Study The Effect And Mechanism Of Endothelin-1 On The Trabecular Meshwork Aqueous Humor Outflow Pathway

Glaucoma, a leading cause of irreversible blindness in the world, is a group of optic neuropathy. Pathological increased introcular pressure (IOP), a result of abnormally increased resistance to drainage of aqueous humor from trabecular meshwork, is the most risk factor for glaucoma neuropathies.It reported that aqueous humor outflow resistance can be enhanced by the phagocytosis function defect of trabecular meshwork, also the extracellular matrix overexpression and deposition in trabecluar meshwork tissue, and the abnormal altering of the actin cytoskeleton and cell-extracellular matrix and cell-cell adhesions within the trabecular meshwork.Endothelin-1(ET-1), a micromolecule bioactivity peptide, is involved in variety of physiological and/or pathophysiological functions, such as maintaining vascular tone, regulating the constriction and dilatation of vascular and cell karyogenetic division. Meanwhile, ET-1 is also recognized as an important pro-fibrotic factor in maintaining fibrosis of various tissues. ET-1 can increase the production and deposition of extracellular matrix (ECM) in several cell types including fibroblasts, cardiac myocytes, and smooth muscle cells and plays an important role in hypertension, fibrosis of renal and cardiac myocytes. Meanwhile, ET-1 can lead to the cytoskeleton reorganization of vascular endothelial cell and pulmonary epithelial cells via activating the rho kinase, and ET-1 induces oxidative stress and inflammation by releasing of many inflammatory mediators.Recently, many reports indicated that the value of ET-1 is particularly high in the aqueous humour in POAG patient, normal tension glaucoma patient and glaucoma animal model. ET-1 is an intraocular pressure regulator and high associated with glaucoma However, the effect of ET-1 on the extracellular matrix production and deposition in trabecular meshwork, the phagocytosis function of trabecular meshwork, the F-actin cytoskeleton reorganization of the trabecular meshwork are unknown.To investigated the effect and mechanism of ET-1 on the human trabecular meshwork aqueous humor outflow pathway, this study were divided into four sections:1:we will focus on investigating the effect of ET-1 on extracellular matrix---fibronectin(FN) and collagen typeⅣ(ColⅣ) expression in cultured human trabecular meshwork cells(HTMCs) and in trabecular meshwork tissue of perfusion cultured human eye anterior segment. The effect of ET-1 on the IOP of perfusion cultured human eye anterior segment.2:we will further study the molecular mechanism of connective tissue growth factor (CTGF) in extracellular matrix production induced by ET-1.3:we investigate the effects of ET-1 on the phagocytosis function of trabecular meshwork.4:we study the effect of ET-1 on the cytoskeleton protein F-actin expression and organization. We try to understand the role of ET-1 regulate aqueous humor outflow and provide a theoretic evidence for lowing intraocular pressure in POAG.PartⅠEeffects of Endothelin-1 on the expression of Fibronectin and Collagen IV in human Trabecular meshwork Cells Objective 1.Observe the effect of ET-1 and ETR antagonist on the expression of FN and ColⅣin cultured human trabecular meshwork cells (HTMCs) in vitro.2.Observe the effect of ET-1 on the intraocular pressure and expression of and FN, ColⅣin trabecular meshwork of Perfusion Cultured Human Eye Anterior SegmentsMethods 1. HTMCs were cultured, subcultured in vitro and identified with anti-NSE, FN, LN and FⅧRag immunocytochemistry stain; 2. Observe the effect of ET-1 on the expression of ColⅣand FN in cultured human trabecular meshwork in vitro. The third passage HTMCs were randomly divided into four groups, control group, low-dose ET-1 (10-9 mol/L) treatment group, middle-dose ET-1 (10-8 mol/L) treatment group and high-dose ET-1 (10-7 