17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways

AIM:To investigate the impact of 17β-estradiol on the collagen gels contraction(CGC)and inflammation induced by transforming growth factor(TGF)-βin human Tenon fibroblasts(HTFs).METHODS:HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-β(5 ng/mL),17β-estradiol(12.5 to 100μmol/L),or progesterone(12.5 to 100μmol/L).Then,the collagen gel diameter was determined to assess the contraction,and the development of stress fibers was analyzed using immunofluorescence staining.Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)being released into culture supernatants.Enzyme-linked immunosorbent assay(ELISA)and reverse transcription-quantitative polymerase chain reaction(RT-PCR)were used to detect interleukin(IL)-6,monocyte chemoattractant proteins(MCP)-1,and vascular endothelial growth factor(VEGF)in HTFs at the translational and transcriptional levels.The phosphorylation levels of Sma-and Mad-related proteins(Smads),mitogen-activated protein kinases(MAPKs),and protein kinase B(AKT)were measured by immunoblotting.Statistical analysis was performed using either the Tukey-Kramer test or Student’s unpaired t-test to compare the various treatments.RESULTS:The CGC caused by TGF-βin HTFs was significantly inhibited by 17β-estradiol(25 to 100μmol/L),and a statistically significant difference was observed when comparing the normal control group with 17β-estradiol concentrations exceeding 25μmol/L(P<0.05).The suppressive impact of 17β-estradiol became evident 24h after administration and peaked at 72h(P<0.05),whereas progesterone had no impact.Moreover,17β-estradiol attenuated the formation of stress fibers,and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-β.The expression of MCP-1,IL-6,and VEGF mRNA and protein in HTFs were suppressed by 100μmol/L 17β-estradiol(P<0.01).Additionally,the phosphorylation of Smad2 Smad3,p38,and extracellular signal-regulated kinase(ERK)were downregulated(P<0.01).CONCLUSION:17β-estradiol significantly inhibits the CGC and inflammation caused by TGF-βin HTFs.This inhibition is likely related to the suppression of stress fibers,inhibition of MMPs,and attenuation of Smads and MAPK(ERK and p38)signaling.17β-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.

Triptolide inhibits TGF-β-induced matrix contraction and fibronectin production mediated by human Tenon fibroblasts

AIM： To determine if triptolide influences the contractility and fibronectin production in human Tenon fibroblasts（HTFs）. METHODS： HTFs were cultured in type I collagen gels with or without transforming growth factor beta（TGF-β） and/or triptolide. The diameter of the collagen gel was used to measure contraction. Immunoblot analysis was used to quantify myosin light chain（MLC） phosphorylation and integrin expression. Laser confocal fluorescence microscopy was used to monitor the formation of actin stress fibers. Fibronectin production was measured with an enzyme immunoassay. RESULTS： Triptolide inhibition of contraction in TGF-β-induced collagen gel mediated by HTFs was dosedependent and statistically significant at 3 nmol/L（P〈0.05） and maximal at 30 nmol/L and significantly time dependent at 2 d（P〈0.05）. Triptolide reduced TGF-β-induced expression of integrins α5 and β1, phosphorylation of MLC, and formation of stress fibers in HTFs. Furthermore, the inhibition of triptolide on the attenuated TGF-β-induced production of fibronectin by HTFs was concentration-dependent and significant at 1 nmol/L（P〈0.05） and maximal at 30 nmol/L. CONCLUSION： Triptolide suppress the contractility of HTFs induced by TGF-β and the production of fibronectin by these cells. It is promising that triptolide treatment may possibly inhibit scar formation after glaucoma filtration surgery.

Anti-scarring effects of butaprost on human subconjunctival Tenon's fibroblasts

AIM： To investigate the toxicity of the E-prostanoid 2（EP2） receptor agonist, butaprost against human subconjunctival（Tenon＇s capsule） fibroblasts, and to determine the underlying mechanism. METHODS： We isolated Tenon＇s fibroblasts from the subconjunctival area of healthy subjects and evaluated the types of EP receptors expressed using quantitative realtime reverse transcription polymerase chain reaction（RTPCR）. The toxicity of butaprost against the fibroblasts was evaluated using methyl thiazolyl tetrazolium and lactic dehydrogenase assays. The inhibition of conjunctival fibroblast proliferation by butaprost was assessed by measuring α-actin levels. The underlying mechanism was assessed by measuring intracellular cyclic adenosine monophosphate（c AMP） levels. Intergroup differences were statistically analyzed using an independent t-test. Densitometry of the Western blot bands was performed using the Image J software. RESULTS： Quantitative real-time RT-PCR revealed that the fibroblast EP2 receptor levels were higher than those of the other EP receptors. Butaprost did not show toxicity against Tenon＇s tissue, but inhibited conjunctival fibroblast proliferation by reducing collagen synthesis. EP2 receptor activation enhanced the c AMP cascade, which might be an important mechanism underlying this effect.CONCLUSION： Butaprost effectively reduces the subconjunctival scarring response. Given the significanceof wound healing modulation in blebs, butaprost＇s inhibitory effect on subconjunctival Tenon＇s fibroblasts may be beneficial in managing postoperative scarring in glaucoma surgery.

Involvement of Rho-associated coiled-coil kinase signaling inhibition in TGF-β1/Smad2,3 signal transduction in vitro

AIM：To research the effect of Y-27632,a selective Rhoassociated coiled-coil kinase（ROCK） inhibitor,on TGF-β1/Smad2,3 signal transduction in ocular Tenon＇s capsule fi broblasts（OTFs）.METHODS：Primary ocular Tenon＇s capsule fibroblasts had been cultured in vitro.The effect of Y27632 on proliferation of OTF stimulated by lysophosphatidic acid（LPA） was evaluated by MTT colorimetric assay so as to sift out the proper concentrations range of Y-27632 for the next experiment.Real time-polymerase chain reactor（RT-PCR） was to analyze the changes of Smad2 and Smad3 genes of cells affected by Y-27632,though unaffected by transforming growth factorbeta1（TGF-β1）.Proteins of Smad2,Smad3,phosphorylated Smad2（Ser245/250/255）,and phosphorylated Smad3（Ser423/425/203） were respectively quantifi ed by Western blot after OTFs were successively incubated by TGF-β1 and Y-27632.Meanwhile,α-smooth muscular actin（α-SMA） protein was also quantified after the small intervening gene fragments of human Smad2 and Smad3 were designed,synthesized,and then transfected to OTFs.RESULTS：Y-27632 signifi cantly inhibited OTFs proliferation stimulated by LPA.Also Y-27632 signifi cantly suppressed the expressions of Smad2 m RNA,Smad2,3 proteins expressions,Smad3 phosphorylation at the carboxylic terminals of Ser423/425/203 which had been radically promoted by TGF-β1.Si RNA-Smad2,3 suppressed α-SMA expressions,but less effectively than Y-27632.CONCLUSION：The inhibition of ROCK signaling may be a potential therapeutic candidate for the treatment of the fi ltration channel fi brosis.