RESUMEN
Glaucoma is a disease that damages the optic nerve, frequently leading to blindness. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, which is expected to affect 80 million people by 2020, causing bilateral blindness in over 10 million individuals. Because pathological changes to Schlemm's canal (SC) may account for significant resistance to outflow, there is considerable interest in characterizing and evaluating the Schlemm's canal as a target for glaucoma therapeutics. In conventional, two-dimensional culture, human Schlemm's canal (HSC) cells lose spatial, mechanical and biochemical cues, resulting in altered gene expression and cell signaling than observed in vivo, compromising the clinical relevance of data obtained from such systems. Here, we report, for the first time, that 3D culture of HSC cells on microfabricated scaffolds with defined physical and biochemical cues, rescued expression of key HSC markers, VE-cadherin and PECAM1, and mediated pore formation, crucial for the Schlemm's canal regulation of IOP. We demonstrated that following treatment with the glaucopathogenic agent, TGF-ß2, HSC cells undergo an endothelial-mesenchymal transition, which together with the increase in extracellular matrix (ECM) proteins might account for the decrease in outflow facility observed in patients with high TGF-ß2 levels in their aqueous humor. We also demonstrated that unlike 2D cultures, 3D cultures of HSC cells are amenable to gene transfer. Thus, our data imply that 3D culture of HSC cells may be used as a platform to advance our understanding of HSC physiology and pathology and as a model for high-throughput drug and gene screening.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Endotelio/citología , Ojo/citología , Glaucoma/tratamiento farmacológico , Ingeniería de Tejidos/métodos , Actinas/análisis , Antígenos CD/análisis , Biomimética , Cadherinas/análisis , Células Cultivadas , Técnicas de Cocultivo/métodos , Endotelio/efectos de los fármacos , Ojo/efectos de los fármacos , Ojo/patología , Glaucoma/patología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Andamios del Tejido/química , Factor de Crecimiento Transformador beta2/análisisRESUMEN
The mouse eye has physiological and genetic advantages to study conventional outflow function. However, its small size and shallow anterior chamber presents technical challenges to efficient intracameral delivery of genetic material to conventional outflow cells. The goal of this study was to optimize methods to overcome this technical hurdle, without damaging ocular structures or compromising outflow function. Gene targeting was monitored by immunofluorescence microscopy after transduction of adenovirus encoding green fluorescent protein driven by a CMV promoter. Guided by a micromanipulator and stereomicroscope, virus was delivered intracamerally to anesthetized mice by bolus injection using a 33 gauge needle attached to Hamilton syringe or infusion with glass micropipette connected to syringe pump. The total number of particles introduced remained constant, while volume of injected virus solution (3-10 µl) was varied for each method and time of infusion (3-40 min) tested. Outflow facility and intraocular pressure were monitored invasively using established techniques. Unlike bolus injections or slow infusions, introduction of virus intracamerally during rapid infusions (3 min) at any volume tested preferentially targeted trabecular meshwork and Schlemm's canal cells, with minimal transduction of neighboring cells. While infusions resulted in transient intraocular pressure spikes (commensurate with volume infused, Δ40-70 mmHg), eyes typically recovered within 60 min. Transduced eyes displayed normal outflow facility and tissue morphology 3-6 days after infusions. Taken together, fast infusion of virus solution in small volumes intracamerally is a novel and effective method to selectively deliver agents to conventional outflow cells in living mice.
