The Selective Cysteinyl Leukotriene Receptor 1(CysLT1R) Antagonist Montelukast Regulates Extracellular Matrix Remodeling

1 IntroductionPostoperative scarring is one of the main reasons for failure of glaucoma filtration surgery.Wound healing in the conjunctiva is characterized by inflammation followed by re-epithelialization,synthesis of new extracellular matrix,wound contraction,and the formation of a subconjunctival fibrous scar.Excessive contraction of subconjunctival tissue at the wound site can result in excessive scarring.Multiple lines of evidence has displayed that human Tenon capsule fibroblasts(HTFs)located in the incision area are associated with scar formation.Physiological activities such as proliferation,migration,synthesis and deposition of extracellular matrix(ECM)of HTFs play critical roles in scar formation and obstruction of the filtration passage.The wound healing response at the subconjunctival filtering bleb site is regulated by various cytokines,such as the transforming growth factor TGF-β1.TGF-β1 has become a major target of evolving antifibrotic strategies.Montelukast,a selective antagonist of cysteinyl leukotriene receptor 1(CysLT1R).The pharmacological role of montelukast is achieved by preventing CysLT-mediated bronchoconstriction,increased vascular permeability and recruitment of inflammatory cells to the airway.However,little information with regard to antifibrotic properties of montelukast has been reported before.2 PurposeIn the present study,we attempted to examine the effects of montelukast on TGF-β1-induced extracellular matrix(ECM)synthesis in primary cultured human Tenon’s fibroblasts.3 Methods3.1 Cell culture.Tenon ’s capsule fibroblasts were isolated from subconjunctival tissue of 10 individuals undergoing eyelid or strabismus surgery as previously described.Cells were maintained in Dulbecco’s modified Eagle’s medium(DMEM)containing 10%fetal bovine serum(FBS),1%L-glutamine(2 mM),and 1%antibiotics(penicillin and streptomycin)at 37℃ in 5%CO2 atmosphere.All experiments used HTFs from passage 3 to 6 cultures.3.2.Reverse transcriptase polymerase chain reaction(RT-PCR)Total intracellular RNA was isolated from cultured HTFs using the Qiazol reagent according to manufacturer’s instructions.The cDNA was then synthesized.Then,We examined the expression of CysLT1R mRNA in HTFS.3.3 Assay of collagen gel contractionFree-floating collagen was prepared as previously described.0.5 mL of the mixture was added to each well of the 24-well cell culture plate.After gelation of the mixture,the collagen gels were detached from the wells of the culture plate.0.5 ml serum-free DMEM containing TGF-β1 and/or montelukast was then added on top of each gel.The diameter of the gels was measured daily to calculate the extent of gel contraction.3.4 Western blot analysisTotal cell proteins from HTFs which were treated with TGF-β1 and/or montelukast were extracted using cell lysis buffer with EDTA free protease inhibitor cocktail.The protein level of CysLT1R、MMP-1、MMP-3、FAK、paxillin、p-FAK,p-paxillin and a-SMA was detected by Western blot method.3.5 Immunofluorescence microscopyImmunofluorescence staining was used to detect the expression of CysLTIR in the cells.3.6 Determination of fibronectin and the C peptide of procollagen type ⅠFibronectin and the C peptide of procollagen type I in the cultured medium were measured with ELISA as previously described.4 Results4.1 CysLTIR was expressed in HTFsExpression patterns of CysLT1R in HTFs haven’t been reported before.Therefore,we firstly examined whether CysLTIR is expressed in HTFs.The RT-PCR results indicated that CysLTIRwas indeed expressed in HTFs.The protein expression of CysLTIR in HTFs was verified by western blot analysis.Human A431 cells were used as a positive control,as the expression of CysLTIR in these cells has been reported before.Consistently,immunofluorescence staining experiments displayed that CysLTIR was expressed in HTFs.4.2 TGF-β1 increased the expression of CysLTIR in HTFsWe next determined the expression patterns of CysLTIR in response to TGF-β1 treatment.Interestingly,we found that TGF-β1 treatment resulted in a sustainable increase in the expression of CysLTIR in a concentration dependent manner from 0.5 to 5 ng/ml at both mRNA and protein levels,suggesting that CysLT1R might be associated with the psychological function of TGF-β1 in HTFs.4.3 Effects of montelukast on TGF-β1-induced collagen gel contraction mediated by HTFsMontelukast is a selective antagonist of CysLT1R.The molecular structure of montelukast is shown.HTFs were incubated with montelukast at the concentration of 0,1,10,50 μM for 3 days in the presence or absence of TGF-β1(2.5 ng/ml).Results showed that montelukast ameliorated TGF-β1-induced gel contraction in a dose-dependent manner.The stimulatory effect of TGF-β1 on collagen gel contraction was thus inhibited by 30%,48%,or 55%by montelukast at the concentration of 1,10,or 50μM,respectively.Moreover,HTFs were incubated with 50 pM montelukast for various times.Results showed that montelukast inhibited TGF-β1-induced gel contraction in a time dependent manner.4.4 Effects of montelukast on the expression of MMPs and matrix productionCell-mediated collagen contraction is mediated by matrix metalloproteinases(MMPs).We then evaluated the effects of montelukast on the expression of MMPs.Western blot analysis results revealed that TGF-β1 treatment resulted in a significant increase in the expression of MMP-1 and MMP-3,which were prevented by montelukast in a dose-dependent manner.In addition to ameliorating matrix contraction,MMP inhibition has been reported to reduce matrix production without cell toxicity.We next examined whether the inhibitory effects of montelukast on MMPs expression affected matrix production.The release of fibronectin and collagen type I from HTFs were determined by the use of specific EIAs.Results showed that the release of fibronectin and the secreted C-terminal propeptide of collagen type I(CICP)-induced by TGF-β1(2.5ng/ml)was inhibited by montelukast in a concentration-dependent manner.4.5 Effects of montelukast on phosphorylation of FAK,paxillin and a-SMA expression in HTFsMechanism of cell-mediated collagen contraction is complex.Activation of focal adhesion kinase(FAK)and paxillin by phosphorylation and the expression ofα-smooth muscle actin(a-SMA)have been involved in this process.Therefore,we next examined whether montelukast might affect the phosphorylation of FAK and paxillin as well as the expression of a-SMA in HTFs.TGF-β1(2.5ng/mL)induced the phosphorylation of FAK and paxillin in HTFs,which was inhibited by montelukast in a concentration-dependent manner.However,the abundance of FAK or paxillin was not affected by TGF-β1 or montelukast.Western blot results demonstrated that neither TGF-β1 nor montelukast affected the expression of α-SMA.5 conclusion5.1 We found that CysLT1R is expressed in HTFs.5.2 The expression of CysLTIR in HTFs is increased in response to TGF-β1 treatment,suggesting its potential role in TGF-β1 induced excessive production of extracellular matrix and subconjunctival fibrosis。5.3 Montelukast ameliorated TGF-β1-induced MMP-1 and MMP-3 release as well as the release of fibronectin and collagen type I from HTFs which reduced the contraction and deposition of extracellular matrix.5.4 Montelukast was reported to attenuate FAK and paxillin phosphorylation,Thereby attenuating TGF-β1-induced extracellular matrix contraction.5.5 Montelukast might be an effective agent for inhibition of scar formation during subconjunctival wound healing after such surgery.