Effects of aptamers on TGF-β2activity in HTFs were extremely different mainlydue to their sequences and structures.