Asunto(s)
Ojo/metabolismo , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Inyecciones Intraoculares/métodos , Transducción Genética/métodos , Adenoviridae/genética , Animales , Segmento Anterior del Ojo/metabolismo , Humor Acuoso/metabolismo , Citomegalovirus/genética , Terapia Genética/métodos , Glaucoma/terapia , Inyecciones Intraoculares/efectos adversos , Inyecciones Intraoculares/instrumentación , Presión Intraocular , Ratones , Ratones Endogámicos C57BL , Microinyecciones/efectos adversos , Microinyecciones/instrumentación , Microinyecciones/métodos , Microscopía Fluorescente , Regiones Promotoras Genéticas/genética , Malla Trabecular/metabolismoRESUMEN
The two cell types that populate the human conventional outflow pathway, Schlemm's canal (SC) and trabecular meshwork (TM) regulate intraocular pressure. In culture, SC and TM cells have been useful tools toward understanding their respective roles in conventional outflow homeostasis. Unfortunately, currently available protein markers that distinguish SC from TM cells are limited, motivating the present study. Antibodies that specifically recognize different vascular endothelial markers were used to probe lysates from mature cell monolayers subjected to SDS-PAGE followed by western blot analyses. Results show that SC and TM cells both expressed many of the endothelial candidate proteins investigated, such as Robo1/4, Tie2/TEK, VEGF-R1/R2, VCAM-1, eNOS and neuropilin-1. In contrast, all SC cell strains tested (n=11) expressed two proteins, fibulin-2 and vascular endothelial (VE) cadherin, not expressed by TM cells. To examine changes in VE-cadherin expression and cell-cell junction formation, indicated by transendothelial electrical resistance (TEER), SC cells were seeded onto filters at confluence and growth factors were withdrawn. Culturing cells in media containing adult bovine serum rather than fetal bovine serum resulted in a 75% mean increase in TEER and 67% corresponding average increase in VE-cadherin expression (p<0.05). While both TM and SC cells form monolayers, are contact inhibited, share some endothelial responsibilities and several endothelial protein markers, SC cells uniquely express at least two proteins which likely reflect a distinction in cellular responsibilities in vivo. One of these responsibilities, maintenance of the blood-aqueous barrier, can be modeled in culture upon withdrawal of growth factors from SC cell monolayers.
Asunto(s)
Antígenos CD/metabolismo , Biomarcadores/metabolismo , Cadherinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Endotelio Vascular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Limbo de la Córnea/metabolismo , Western Blotting , Diferenciación Celular , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Integrina alfa6/metabolismo , Limbo de la Córnea/citología , Malla Trabecular/citología , Malla Trabecular/metabolismoRESUMEN
Primary open-angle glaucoma is associated with elevated intraocular pressure, which in turn is believed to result from impaired outflow of aqueous humour. Aqueous humour outflow passes mainly through the trabecular meshwork (TM) and then through pores formed in the endothelium of Schlemm's canal (SC), which experiences a basal-to-apical pressure gradient. This gradient dramatically deforms the SC endothelial cell and potentially contributes to the formation of those pores. However, mechanical properties of the SC cell are poorly defined. Using optical magnetic twisting cytometry and traction force microscopy, here we characterize the mechanical properties of primary cultures of the human SC cell, and for the first time, the scope of their changes in response to pharmacological agents that are known to modulate outflow resistance. Lysophosphatidic acid, sphingosine-1-phosphate (S1P) and thrombin caused an increase in cell stiffness by up to 200 per cent, whereas in most cell strains, exposure to latrunculin A, isoproterenol, dibutryl cyclic-AMP or Y-27632 caused a decrease in cell stiffness by up to 80 per cent, highlighting that SC cells possess a remarkably wide contractile scope. Drug responses were variable across donors. S1P, for example, caused 200 per cent stiffening in one donor strain but only 20 per cent stiffening in another. Isoproterenol caused dose-dependent softening in three donor strains but little or no response in two others, a finding mirrored by changes in traction forces and consistent with the level of expression of ß(2)-adrenergic receptors. Despite donor variability, those drugs that typically increase outflow resistance systematically caused cell stiffness to increase, while in most cases, those drugs that typically decrease outflow resistance caused cell stiffness to decrease. These findings establish the endothelial cell of SC as a reactive but variable mechanical component of the aqueous humour outflow pathway. Although the mechanism and locus of increased outflow resistance remain unclear, these data suggest the SC endothelial cell to be a modulator of outflow resistance.
Asunto(s)
Humor Acuoso/fisiología , Células Endoteliales/fisiología , Mecanotransducción Celular/fisiología , Reología/métodos , Malla Trabecular/fisiología , Células Cultivadas , Humanos , Resistencia al Corte/fisiología , Estrés MecánicoRESUMEN
To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.
