| ObjectiveOPN (osteopontin, OPN) is a multi-functional phosphorylated glycoprotein and plays animpotant role in promoting scar formation through stimulating fibroblast proliferationand angiogenesis. The article is aimed to export the modulating function of OPN in HTFs(human tenon’s fibroblasts, HTFs) in vitro and its influence in surgical region of theglaucoma filtration surgery in vivo. More insight of the mechanism is investigated thathow OPN plays its biological activities in glaucoma postoperative scar formation. Theefficacy of1A12, which is a self-developed monoclonal antibody against OPN, wasevaluated both in vitro and in vivo and we hope to provide fresh ideas for treating scarformation in glaucoma filtration surgery.Methods1、The role of OPN in HTFs and its mechanism (in vitro studies)With the intervention of OPN in different concentration (0，0.05μM，0.5μM and5μM),OPN combined with1A12(5μM OPN+20μg/ml1A12), the proliferation rate of HTFswas measured by MTT and the motility of HTFs was surveyed by cell scratch method.Real-time PCR was applied to measure the mRNA of VEGF and TGF-β in HTFs in thepresence of OPN with different concentration (0，0.05μM，0.5μM and5μM), OPNcombined with PD98059and LY294002, which were signal pathways inhibitors ofMAPK/ERK and PI3K/AKT respectively, and OPN combined with1A12(5μMOPN+20μg/ml1A12). Western blotting was used to measure the protein of Collagen-I aswell as p-ERK, p-AKT, p-IKKα and p-NFκB in the signal transduction pathways. It wasdiscussed that the mechanism of the signal pathways through which OPN modulates theproliferation and motility of HTFs.2、The role of OPN in surgical region of glaucoma filtration surgeryin rabbit model and its mechanism (in vivo studies)The rabbit models were established with glaucoma filtration surgery and wereallocated randomly into four groups. Group NS:0.1ml NS was injectedsubconjunctivally during operation and1,2, and7days after operation; Group MMC: a cotton ball with0.4mg/ml MMC was putted sub-tenon’s capsule for5minutes duringoperation and0.1ml NS was injected1,2, and7days after operation; Group OPN:0.1ml(2.5μg) OPN was injected subconjunctivally during operation and1,2, and7days afteroperation; Group1A12:0.1ml (10μg)1A12was injected subconjunctivally duringoperation and1,2and7days after operation. The characteristics such as area and heightof filtering bleb as well as intraocular pressure (IOP) were measured dynamically afteroperation. The samples were removed and histopathological changes were observed andevaluated by haematoxylin-eosin (HE), Masson trichrome staining, reticular fiberstaining, α-smooth muscle actin (α-SMA) immunocytochemistry and Ki67immunocytochemistry. Real-time PCR was applied to detect mRNA of OPN, VEGF,TGF-β, collagen-I and western blotting was applied to detect protein of collagen-I,p-ERK and p-AKT in the tissues of operational region in four groups in differenttime-points.Results1、OPN induced proliferation and motility of HTFs and the effectswere inhibited by1A12.OPN induced the proliferation of HTFs indicated by MTT and enhanced the motilityof HTFs showed by cell scratch method. The effects were concentration-dependent (0，0.05μM，0.5μM and5μM). With the presence of anti-OPN1A12(5μM OPN+20μg/ml1A12), the effects of OPN in HTFs were inhibited.The amount of mRNA of VEGF and TGF-β was little detected by real-time PCR inHTFs. There were no significant differences in mRNA of VEGF and TGF-β when treatedwith OPN in different concentration. The expression of Collagen-I protein wasaugmented concentration-dependently when treated with OPN (0，0.05μM，0.5μM and5μM). The effect was inhibited by PD98059and LY294002, which were signal pathwaysinhibitors of MAPK/ERK and PI3K/AKT respectively, as well as anti-OPN1A12. Thep-ERK and p-AKT protein were up-regulated with the increasing concentration of OPNand were down-regulated by PD98059and LY294002, as well as1A12. The p-IKKα andp-NFκB protein remained stable with the altering concentration of OPN and was stillunchanged when treated with PD98059, LY294002and1A12respectivly. 2、OPN enhanced scar formation in the glaucoma filtration surgeryof rabbit models and the effect was inhibited by1A12.In the rabbit models, the time needed for the relative area and height of bleb toreduce was shortest in Group OPN, next in Group NS, last in Group MMC and Group1A12. There were no significant differences between Group MMC and Group1A12.However, the wall of filtrating bleb was relatively thick in Group1A12, close to thenormal conjunctival tissue while the wall of filtrating bleb in Group MMC was pale, thinand cystic. There were no significant differences in IOP before and1d,3d and5d afteroperation. IOP of Group OPN was significantly higher than of the other three groups in7d time-point. There were no significant differences between Group NS and Group OPNin14d and21d time-points while the IOP of which were higher than Group MMC andGroup1A12respectively. There were no significant IOP differences in28d time-point.It was observed that the collagen fiber of sclera was denser in Group OPN than inthe other three groups showed by Masson trichrome staining and reticular fiber staining.The score of α-SMA immunocytochemistry in Group OPN was significantly higher thanthat of Group MMC and Group1A12. The score of Ki67immunocytochemistry in GroupOPN was significantly higher than that of the other three groups.The amount of mRNA of VEGF and TGF-β was little detected by real-time PCR.There were no significant differences in mRNA of VEGF and TGF-β in four groups.Compared with Group NS, the mRNA and protein of collagen-I were up-regulated inGroup OPN and were down-regulated in Group MMC and Group1A12. The effects weremore obvious in the early phase (Day3). The protein of p-ERK and p-AKT wereup-regulated in Group OPN and were down-regulated in Group MMC and Group1A12compared with Group NS. The results of in vivo experiment matched up with the resultsof in vitro experiment highly.ConclusionsOPN is able to enhance the proliferation and motility of HTFs. The effect isconcentration-dependent and can be inhibited by anti-OPN1A12. OPN is closely relatedwith the signal pathways of MAPK/ERK and PI3K/AKT. Through activating signalpathways of MAPK/ERK and PI3K/AKT, OPN may stimulate the downstream cellbiological effect of the pathways. OPN induces collagen-I expression in HTFs by activating MAPK/ERK and PI3K/AKT signal pathways.1A12, which is the monoclonalantibody of OPN, can inhibit the effects of OPN in HTFs. It is observed in vivo that OPNenhances scar formation while1A12alleviates it. MAPK/ERK and PI3K/AKT signalpathways are activated when the expression of collagen-I is up-regulated by OPN. Theeffects are also inhibited by1A12. To the best of our knowledge, this is the first reportproposing the idea that anti-OPN1A12, which is self-developed, can reduce scarformation and improve surgical outcome in glaucoma filtration surgery.1A12mightserve as a new valuable anti-scarring agent in glaucoma filtration surgery in the future. |
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