RESUMO
Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.
Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Guias como Assunto , Malha Trabecular/citologia , Fatores Etários , Animais , Biomarcadores/metabolismo , Consenso , Feto , Humanos , Doadores de Tecidos , Preservação de Tecido , Coleta de Tecidos e Órgãos , Malha Trabecular/metabolismoRESUMO
Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 +/- 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R(f)) in recipient to donor cell was 0.47 +/- 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R(f) by approximately 60% to 0.20 +/- 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 microM) also reduced LY dye transfer by approximately 60%, reducing R(f) from 0.41 +/- 0.05 (n = 15) to 0.17 +/- 0.05 (n = 20) after 10 min. Junctional currents were lowered by approximately 50% (n = 6) after 10-min perfusion with 500 microM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 microM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.
Assuntos
Corpo Ciliar , Células Epiteliais/metabolismo , Junções Comunicantes/metabolismo , Animais , Humor Aquoso/metabolismo , Bucladesina/metabolismo , Bovinos , Células Cultivadas , Corpo Ciliar/citologia , Corpo Ciliar/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/citologia , Corantes Fluorescentes/metabolismo , Heptanol/metabolismo , Isoquinolinas/metabolismo , Técnicas de Patch-Clamp , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
PURPOSE: Mutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. We investigated the potential use of small interfering RNAs (siRNAs) to suppress the expression of KE and MYOC and the related cytotoxicity of mutant myocilins in vitro. METHODS: cDNAs of the human keratoepithelin (KE) gene and myocilin (MYOC) gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP fusion protein by KE-specific shRNAs was evaluated by fluorescence microscopy and western blotting. Suppression of MYOC-EGFP fusion protein by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of western blots. The cellular stress response of TM cells induced by misfolded mutant myocilins was evaluated with a BiP promoter-driven luciferase reporter assay. RESULTS: One shRNA (targeting the coding sequence starting at 1,528 bp of KE) reduced the expression of KE-EGFP in HEK293 cells approximately by 50% whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed more than 80% of the expression of fusion protein. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of MYOC (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP fusion protein, ranged from 78% to 90% on average. The activation of the BiP gene (a cellular stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs. CONCLUSIONS: KE-specific or MYOC-specific shRNAs effectively suppressed the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins in vitro. RNA interference may have future therapeutic implications in suppressing these genes.