Asunto(s)
Exosomas/química , Malla Trabecular/citología , Humor Acuoso/fisiología , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Dexametasona/farmacología , Proteínas del Ojo/metabolismo , Glicoproteínas/metabolismo , Humanos , Ionomicina/farmacología , Glicoproteínas de Membrana/metabolismo , Neuropilina-1/metabolismo , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismoRESUMEN
Oxidative stress and TGFbeta-induced disturbance of cells and tissues are implicated in initiation and progression of pathophysiology of cells/tissues. Using primary human trabecular meshwork (TM) cells from normal and glaucomatous subjects, this study demonstrated that peroxiredoxin (PRDX) 6, an antioxidant, offsets the deleterious effects of oxidative stress on TM cells by optimizing ROS and TGFbeta levels. An analysis of glaucomatous TM cells revealed a reduced expression of PRDX6 mRNA and protein. Biochemical assays disclosed enhanced levels of ROS, as well as high levels of TGFbetas and these cells expressed elevated extracellular matrix (ECM) and Tsp1 proteins with reduced MMP2; conditions implicated in the pathophysiology of glaucoma. Non-glaucomatous TM cells exposed to TGFbetas/ROS showed similar features as in glaucomatous cells. The abnormalities induced were reversed by delivery of PRDX6. The data provide evidence that oxidative stress-induced abnormality in TM may be related to reduced PRDX6 expression and provide a foundation for antioxidant-based therapeutics for treating glaucoma.
Asunto(s)
Glaucoma/metabolismo , Estrés Oxidativo , Peroxiredoxina VI/metabolismo , Malla Trabecular/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Actinas/metabolismo , Células Cultivadas , Senescencia Celular , Daño del ADN , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Glaucoma/patología , Humanos , Peróxido de Hidrógeno/toxicidad , Metaloproteinasa 2 de la Matriz/metabolismo , Oxidantes/toxicidad , Estrés Oxidativo/efectos de los fármacos , Peroxiredoxina VI/genética , Fenotipo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Trombospondina 1/metabolismo , Malla Trabecular/efectos de los fármacos , Malla Trabecular/patología , Transducción Genética , Factor de Crecimiento Transformador beta/metabolismo , Transglutaminasas/metabolismo , Tropomiosina/metabolismoRESUMEN
Aquaporin-1 (AQP1) channels are expressed by trabecular meshwork (TM) and Schlemm's canal cells of the conventional outflow pathway where fluid movement is predominantly paracellular, suggesting a non-canonical role for AQP1. We hypothesized that AQP1 functions to protect TM cells during periods of mechanical strain. To test this idea, primary cultures of confluent human TM cells on Bioflex membranes were exposed to static and cyclic stretch for 8 and 24h using the Flexcell system. AQP1 expression in TM cells was assessed by SDS-PAGE and Western blot using anti-AQP1 IgGs. AQP1 protein bands were analyzed using densitometry and normalized to beta-actin expression. Cell damage was monitored by measuring lactate dehydrogenase (LDH) and histone deacetylase appearance in conditioned media. Recombinant expression of AQP1 in TM cell cultures was facilitated by transduction with adenovirus. Results show that AQP1 expression significantly increased 2-fold with 10% static stretch and 3.5-fold with 20% static stretch at 8h (n=4, p<0.05) and 24h (n=6, p<0.05). While histone deacetylase levels were unaffected by treatments, release of LDH from TM cells was the most profound at the 20% static stretch level (n=4, p<0.05). Significantly, cells were refractory to the 20% static stretch level when AQP1 expression was increased to near tissue levels. Analysis of LDH release with respect to AQP1 expression revealed an inverse linear relationship (r(2)=0.7780). Taken together, AQP1 in human TM appears to serve a protective role by facilitating improved cell viability during conditions of mechanical strain.
Asunto(s)
Acuaporina 1/fisiología , Malla Trabecular/citología , Adolescente , Adulto , Acuaporina 1/metabolismo , Tamaño de la Célula , Supervivencia Celular/fisiología , Células Cultivadas , Homeostasis/fisiología , Humanos , Lactante , L-Lactato Deshidrogenasa/metabolismo , Mecanotransducción Celular/fisiología , Estrés Mecánico , Malla Trabecular/metabolismo , Malla Trabecular/fisiologíaRESUMEN
AIM: The aim of the study was to compare selenium levels in plasma and aqueous humour in subjects with and without primary open-angle glaucoma (POAG). METHODS: Forty-seven POAG cases and 54 controls in this case-control study were recruited from surgery patients at the University Physician's Ophthalmology Clinic in Tucson, Arizona, USA. Aqueous humour and plasma selenium were determined by high-performance liquid chromatography ion channel plasma mass spectrometry (HPLC ICP-MS). Potential confounders were assessed via a questionnaire. Biological samples were collected and processed at surgery and analysed for selenium content after collection was complete. Outcome measures included the odds of glaucoma in relationship to plasma selenium, aqueous humour selenium, and the ratio of levels of aqueous humour selenium to plasma selenium. RESULTS: Tertile of selenium and its relationship to POAG was examined. After adjustment for common glaucoma risk factors, the odds of glaucoma in the highest tertile of plasma selenium (OR = 11.3; p = 0.03) and the middle tertile of aqueous humour selenium (OR = 0.06; p = 0.02) was significantly associated with glaucoma. CONCLUSION: Although a causal pathway cannot be inferred from our analysis, our data, added to that of others, suggest that the pathology is selenium-related.
