RESUMEN
Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.
Asunto(s)
Humor Acuoso/metabolismo , Presión Intraocular , Mecanotransducción Celular , Malla Trabecular/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Anciano , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Nitritos/metabolismo , Regiones Promotoras Genéticas , Estrés MecánicoRESUMEN
Purpose: While nitric oxide (NO) donors are emerging as treatments for glaucoma, the mechanism by which NO lowers intraocular pressure (IOP) is unclear. NO activates the enzyme guanylyl cyclase (GC) to produce cyclic guanosine monophosphate. We studied the ocular effects of inhaled and topically applied NO gas in mice and lambs, respectively. Methods: IOP and aqueous humor (AqH) outflow were measured in WT and GC-1α subunit null (GC-1-/-) mice. Mice breathed 40 parts per million (ppm) NO in O2 or control gas (N2/O2). We also studied the effect of ocular NO gas exposure (80, 250, 500, and 1000 ppm) on IOP in anesthetized lambs. NO metabolites were measured in AqH and plasma. Results: In awake WT mice, breathing NO for 40 minutes lowered IOP from 14.4 ± 1.9 mm Hg to 10.9 ± 1.0 mm Hg (n = 11, P < 0.001). Comparable results were obtained in anesthetized WT mice (n = 10, P < 0.001). In awake or anesthetized GC-1-/- mice, IOP did not change under similar experimental conditions (P ≥ 0.08, n = 20). Breathing NO increased in vivo outflow facility in WT but not GC-1-/- mice (+13.7 ± 14.6% vs. -12.1 ± 9.4%, n = 4 each, P < 0.05). In lambs, ocular exposure to NO lowered IOP in a dose-dependent manner (-0.43 mm Hg/ppm NO; n = 5 with 40 total measurements; P = 0.04) without producing corneal pathology or altering pulmonary and systemic hemodynamics. After ocular NO exposure, NO metabolites were increased in AqH (n = 8, P < 0.001) but not in plasma. Conclusions: Breathing NO reduced IOP and increased outflow facility in a GC-dependent manner in mice. Exposure of ovine eyes to NO lowers IOP.
Asunto(s)
Humor Acuoso/fisiología , Guanilato Ciclasa/fisiología , Presión Intraocular/efectos de los fármacos , Óxido Nítrico/farmacología , Administración por Inhalación , Administración Tópica , Animales , Modelos Animales de Enfermedad , Femenino , Guanilato Ciclasa/deficiencia , Masculino , Ratones , Ratones Transgénicos , Óxido Nítrico/administración & dosificación , OvinosRESUMEN
Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1-/-) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm's canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1-/- outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1-/- eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology.
Asunto(s)
Caveolas/metabolismo , Caveolina 1/metabolismo , Glaucoma/metabolismo , Presión Intraocular , Malla Trabecular/metabolismo , Animales , Caveolas/patología , Caveolina 1/genética , Glaucoma/genética , Glaucoma/patología , Glaucoma/fisiopatología , Humanos , Ratones , Ratones Noqueados , Malla Trabecular/patología , Malla Trabecular/fisiopatologíaRESUMEN
Visual impairment due to glaucoma currently impacts 70 million people worldwide. While disease progression can be slowed or stopped with effective lowering of intraocular pressure, current medical treatments are often inadequate. Fortunately, three new classes of therapeutics that target the diseased conventional outflow tissue responsible for ocular hypertension are in the final stages of human testing. The rho kinase inhibitors have proven particularly efficacious and additive to current therapies. Unfortunately, non-contact technology that monitors the health of outflow tissue and its response to conventional outflow therapy is not available clinically. Using optical coherence tomographic (OCT) imaging and novel segmentation software, we present the first demonstration of drug effects on conventional outflow tissues in living eyes. Topical netarsudil (formerly AR-13324), a rho kinase/ norepinephrine transporter inhibitor, affected both proximal (trabecular meshwork and Schlemm's Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract. Hence, increased perfusion of outflow tissues was reliably resolved by OCT as widening of the trabecular meshwork and significant increases in cross-sectional area of Schlemm's canal following netarsudil treatment. These changes occurred in conjunction with increased outflow facility, increased speckle variance intensity of outflow vessels, increased tracer deposition in conventional outflow tissues and decreased intraocular pressure. This is the first report using live imaging to show real-time drug effects on conventional outflow tissues and specifically the mechanism of action of netarsudil in mouse eyes. Advancements here pave the way for development of a clinic-friendly OCT platform for monitoring glaucoma therapy.