mol/L) treatment group. To eveluate the effect of endothelin recptor antagonist on the expression of ColⅣand FN in HTMCs, the HTMCs were randomly divided into four groups, control group, ET-1(10-7 mol/L) treatment group, ETAR antagonist (1×10-7mol/L BQ123+10-7 mol/L ET-1) treatment group and ETBR antagonist (1×10-7 mol/L BQ788+10-7 mol/L ET-1) treatment group, each group was treated for 72h, The expression of ColⅣand FN of HTMCs were detected by Western-blot and immunofluorescence stain.3. To evaluate the effect of ET-1 on the expression of the ColⅣand FN in trabecular meshwork in perfusion cultured human eye anterior segment. Established perfusion cultured human eye anterior segment model,3 pair eyes were divided into control eye(left eye) and ET-1 treated eye(right eye), ET-1 treated eyes were perfused with 10-7mol/L ET-1 for 72h, and the intraocular pressure were recoded after 6h,12h,24h, 30h,36h,42h,48h,54h,60h,66h,72h treated, then the human eye anterior segment fixed with 4%paraformaldehyde, The ColⅣand FN were detected by immunofluorescence stain.Results 1. Anti-NSE, FN, LN were positive and FⅧRag immunocytochemistry stains were negative in HTMCs. HTMCs were cultured and subcultured successfully in vitro.2. The expression of FN significantly increased after treated with 10-7～10-9mol ET-1 (P<0.05); And The expression of ColⅣin HTMCs significantly increased after treated with 10-8～10-9mol ET-1 (P<0.05). Compared with ET-1 treated group, the expression of FN and Col IV decreased after treated with ETAR antagonist (P<0.05) but not ETBR antagonist (P>0.05). 3. The expression of FN and Col IV significantly increased in trabecular meshwork of perfusion cultured human eye anterior segment after treated with ET-1. The basal IOP rang from 15-20mmHg, the IOP increased and the maximum IOP reached to 27mmHg after treated with ET-1.Conclusions 1. ET-1, via ETA receptor, increases the expression of FN and ColⅣin cultured HTMCs in vitro.2. ET-1 increases the expression of FN and ColⅣin trabecular meshwork of perfusion cultured human eye anterior segment, and also increase the IOP of human eye anterior segment.PartⅡEeffects of CTGF on the expression of CoL IV and FN induced by endothelin-1 in human Trabecular meshwork CellsObjective 1.Observe the effect of CTGF on the expression of FN and ColⅣin human trabecular meshwork cells induced by ET-1.Methods 1. HTMCs were cultured and subcultured in vitro. The HTMCs were randomly divided into four groups:control group,24h,48h and 72h ET-1 (10-7mol/L) treatment group. The HTMCs were collected at different times and the mRNA of CTGF, FN and ColⅣwere detected by RT-PCR.2. Designed and synthesized CTGF antisense oligonucleotide (CTGF-ASODN) sequence and screening the most effective CTGF-ASODN sequence.3. The third passage HTMCs were randomly divided into five groups, control group, ET-1 group(10-7 mol ET-1), CTGF-ASODN group(100nmol CTGF-ASODN), ET-1+ CTGF-ASODN group(10-7mol ET-1+100nmol CTGF-ASODN+liposome 2000) and liposome 2000 control group(10-7mol ET-1+liposome 2000). The expression of CTGF, FN and Col IV were detected by Western-blot after cultured 24h. Results 1. The mRNA of CTGF, FN and Col IV in HTMCs significantly increased (P<0. 05), the mRNA of CTGF reached to maximum at 48h and FN, Col IV reached to maximum at 72h after treated with ET-1.2. Compared with ET-1 group, the expression of FN and Col IV in ET-1+CTGF-ASODN group decreased significantly (P<0.05), but not decreased in the CTGF-ASODN and liposome control group(P>0.05)Conclusions CTGF downregulation inhibited the expression of FN and Col IV in HTMCs induced by ET-1. CTGF is a key downstream mediator of ET-1.PartⅢThe Effect of Endothelin-1 on the Phagocytic Function in Cultured Human Trabecular Meshwork CellsObjective 1. Observe the Kinetics of phagocytosis in HTMCs 2. Study the effect of ET-1 on the phagocytic function in cultured HTMCs.Methods 1. HTMCs were cultured and