Asunto(s)
Humor Acuoso/química , Glaucoma de Ángulo Abierto/metabolismo , Selenio/análisis , Malla Trabecular/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Cromatografía Líquida de Alta Presión , Femenino , Glaucoma de Ángulo Abierto/sangre , Glaucoma de Ángulo Abierto/inducido químicamente , Humanos , Masculino , Persona de Mediana Edad , Medición de Riesgo , Selenio/efectos adversos , Selenio/sangre , Encuestas y CuestionariosRESUMEN
Mutations in myocilin result in ocular hypertension, likely due to decreased drainage of aqueous humor through the trabecular meshwork. Since less myocilin is found in the aqueous humor of those with disease-causing mutations, understanding myocilin's role in the aqueous humor is of clinical importance. Recently, myocilin was shown to exit cultured trabecular meshwork cells in association with shed vesicles called exosomes. To examine relevance of this finding in a physiological setting, the present study examined three different types of ocular samples for the presence of myocilin-associated exosomes. Using differential centrifugation steps, we found myocilin associated with exosomes isolated from effluent collected from human anterior segments in organ culture and aqueous humor obtained from human cadaveric eyes or from patients undergoing excisional surgery. Similar to results with cultured cells, myocilin associated predominately with exosomes in fresh samples, appeared mostly soluble at later times, and had biochemical properties (density of 1.13-1.19 g/ml in linear sucrose gradient) similar to those characteristics of exosomes. These data indicate that exosomes are present and may facilitate the transport of myocilin into the extracellular space of human ocular cells.
Asunto(s)
Humor Acuoso/química , Vesículas Citoplasmáticas/química , Proteínas del Citoesqueleto/análisis , Proteínas del Ojo/análisis , Glicoproteínas/análisis , Glaucoma/metabolismo , HumanosRESUMEN
Mutations in myocilin (MYOC) associate with glaucoma and ocular hypertension. Unfortunately, the specific role of MYOC, a widely expressed protein of unknown function, in ocular hypertension is unknown. Since MYOC localizes both to intracellular membranes and to the cytosol, we tested the hypothesis that MYOC is a cytosolic protein that associates with cellular membranes via its coiled-coil domain. Using green fluorescent protein (GFP) chimeras in expression and metabolic labeling studies, we observed that MYOC's putative signal peptide failed to traffic GFP into the secretory machinery and out of transfected cells. Next, we tested which of MYOC's three folding domains were responsible for targeting. In cell fractionation and immunofluorescence microscopy studies, the coiled-coil, but not the helix-turn-helix or olfactomedin domains, was necessary and sufficient to target GFP chimeras to cell membranes. Interestingly, a vesicular phenotype required sequential addition of the helix-turn-helix and olfactomedin domains to the coiled-coil. Taken together, these data indicate that the coiled-coil domain, not the putative signal sequence, is responsible for the targeting of MYOC to the secretory machinery.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/metabolismo , Glicoproteínas/metabolismo , Membranas Intracelulares/metabolismo , Malla Trabecular/metabolismo , Animales , Células COS , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Proteínas del Ojo/genética , Glicoproteínas/genética , Proteínas Fluorescentes Verdes , Microscopía Confocal , Microscopía Fluorescente , Estructura Terciaria de Proteína , Malla Trabecular/citología , TransfecciónRESUMEN
Organ culture of human anterior segments is a powerful tool for understanding trabecular meshwork biology. However, data from a significant percentage of cultured anterior segments are unusable because tissues fail to meet quality control requirements, such as having adequate trabecular meshwork histology. The purpose of the present study was to evaluate a novel, real time method for assessing the viability of conventional drainage tissues in the human anterior segment perfusion model. Twenty-two human anterior segments were perfusion cultured using standard techniques for one week while measuring outflow facility and central corneal thickness (CCT). After perfusion-fixation, toludine blue-stained histological sections of drainage tissues from all four quadrants of each anterior segment were graded and endothelial cell nuclei from cornea centers were stained with 4',6-diamidino-2-phenylindole and counted. We found that most anterior segments with a stable outflow facility had a CCT that decreased over time, while anterior segments with an unstable outflow facility had CCT measurements that failed to decrease over time (P<0.01). When comparing CCT measurements to histological appearance of outflow tissues, we found that in 11/11 cases, anterior segments with an acceptable histological score had a negative CCT slope (P<0.01). Conversely in 3/4 instances, anterior segments with an unacceptable histological score had a positive CCT slope. Lastly, we observed a significant relationship between CCT measurements and corneal endothelial density (P<0.01). Thus, the simple procedure of measuring CCT during anterior segment perfusion provides a second useful measure to assess the viability of the anterior segment during the perfusion process.