Asunto(s)
Benzoatos/farmacología , Ojo/efectos de los fármacos , Fenómenos Fisiológicos Oculares/efectos de los fármacos , beta-Alanina/análogos & derivados , Animales , Humor Acuoso/efectos de los fármacos , Humor Acuoso/metabolismo , Humor Acuoso/fisiología , Benzoatos/administración & dosificación , Ojo/metabolismo , Colorantes Fluorescentes/metabolismo , Procesamiento de Imagen Asistido por Computador , Presión Intraocular/efectos de los fármacos , Ratones , Tomografía de Coherencia Óptica , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , Malla Trabecular/fisiología , beta-Alanina/administración & dosificación , beta-Alanina/farmacologíaRESUMEN
PURPOSE: Mice are commonly used to study conventional outflow physiology. This study examined how physical factors (hydration, temperature, and anterior chamber [AC] deepening) influence ocular perfusion measurements in mice. METHODS: Outflow facility (C) and pressure-independent outflow (Fu) were assessed by multilevel constant pressure perfusion of enucleated eyes from C57BL/6 mice. To examine the effect of hydration, seven eyes were perfused at room temperature, either immersed to the limbus in saline and covered with wet tissue paper or exposed to room air. Temperature effects were examined in 12 eyes immersed in saline at 20 °C or 35 °C. Anterior chamber deepening was examined in 10 eyes with the cannula tip placed in the anterior versus posterior chamber (PC). Posterior bowing of the iris (AC deepening) was visualized by three-dimensional histology in perfusion-fixed C57BL/6 eyes and by spectral-domain optical coherence tomography in living CD1 mice. RESULTS: Exposure to room air did not significantly affect C, but led to a nonzero Fu that was significantly reduced upon immersion in saline. Increasing temperature from 20 °C to 35 °C increased C by 2.5-fold, more than could be explained by viscosity changes alone (1.4-fold). Perfusion via the AC, but not the PC, led to posterior iris bowing and increased outflow. CONCLUSIONS: Insufficient hydration contributes to the appearance of pressure-independent outflow in enucleated mouse eyes. Despite the large lens, AC deepening may artifactually increase outflow in mice. Temperature-dependent metabolic processes appear to influence conventional outflow regulation. Physical factors should be carefully controlled in any outflow studies involving mice.
Asunto(s)
Cámara Anterior/metabolismo , Presión Intraocular/fisiología , Malla Trabecular/metabolismo , Animales , Cámara Anterior/citología , Humor Acuoso/metabolismo , Enucleación del Ojo , Femenino , Imagenología Tridimensional , Masculino , Ratones , Ratones Endogámicos C57BL , Tomografía de Coherencia ÓpticaRESUMEN
PURPOSE: Endothelial nitric oxide (NO) synthase is regulated by shear stress. At elevated intraocular pressures when the Schlemm's canal (SC) begins to collapse, shear stress is comparable with that in large arteries. We investigated the relationship between NO production and shear stress in cultured human SC cells. METHODS: Schlemm's canal endothelial cells isolated from three normal and two glaucomatous human donors were seeded into Ibidi flow chambers at confluence, cultured for 7 days, and subjected to steady shear stress (0.1 or 10 dynes/cm(2)) for 6, 24, or 168 hours. Cell alignment with flow direction was monitored, and NO production was measured using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and Griess reagents. Human trabecular meshwork (TM) and umbilical vein endothelial cells (HUVECs) were used as controls. RESULTS: Normal SC strains aligned with the direction of flow by 7 days. Comparing 0.1 vs. 10 dynes/cm(2), NO levels increased by 82% at 24 hours and 8-fold after 7 days by DAF-FM, and similar results were obtained with Griess reagent. Shear responses by SC cells at 24 hours were comparable with HUVECs, and greater than TM cells, which appeared shear-insensitive. Nitric oxide production by SC cells was detectable as early as 6 hours and was inhibited by 100 µM nitro-L-arginine methyl ester. Two glaucomatous SC cell strains were either unresponsive or lifted from the plate in the face of shear. CONCLUSIONS: Shear stress triggers NO production in human SC cells, similar to other vascular endothelia. Increased shear stress and NO production during SC collapse at elevated intraocular pressures may in part mediate IOP homeostasis.