In vitro localization of TIGR/MYOC in trabecular meshwork extracellular matrix and binding to fibronectin.

PURPOSE: To determine whether trabecular meshwork-inducible glucocorticoid response/myocilin (TIGR/MYOC) protein associates with the extracellular matrix (ECM) of human trabecular meshwork (HTM) cells. METHODS: The extracellular localization of TIGR/MYOC was examined by immunofluorescence microscopy in HTM cultures treated with and without dexamethasone and ascorbate and in a transformed HTM cell line, TM-1, transiently transfected with TIGR/MYOC cDNA. Antibodies to TIGR/MYOC, fibronectin, laminin, type IV collagen, or thrombospondin were used to determine the extracellular localization of TIGR/MYOC. Solid phase binding assays using 125I-recombinant TIGR/MYOC and types I and IV collagens, fibronectin, and laminin were done to examine the association of TIGR/MYOC with these proteins and to identify a specific TIGR/MYOC binding site within fibronectin. The domains of fibronectin tested were the fibrin/collagen binding domain, the RGD domain, and the Heparin II (Hep II) domain. RESULTS: TIGR/MYOC colocalized with fibronectin, laminin, and type IV collagen, but not thrombospondin in both dexamethasone and dexamethasone/ascorbate-treated HTM cultures and in TM-1 cultures transfected with TIGR/MYOC cDNA. In solid phase binding assays, 125I-TIGR/MYOC bound fibronectin but not laminin or type IV collagen. Binding to fibronectin could be competed with excess TIGR/MYOC or fibronectin. Specific binding was found for the Hep II domain of fibronectin. CONCLUSIONS: TIGR/MYOC can associate with components of the ECM via interactions with the Hep II domain of fibronectin. The interactions with the Hep II domain of fibronectin could alter cell-matrix interactions in the TM and provides an interesting lead to explore the role(s) of TIGR/MYOC in both steroid-induced and primary open angle glaucoma.     

Immunohistochemical localization of MYOC/TIGR protein in the trabecular tissue of normal and glaucomatous eyes

PURPOSE: To examine immunohistochemically the localization of myocilin/trabecular meshwork inducible glucocorticoid response (MYOC/TIGR) protein in the glaucomatous and normal trabecular meshworks. METHODS: Trabecular tissues were used from one eye with late-onset goniodysgenetic glaucoma, three with primary open angle glaucoma (one of which had the MYOC/TIGR gene mutation), two with exfoliation glaucoma and one without glaucoma. For light microscopic immunohistochemistry, frozen sections were stained by the avidin-biotin complex method using anti-MYOC/TIGR polyclonal antibody. For electron microscopic immunohistochemistry, the pre-embedding method using the same antibody was performed. Double immunostaining using both anti-MYOC/TIGR and anti-type VI collagen antibodies was done by the immunofluorescence method. RESULTS: With light microscopy, immunoreactivity was seen in the whole trabecular meshwork of each of the specimens. No notable differences were detected in staining among the types of glaucoma, or between the eyes with and those without the gene mutation. Under electron microscopy, immunoreaction products were observed not only in the cytoplasm of the trabecular cells but also in the extracellular matrix, where staining was associated with the long-spacing collagen, fine granular materials and possibly microfibrils. With double immunohistochemistry, MYOC/TIGR was colocalized with type VI collagen in the trabecular meshwork. CONCLUSIONS: In glaucomatous and normal trabecular meshworks, the MYOC/TIGR protein is distributed in the extracellular matrix colocalizing with type VI collagen.     

Current perspectives on the TIGR/MYOC gene (Myocilin) and glaucoma

Over the past several years, many groups worldwide have confirmed the presence of probable disease-causing mutations in the coding region of the (TIGR/MYOC) gene associated with glaucoma. Disease-associated point mutations are often found in the third exon of TIGR/MYOC and are predicted to exert a substantial influence on protein structure. Although there has been speculation as to the mechanisms involved in the pathogenic effects for a number of the mutations, the processes leading to the development of glaucoma involving TIGR/MYOC remain to be elucidated. In addition to ongoing mutation studies, efforts are underway to follow up on TIGR/MYOC gene regulation studies in human trabecular meshwork cells and other possibly relevant cell types. Potentially by altering gene regulation, a major variant (-1000 G/C), present in 15-20% of individuals, appears to be associated with a more rapid progression of glaucomatous disease. This article addresses several of these areas of research on the TIGR/MYOC gene and glaucoma, briefly presenting currently available evidence and considering or updating information presented previously.     