Asunto(s)
Segmento Anterior del Ojo/fisiología , Técnicas de Cultivo de Órganos/métodos , Anciano , Anciano de 80 o más Años , Segmento Anterior del Ojo/anatomía & histología , Segmento Anterior del Ojo/citología , Recuento de Células , Supervivencia Celular/fisiología , Córnea/anatomía & histología , Córnea/citología , Córnea/fisiología , Células Endoteliales/fisiología , Endotelio Corneal/citología , Endotelio Corneal/fisiología , Humanos , Persona de Mediana Edad , Modelos Biológicos , Esclerótica/anatomía & histología , Esclerótica/fisiología , Malla Trabecular/anatomía & histologíaRESUMEN
PURPOSE: Elevated intraocular pressure in those with glaucoma appears to be a function of increased resistance to movement of aqueous humor through the conventional outflow pathway. The majority of resistance in both normal and glaucomatous eyes is generated in the region between the juxtacanalicular trabecular meshwork and the inner wall of Schlemm's canal. To accommodate transient elevations in pressure, we hypothesize that conventional outflow increases rapidly due to changes in complexity of intercellular junctions between cells of the inner wall of Schlemm's canal. METHODS: To test this hypothesis we examined specifically the effects of hydrostatic pressure gradients and the calcium chelator, Na2EDTA, on permeability of cultured human Schlemm's canal cell monolayers in isolation. Human Schlemm's Canal cells were isolated, cultured and then seeded onto permeable supports and maintained in culture to allow intercellular junctions to mature. With a minimum net transendothelial electrical resistance of 10 Ohm cm2, cells were placed into an Ussing-type chamber and hydraulic conductivity was calculated from pressure and flow measurements that were continuously recorded. Simultaneously, transendothelial electrical resistance was measured manually at fixed intervals. In parallel experiments, cell margins were monitored in real time by videomicroscopy. RESULTS: During the baseline measurement period when cells were exposed to pressure but not Na2EDTA, hydraulic conductivity was constant but transendothelial electrical resistance decreased continuously at rate of 0.24 Ohm cm2/minute. After Na2EDTA treatment, no significant change in transendothelial electrical resistance was measured while, hydraulic conductivity of Schlemm's Canal monolayers increased significantly by 125%; corresponding to noticeable intercellular separations. Restoration of cell-cell contact was observed by videomicroscopy 30 minutes following washout of Na2EDTA and functionally after 2 hours. CONCLUSIONS: Responses of Schlemm's Canal cells to pressure and calcium chelators in vitro are consistent with a role for calcium sensitive junctions in outflow resistance in vivo.
Asunto(s)
Segmento Anterior del Ojo/metabolismo , Quelantes/farmacología , Ácido Edético/farmacología , Presión Hidrostática , Uniones Intercelulares/fisiología , Animales , Segmento Anterior del Ojo/citología , Segmento Anterior del Ojo/efectos de los fármacos , Células Cultivadas , Perros , Conductividad Eléctrica , Impedancia Eléctrica , Humanos , Microscopía por Video , Permeabilidad , Esclerótica/citologíaRESUMEN
A(1) adenosine receptors (ARs) reduce, and A(2)ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A(1)AR and A(2)AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A(1), A(2A) and A(3)AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A(1)ARs, CGS 21680 and DPMA for A(2A)ARs, and Cl-IB-MECA and IB-MECA for A(3)ARs. Stimulation of A(1), A(2A) and A(3)ARs elevated Ca(2+), measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A(1), A(2A) and A(3)AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A(1), A(2A) and A(3)ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca(2+) and volume, but likely by actions on separate cell targets.
