Pparγagnoists Inhibit Cell Growth And TGF-β₂ Induced Extracellular Matrix Production In Human Tenon's Fibroblasts

Glaucoma is a group of ocular disorders usually associated with increased intraoculaxpressure that leads to irreversible functional impairment ofthe optic nerve It ranks as thesecond main cause ofpreventable vision loss in developed countries Filtration surgery isone ofthe most effective therapies for glaucoma,but the therapy often fails because ofsubconjunctival scarring Current antiscarring therapies focus on suppressing theproliferation offibroblasts through anti-metabolic agents such as mitomycin C(MMC)and5-fluorouracil(5-Fu)However,they are associated with several severe complicationsBetter antiscaxring therapeutic agents which are more effective,safe and less complicationare neededIn recent years,many studies have demonstrated that PPARγis involved in theanti-fibrotic effect in many tissues,and is thought to be a promising target for treatment offibrotic diseases Is PPARγparticipated during healing following filtration surgery?MethodsTo explore this hypothesis,in the present study w-e examined the effects oftwo differentPPAR7 agonists 1 5d-PGJ₂ and rosiglitazone on proliferation and extracellular matrixproduction by cultured human Tenon'S fibroblasts1 To detect the expression of PPARγwith immunofluorescence,RT-PCR and Westernblot in HTFs HTFs were isolated and cultured from patients of glaucoma The cells wereidentified by immunofluorescence method2 To test the effects of two different PPARγagonists 1 5d-PGJ₂ and rosiglitazone onproliferation by cultured human Tenon'S fibroblasts(1)Use MTT to test the proliferation,trypan blue exclusion method to evaluate cell viability and flow--cytometric to analyze cellcycle distribution.(2)Real time PCR and Western blot to test the express level of TGF-β3 HTFs were treated with TGF-β₂ at the concentration of 5ng/ml to imitate the processof scarring following filtration surgery.(1)HTFs were treated with 15d-PGJ₂,rosiglitazone and GW9662 at the concentration of l0μM,20μM and 10μM. Theproliferation and extracellular matrix production were test by cell counting,RT-PCR andWestern blot.(2)To test the expression of Smad2/3 and p-Smad2/3 in HTFs byimmunofluorescence,RT-PCR,Western blot and ELISAStatistical analvsis:All data are expressed as mean±SD Comparisons between groups were carried outusing oneway ANOVA,straight line Pearson relevance analyses and ratio compares using χ² test(SPSS 12.0).P values less than 0.05 were considered to be statistically significantResults1 The primary cultured HTFs were spindle and had common characters offibroblast,such as vimentin positive,keratin negative The positive staining of PPARγlocalized inHTFs2 Suppress the proliferation ofthe HTFs cells and secretion of TGF-β(1)Both 15d-PGJ₂ and rosiglitazone can effectively restrain the proliferation and thecell cycle ofthe HTFs cells It is dose-dependent,but has no effect on cell viability TheIC₅₀ of 15d-PGJ₂ is 13.497μM and rosiglitazone is 21.622μM Flow-cytometric analysis ofHTFs cell cycle distribution showed that 15d-PGJ₂ caused an increase in the proportion ofcells in S phase,and a decrease in G₂/M.In contrast,rosiglitazone prevented theprogression of cells from G₁ to S phase(2)Rosiglitazone can effectively suppress the secretion ofTGF-β₁,TGF-β₂,TGF-β₃,but 15d-PGJ₂ cannot3 Suppress TGF-β₂ induced the proliferation ofthe HTFs cells and secretion of ECM(1)Both 15d-PGJ₂ and rosiglitazone can effectively suppress TGF-β₂ induced theproliferation ofthe HTFs cells and secretion of ECM Treatment with GW9662significantly attellLIated the inhibition of TGF-β₂ induced HTFs proliferation,α-SMA andcollagen I expression by 15d-PGJ₂,but had no evident effect on the fibronectin expressionIn contrast,treatment with GW9662 significantly attenuated the inhibition of TGF-β₂induced fibronectin and collagen I expression by rosiglitazone,but had no evident effect onthe HTFs proliferation andα-SMA expression(2)TGF-β₂ can dose-dependently raise the expression of p-Smad2/3,which can besuppressed by 15d-PGJ₂ and rosiglitazone GW9662 can significantly attenuated this effectby 15d-PGJ₂,but only partly of rosiglitazone The result showed that GW9662 had noeffect on TGF-β₂ and Smad2/3Conclusions1 PPARγis definitely expressed in primary cultured HTFs2 1 5d-PGJ₂ caused an increase in the proportion of cells in S phase,and a decrease inG₂/M In contrast,rosiglitazone prevented the progression of cells from G₁ to S phaseBoth were dose-dependent and had no significant cytotoxicity3 Rosiglitazone can effectively suppress the secretion ofTGF-β₁,TGF-β₂,TGF-β₃,but15d-PGJ₂ cannot 4 Both 15d-PGJ₂ and rosiglitazone can effectively suppress TGF-β₂ induced theproliferation of the HTFs cells and secretion of ECM Both PPART-dependent and—independent effect involved in this process. The PPARγ-dependent effect is via regulationof TGF-β/Smad pathway which is similar to PPARγ/TGF-β/Smad pathway．5 Rosiglitazone had some side effects such as heart toxicity,macula edema. In thisregard,15d-PGJ₂ represents a promising new-agent to prevent the scarring after filtrationsurgery. But it also need more experiments to test the exactly effect in vivo.