A review of genetic and structural understanding of the role of myocilin in primary open angle glaucoma

Primary open angle glaucoma (POAG) is the most common form of glaucoma and the second leading cause of blindness in the world. Discovery of the candidate gene MYOC (TIGR/MYOC) encoding the protein myocilin, believed to have a role in cytoskeletal function, might play a key role in understanding the pathogenesis of POAG. MYOC is expressed in many ocular tissues, including trabecular meshwork (TM), a specialised eye tissue essential in regulating intraocular pressure (IOP). Later it was shown to be the trabecular meshwork inducible-glucocorticoid response protein (TIGR). Mutations in MYOC have been identified as the cause of hereditary juvenile-onset open-angle glaucoma (JOAG). The unprocessed myocilin with signal peptide is a 55-kDa protein with 504 amino acids. Mature myocilin is known to form multimers. Wild type myocilin protein is normally secreted into the trabecular extracellular matrix (ECM) and there appears to interact with various ECM materials. It is believed that the deposition of high amounts of myocilin in trabecular ECM could affect aqueous outflow either by physical barrier and/or through cell-mediated process leading to elevation of IOP. The N-terminal region of the myocilin has sequence similarity to myosin (muscle protein) and the C-terminal of the protein has an olfactomedin-like domain. Structural and genetic studies of the MYOC gene and its protein product along with molecular modeling could lead to better understanding of the pathogenesis of POAG. This review highlights the current understanding of myocilin and the relevance of genetic and structural work.     

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.     

The myocilin (MYOC) gene expression in the human trabecular meshwork

PURPOSE: We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it 'myocilin'. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes. METHODS: The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes. RESULTS: In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall. CONCLUSIONS: Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.     

Recombinant TIGR/MYOC increases outflow resistance in the human anterior segment

PURPOSE: To determine the effect of human recombinant TIGR/myocilin (MYOC) protein on outflow resistance in the human anterior segment. METHODS: A cDNA for MYOC was inserted into a bacterial expression system and purified with nickel ion affinity chromatography. The anterior segments of 12 pairs of human eyes were placed in perfusion organ culture. One eye received an anterior chamber exchange with partially purified recombinant MYOC (25 microgram), whereas the other eye received either heat-denatured recombinant MYOC (25 microgram), partially purified ss-galactosidase (25 or 250 microgram), or partially purified control proteins isolated from a null expression lysate (25 microgram). Eyes were fixed up to 72 hours after infusion, and immunohistochemistry was performed using anti-MYOC polyclonal antibody. RESULTS: Recombinant MYOC caused an increase in IOP over 12 hours, increasing outflow resistance 94%, whereas the fellow eye infused with null expression sample increased 12% (n = 7; P = 0.0005). When compared with recombinant MYOC, neither heat-denatured MYOC, recombinant ss-galactosidase, bovine serum albumin, nor fetal calf serum caused an increase in outflow resistance. MYOC IOP remained above baseline levels for 48 to 72 hours. Immunohistochemistry results confirmed the presence of recombinant MYOC in the trabecular meshwork. CONCLUSIONS: Recombinant MYOC increased outflow resistance in human anterior segments, whereas control proteins did not. MYOC may increase outflow resistance by specific interactions within the trabecular meshwork.     

Expression analysis of the matrix GLA protein and VE-cadherin gene promoters in the outflow pathway

PURPOSE: To test the ability of promoter fragments from the matrix Gla protein (MGP) and vascular endothelial-cadherin (VE-cad) genes to target gene expression in a specific manner in the cells of the outflow pathway, by using adenoviral-mediated gene transfer in organ culture. METHODS: Perfused anterior segments of human eyes were infected with replication-deficient recombinant adenoviruses expressing the beta-galactosidase reporter gene driven by the cytomegalovirus (CMV; control, n = 6), MGP (n = 6), or VE-cad (n = 12) promoters. Forty-eight hours after infection, the anterior segments were fixed and stained for beta-galactosidase activity. The distribution of beta-galactosidase expression was analyzed in paraffin-embedded sections. RESULTS: The MGP promoter fragment resulted in beta-galactosidase expression by the cells of the conventional outflow pathway and did not show any activity in the corneal endothelium or other cells posterior to the scleral spur. Adenovirus containing the VE-cad promoter fragment showed functionality of the promoter in vascular endothelial cells, but failed to produce any detectable expression in the cells of the outflow pathway. CONCLUSIONS: Directed expression by the MGP gene promoter specifically to the trabecular meshwork (TM) provides a new tool for specific gene transfer to the outflow pathway. Results with the VE-cad promoter fragment indicate possible differences in the regulation of this gene between vascular and Schlemm's canal endothelial cells. Taken together, these data demonstrate the feasibility of targeted gene expression to the outflow pathway cells using tissue specific promoters.     