Asunto(s)
Humor Acuoso/efectos de los fármacos , Humor Acuoso/fisiología , Agonistas del Receptor Purinérgico P1 , Malla Trabecular/efectos de los fármacos , Malla Trabecular/fisiología , Adenosina/metabolismo , Agonistas del Receptor de Adenosina A1 , Agonistas del Receptor de Adenosina A2 , Agonistas del Receptor de Adenosina A3 , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Humanos , Líquido Intracelular/metabolismo , Canales Iónicos/efectos de los fármacos , Malla Trabecular/citologíaRESUMEN
Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB(1) class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB(1) receptor-specific oligodeoxynucleotide primers, cannabinoid CB(1) receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB(1) receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB(1) receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB(1) immunoglobulin-G in tissue sections localized cannabinoid CB(1) receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [(35)S]GTP gamma S binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [(35)S]GTP gamma S binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 microM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB(1) receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB(1) receptors by CP-55,940 resulted in a 2.3+/-0.3 and 1.7+/-0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB(1) mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB(1) receptors in the trabecular meshwork and increase aqueous outflow.
Asunto(s)
Cuerpo Ciliar/metabolismo , Receptores de Droga/metabolismo , Malla Trabecular/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Cannabinoides/antagonistas & inhibidores , Cannabinoides/farmacología , Bovinos , Separación Celular , AMP Cíclico/metabolismo , Ciclohexanoles/farmacología , Endocannabinoides , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Presión Intraocular/efectos de los fármacos , Ligandos , Piperidinas/farmacología , Alcamidas Poliinsaturadas , Pirazoles/farmacología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Cannabinoides , Receptores de Droga/agonistas , Receptores de Droga/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , RimonabantRESUMEN
PURPOSE: Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS: An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS: AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS: In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.
Asunto(s)
Acuaporinas/fisiología , Malla Trabecular/citología , Adenovirus Humanos/genética , Adolescente , Anciano , Acuaporina 1 , Antígenos de Grupos Sanguíneos , Permeabilidad de la Membrana Celular , Tamaño de la Célula/fisiología , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Immunoblotting , Recién Nacido , Persona de Mediana Edad , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , Proteínas Recombinantes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Malla Trabecular/metabolismoRESUMEN
PURPOSE: Corneal endothelial fluid transport is dependent on HCO(3)(-) and CO(2) fluxes. CO(2) permeability (P:CO(2)) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO(2). An AQP1 knockout mouse model, however, showed no evidence for CO(2) transport through AQP1 in erythrocytes or lung. Because HCO(3)(-) and CO(2) fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO(2). METHODS: BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1. RESULTS: Expression of AQP1 and osmotic water permeability (control P(f) = 0.046 +/- 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P(f). Adenoviral empty virus (EV) infection did not change AQP1 expression or P(f). PCO(2) was determined by measuring the rate of intracellular pH decrease after exposure to CO(2)/HCO(3)(-)-rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO(2) of BCEC (0.0036 +/- 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P(f) could also be reduced more than 50% by 3 to 5 minutes' exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease. CONCLUSIONS: AQP1 does not contribute to PCO(2) in corneal endothelial cells.
Asunto(s)
Acuaporinas/biosíntesis , Dióxido de Carbono/metabolismo , Endotelio Corneal/metabolismo , Adenoviridae/genética , Animales , Acuaporina 1 , Acuaporinas/genética , Transporte Biológico , Bovinos , Células Cultivadas , Endotelio Corneal/virología , Colorantes Fluorescentes/metabolismo , Expresión Génica , Immunoblotting , Oligonucleótidos Antisentido , Permeabilidad , ARN Mensajero/metabolismo , Transfección , Agua/metabolismoRESUMEN
PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow.