subcultured in vitro. The third passage of HTMCs were incubated with fluorescent red labed latex beads for 0,4,8,12,24,48,72hours, The phagocytic kinetics of HTMCs were continuously evaluated by counting the numbers of latex beads in HTMCs using a fluorescence microscope.2. To evaluate the effect of ET-1 on the Phagocytic Function of HTMCs, the third passage of HTMCs were divided into 4 groups:control group, Low dose group:treated with 10-9 mol/L ET-1, middle-dose group:treated with 10"8 mol/L ET-1, High dose group:treated with 10-7 mol/L ET-1.3. To evaluate the effect of endothelin receptor on the phagocytic function of HTMCs, the third passage of HTMCs were divided into 4 groups:control group. ET-1 group, treated with 10-8 mol/L ET-1. ETAR antagonist group:treated with 10-8 mol/L ET-1 and 1×10-7 mol/L BQ123. ETBR antagonist group:treated with ET-1 and 1×10-7 mol/L BQ123.each group incubated with latex beads.The numbers of latex beads in HTMCs were counted with a fluorescence microscope. Results 1. The phagocytic kinetics studies revealed that the ingestion of latex beads were detected after incubation 4 hours, while the numbers were increasing with time until 24hours that the ingestion quantity was optimization and until 48 hours when ingestion were saturated, and the HTMCs morphology changed.2. When the HTMCs treated with different dose of ET-1, The numbers of latex beads in HTMCs was significantly reduced in a dose dependent manner (P<0.05).3 When treated with ET-1 and endothelin antagonist, the numbers of latex beads in HTMCs of ET-1 group were lower than control group and the ETAR antagonist group were higher than ET-1 group(P<0.05). However, the numbers of latex beads in HTMCs of ETBR antagonist group have no differenc with ET-1 group (P>0.05).Conclusions:1. ET-1 inhibit the phagocytic function of HTMCs.2. the ETA receptor play an part role in the phagocytic function of HTMCs.PartⅣThe Effects of Endothelin-1 on The Cytoskeleton Protein F-actin in Cultured Human Trabecular Meshwork CellsObjective To observe the effect of ET-1 on the Cytoskeleton protein F-actin of cultured HTMCs. Methods HTMCs were cultured and subcultured in vitro. The HTMCs were randomly divided into four groups, control group, low-dose ET-1(10-9 mol/L) treatment group, middle-dose ET-1 (10-8 mol/L) treatment group and high-dose ET-1 (10-7 mol/L) treatment group. After treated with ET-1, the expression of cytoskeleton Protein F-actin in HTMCs was analysised with Western-blot and the distribution of F-actin was detected with FITC- Phalloidin probe.Results ET-1 dose dependently and significantly increased F-actin in HTMCs(P<0.05). The F-actin stress fiber and periphery actin fiber highly increased and showed mild reorganization after treated with ET-1, and there were much more cell-to-cell and cell-to-extracellular matrix attachments formation in ET-1 treated cells comparable to untreated cells.Conclusions:ET-1 promote the expression of Cytoskeleton Protein F-actin and induced the HTMCs F-actin cytoskeleton reorganization.CONCLUSIONS and SUMMARY1. Our study found that ET-1, via ETA receptor, increase the expression of FN and Col IV in cultured HTMCs. In this study we also established perfusion human anterior segment model successfully. ET-1 promote FN and ColⅣproduction and deposition in TM tissue.2. CTGF downregulation obviously suppress the overexpression of FN and ColⅣinduced by ET-1.The results suggested that CTGF is an important downstream mediator of ET-1.3. ET-1 damaged the HTMCs phagocytic function. ETA receptor inhibitor partly blocks the role of ET-1.4. ET-1 increases the expression of cytoskeleton protein F-actin and induce the cytoskeleton reorganization of cultured HTMCs.ET-1 may be play an important role in regulating IOP and participate in development of POAG through increasing extracellular matrix production, cytoskeleton reorganization and phagocytosis damaging of trabcular meshwork.