内皮素-1体外对人小梁细胞骨架肌动蛋白的影响

目的:观察内皮素-1(endothelin-1,ET-1)对人眼小梁细胞细胞骨架肌动蛋白的影响。方法:培养3代人眼小梁细胞,随机分为4组:对照组(0mol/L ET-1);ET-1低剂量组(10-9mol/L ET-1);ET-1中剂量组(10-8mol/L ET-1);ET-1高剂量组(10-7mol/L ET-1),各组细胞分别处理72h后,应用Western-blot方法检测小梁细胞骨架肌动蛋白(F-actin)的表达,并用FITC-phalloidin荧光探针观察肌动蛋白在细胞内的分布。结果:ET-1作用后人小梁细胞肌动蛋白表达明显增高,在10-9～10-7mol/L浓度范围内呈剂量依耐性,荧光探针示ET-1处理组小梁细胞肌动蛋白应力纤维和周边肌动蛋白明显增多浓集,细胞微丝附着与胞膜,细胞间连接及细胞贴壁部位连接明显增多。结论:ET-1能促进小梁细胞肌动蛋白表达,参与了小梁细胞骨架的重构。     

Effects of endothelin-1 on the cytoskeleton protein F-actin of human trabecular meshwork cells in vitro

AIM: To observe the effect of endothelin-1 (ET-1) on the cytoskeleton protein F-actin of cultured human trabecular meshwork (HTM) cells. · METHODS: Cultured HTM cells 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 trabecular meshwork was analyzed 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 trabecular meshwork cells (P <0.05). The F-actin stress fiber and periphery actin fiber highly increased and manifested 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 HTM cells than that in the untreated HTM cells. · CONCLUSION: ET-1 promotes the expression of cytoske- leton protein F-actin and induced the trabecular meshwork actin cytoskeleton reorganization.     

Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter

PURPOSE: To compare the gene expression profile of trabecular meshwork (TM) and Schlemm's canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway. METHODS: The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5' promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ. RESULTS: gamma-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments. CONCLUSIONS: Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.     

Clinical phenotype of a Japanese family with primary open angle glaucoma caused by a Pro370Leu mutation in the MYOC/TIGR gene.

PURPOSE: To present the phenotype of two patients with primary open angle glaucoma (POAG) caused by a mutation of the myocilin/trabecular meshwork-inducible glucocorticoid response (MYOC/TIGR) gene. METHODS: Complete ocular examinations were performed on the 13-year-old proband, her father, mother, and sister. DNA analysis was performed to detect the mutant gene. RESULTS: The proband and her father were found to have a mutation of the MYOC/TIGR gene. Both patients carried a heterozygous mutation in the 1,109th nucleotide, which corresponds to the 370th amino acid residue of the MYOC/TIGR gene. The clinical characteristics of both patients were: (1) development of POAG at an early age, (2) high peaks of intraocular pressure. and (3) poor response to medical treatment. CONCLUSIONS: The phenotype of these patients with a mutation of the MYOC/TIGR gene agreed with reports of other patients with mutations at other loci in this gene. The discovery of the MYOC/TIGR gene not only makes early detection of glaucoma possible, but also presents a new direction for investigating the pathogenesis of glaucoma.     

Herpes simplex virus mediated gene transfer to primate ocular tissues.

We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and beta-galactosidase activity was measured histochemically. Six cynomolgus monkey eyes received viral injections into the anterior chamber (2 x 10(7) pfu) and/or the vitreous (5 x 10(7) pfu), and the distribution of cells expressing lacZ was evaluated. In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent beta-galactosidase activity. In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells. Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively. We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis. This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys. Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable.     

Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus

AIM:To investigate whether the enhanced green fluorescent protein (EGFP) reporter gene could be transferred into human trabecular meshwork (HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS:The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection (MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1×10⁸ transducing unit (TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS:The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo. Immunohistochemistry staining revealed that 88.19% EGFP-positive trabecular meshwork (TM) cells were observed in the human anterior segment. Nevertheless, the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group (P>0.05).CONCLUSION:EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.     

Effects of endothelin-1 on human trabecular meshwork cell contraction. an in vitro cell culture model

Trabecular meshwork (TM) cells are now considered to play an active role in the aqueous outflow mechanism, since they exhibit smooth muscle-like contractile properties. Endothelin-1 (ET-1), a potent vasoconstrictor peptide, has been proposed to play a role in the local regulation of aqueous outflow and intra-ocular pressure control. We propose an in vitro culture model as a method in the study of ET-1-induced human trabecular meshwork (HTM) cell contractility. Experiments were performed on semi-confluent HTM cells (primary cultures established from normotensive human donor eyes) at the 2nd passage with PBS as control, ET-1, sarafotoxin 6c (a selective endothelin B receptor agonist) and Y-27632 (a selective inhibitor of rho-associated kinase). The contractile status of the cells was evaluated by a morphometric analysis of the cell area, assuming that HTM cells in culture are able to reduce their area as a consequence of cytoskeletal contraction rather than regulatory volume decrease. Our data indicate that image analysis of the HTM cell area can be a reliable method in the study of TM cell contractility, since it utilizes human cells and offers versatile opportunities for the pharmacological evaluation of drugs controlling HTM cell status.