Asunto(s)
Segmento Anterior del Ojo/metabolismo , AMP Cíclico/metabolismo , Endotelio/metabolismo , Presión Hidrostática , Segmento Anterior del Ojo/citología , Humor Acuoso/metabolismo , Separación Celular , Células Cultivadas , Endotelio/citología , Humanos , Persona de Mediana Edad , Malla Trabecular/citología , Malla Trabecular/metabolismoRESUMEN
PURPOSE: An important goal in glaucoma research has been to understand the functional contribution of trabecular meshwork (TM) and Schlemm's canal (SC) endothelia to aqueous humor outflow resistance. To date, TM cells are routinely cultured and used as a model by several laboratories. However, there has been only limited success in isolating SC cells. The current objective was to develop a technique for selective isolation and culture of endothelial cells from human SC. METHODS: The anterior chamber of human cadaveric eyes was cut into eight equal and radially symmetric wedge-shaped pieces. Using a dissecting microscope, a gelatin-coated suture (6-0 sterile nylon monofilament) was gently inserted into the lumen of SC and advanced into the canal. The cannulated pieces of tissue were placed in culture medium and maintained for 3 weeks. Sutures were removed from SC and cells seeded onto 3-cm culture plates. Morphology, growth characteristics, and expression of endothelial surface antigens and other cellular markers were evaluated. RESULTS: Of the 20 pairs of eyes that were cannulated, primary cells were obtained from 13. All SC cell isolates had a fusiform morphology; formed nonoverlapping, linearly arranged monolayers; and were contact inhibited. Schlemm's canal cell isolates reacted with antibodies specific for CD44 (hyaluron receptor), CD54 (intercellular adhesion molecule-1, ICAM-1), tissue-type plasminogen activator, and TM-inducible glucocorticoid-responsive protein-myocilin (TIGR-MYOC). Unlike TM cells, however, TIGR-MYOC protein was not induced in SC cells after long-term dexamethasone treatment. Schlemm's canal cells endocytosed low-density lipoprotein and acetylated low-density lipoprotein, and in the presence of Matrigel organized into multicellular tubelike structures. CONCLUSIONS: Cannulation of SC with gelatin-coated suture material is an effective method for the isolation of human SC cells and provides a cellular model to study the potential role of SC cells in aqueous humor outflow function.
Asunto(s)
Separación Celular/métodos , Endotelio/citología , Esclerótica/citología , Malla Trabecular/citología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Biomarcadores , Técnicas de Cultivo de Célula/métodos , Proteínas del Citoesqueleto/metabolismo , Endotelio/metabolismo , Proteínas del Ojo/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Glicoproteínas/metabolismo , Humanos , Uniones Intercelulares , Persona de Mediana Edad , Esclerótica/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Malla Trabecular/metabolismoRESUMEN
PURPOSE: Prostaglandin F2 alpha (PGF2 alpha) and analogs, such as latanoprost, are thought to lower intraocular pressure (IOP), primarily by increasing uveoscleral outflow. However, outflow through the trabecular meshwork may be increased as well. The authors hypothesize that any effect on the trabecular meshwork is mediated by prostanoid FP receptors (receptors for prostaglandin F2 alpha) in this tissue. METHODS: To test this hypothesis, tissue sections of the human trabecular meshwork and cultures of human trabecular meshwork cells were examined for the presence of FP receptors using immunofluorescence microscopy with affinity-purified antibodies raised against a glutathione-S-transferase (GST)-FPA receptor fusion protein. The presence of the receptor was confirmed by using reverse transcription-polymerase chain reaction (RT-PCR), functional assays of PGF2 alpha-stimulated inositol phosphate hydrolysis, and intracellular calcium measurements. RESULTS: Positive FPA receptor immunolabeling was observed in sections of the human trabecular meshwork and in cultured human trabecular meshwork cells. In both cases, specific labeling could be blocked by preincubation with a GST-FPA receptor fusion protein. Cross-blocking experiments with other receptor fusion proteins did not block specific labeling in cultured trabecular meshwork cells. PGF2 alpha caused a dose-dependent increase in total inositol phosphate accumulation and intracellular calcium release in human trabecular meshwork cells that was consistent with the presence of FP receptors. Using RT-PCR, message-encoding prostanoid FPA receptors were found in total RNA isolated from human trabecular meshwork cells. CONCLUSIONS: Prostanoid FPA receptors exist in human trabecular meshwork cells, as shown by the presence of mRNA, protein, and functional response to PGF2 alpha. This study indicates that functional FP receptors are present in the human trabecular meshwork and that they may be involved in mediating some of the IOP-lowering effects of PGF2 alpha in the eye.