ABSTRACT
Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.
Subject(s)
Aqueous Humor , Digoxin , Intraocular Pressure , Macaca fascicularis , Ocular Hypertension , Animals , Intraocular Pressure/drug effects , Digoxin/pharmacology , Aqueous Humor/metabolism , Aqueous Humor/drug effects , Ocular Hypertension/drug therapy , Ocular Hypertension/physiopathology , Ocular Hypertension/metabolism , Disease Models, Animal , Glaucoma/drug therapy , Glaucoma/metabolism , Glaucoma/physiopathology , Rabbits , Humans , Ciliary Body/drug effects , Ciliary Body/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Male , Trabecular Meshwork/drug effects , Trabecular Meshwork/metabolismABSTRACT
Purpose: To investigate the flow pattern in unconventional outflow and its correlation with conventional outflow in mouse eyes. Methods: Fluorescent microspheres were injected into the anterior chamber of one eye of anesthetized C57BL/6J mice (n = 4), followed by perfused fixation with 4% paraformaldehyde in situ after 45 minutes. Post-euthanasia, the injected eyes were enucleated, further immersion fixed, and dissected into 12 equal radial segments. Both sides of each segment were imaged using a confocal microscope after nuclear counterstaining. Both unconventional and conventional outflow patterns of each eye were analyzed by ImageJ and ZEN 2.3 imaging software. Results: Segmental outflow patterns were observed in both the ciliary body (CB) and the supraciliary space and suprachoroidal space (SCS). In the CB, the tracer intensity was the lowest at 12 o'clock and highest at 9 o'clock, whereas in the SCS it was the lowest at 2 o'clock and the highest at 10 o'clock. Consequently, a segmental unconventional outflow was observed, with the lowest and highest flow regions in the superior and temporal quadrants, respectively. The overall segmental uveoscleral outflow has no correlation with trabecular outflow (P > 0.05). Four different outflow patterns were observed: (1) low-flow regions in both outflows, (2) primarily a high-flow region in conventional outflow, (3) primarily a high-flow region in unconventional outflow, and (4) high-flow regions in both outflows. Conclusions: Uveoscleral outflow is segmental and unrelated to the trabecular segmental outflow. These findings will lead to future studies to identify the best location for the placement of drainage devices and drug delivery.
Subject(s)
Anterior Chamber , Ciliary Body , Mice , Animals , Mice, Inbred C57BL , Coloring Agents , DrainageABSTRACT
Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50-60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT3 receptor was found to be >10-fold higher than that of MT1 and MT2 in porcine ciliary epithelium. Aqueous pre-treatment with MT1/MT2 antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT3 antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl- and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT3 receptors.
Subject(s)
Melatonin , Swine , Melatonin/pharmacology , Melatonin/metabolism , Aqueous Humor/metabolism , Pigment Epithelium of Eye/metabolism , Epithelium/metabolism , Epithelial Cells/metabolism , Carrier Proteins/metabolism , Ciliary Body/metabolism , AnimalsABSTRACT
Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and has been shown to regulate cell contraction, fluid secretion, and ECM remodeling in various cell types, suggesting the potential significance of regulating outflow resistance and IOP. We demonstrated that Ba significantly lowered the IOP by about 5 mmHg in living mice. Consistent with that, Ba increased the outflow facility by up to 90% in enucleated mouse eyes. The effects of Ba on cell volume regulation and contractility were examined in primary human TM (hTM) cells. We found that Ba (1-100 µM) had no effect on cell volume under iso-osmotic conditions but inhibited the regulatory volume decrease (RVD) by up to 70% under hypotonic challenge. In addition, Ba relaxed hTM cells via reduced myosin light chain (MLC) phosphorylation. Using iTRAQ-based quantitative proteomics, 47 proteins were significantly regulated in hTM cells after a 3-h Ba treatment. Ba significantly increased the expression of cathepsin B by 1.51-fold and downregulated the expression of D-dopachrome decarboxylase and pre-B-cell leukemia transcription factor-interacting protein 1 with a fold-change of 0.58 and 0.40, respectively. We suggest that a Ba-mediated increase in outflow facility is triggered by cell relaxation via MLC phosphorylation along with inhibiting RVD in hTM cells. The Ba-mediated changes in protein expression support the notion of altered ECM homeostasis, potentially contributing to a reduction of outflow resistance and thereby IOP.
Subject(s)
Eye Diseases , Flavanones , Animals , Aqueous Humor/metabolism , Eye Diseases/metabolism , Flavanones/metabolism , Flavanones/pharmacology , Intraocular Pressure , Mice , Myosin Light Chains/metabolism , Trabecular Meshwork/metabolismABSTRACT
Rho-kinase (ROCK) inhibitors, a novel class of anti-glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin-1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine-serine-lysine-leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose-dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.
Subject(s)
Antiglaucoma Agents/pharmacology , Enzyme Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Thrombospondins/metabolism , rho-Associated Kinases/metabolism , Animals , Aqueous Humor/metabolism , Cytoskeleton/metabolism , Intraocular Pressure/drug effects , Mice , Phosphorylation , Proteomics , Signal Transduction/drug effects , Trabecular Meshwork/drug effects , rho GTP-Binding Proteins/drug effects , rho GTP-Binding Proteins/metabolismABSTRACT
Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors represent a novel class of anti-glaucoma drugs because of their ocular hypotensive effects. However, the underlying mechanisms responsible for lowering intraocular pressure (IOP) are not completely clear. The protein profile changes in primary human trabecular meshwork (TM) cells after two days treatment with a ROCK inhibitor were studied using label-free SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive TM proteome library. All raw data generated from IDA and SWATH acquisitions were uploaded and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01254).
ABSTRACT
BACKGROUND: Recent studies in Caucasians with transient ischaemic attack or ischaemic stroke have demonstrated significant age-specific associations between cerebral small vessel disease (SVD) burden on magnetic resonance imaging and renal impairment. We aimed to validate these findings in a large cohort of Chinese with ischaemic stroke. METHODS: In 959 Chinese with ischaemic stroke who received a brain magnetic resonance imaging at the University of Hong Kong, we determined the age-specific associations of renal impairment (glomerular filtration rate < 60 mL/min/1.73 m2) with neuroimaging markers of SVD as well as with the SVD score. RESULTS: Although renal impairment was associated with the SVD score in univariate analysis in all patients (odds ratio 1.61, 95% confidence interval 1.24-2.09, P < .0001), these associations were attenuated after adjusting for age and sex (Pâ¯=â¯.38). Similar findings were noted in patients with ischaemic stroke due to SVD and non-SVD subtypes. However, in 222 of 959 patients aged <60, renal impairment was independently associated with an increasing microbleed (adjusted odds ratio 6.82, 2.26-20.59), subcortical (4.97, 1.62-15.24) periventricular white matter hyperintensity (3.96, 1.08-14.51) and global SVD burden (3.41, 1.16-10.04; all P < .05) even after adjusting for age, sex, and vascular risk factors. Nevertheless, there were no associations between renal impairment and individual neuroimaging markers of SVD nor with the SVD score in patients aged ≥60 after adjusting for age and sex (all P > .05). CONCLUSIONS: In Chinese with ischaemic stroke, renal impairment was independently associated with microbleed, white matter hyperintensity and global SVD burden in individuals aged <60, but not in those aged ≥60, suggesting that there may be shared susceptibilities to premature systemic disease.
Subject(s)
Brain Ischemia/ethnology , Cerebral Small Vessel Diseases/ethnology , Glomerular Filtration Rate , Kidney Diseases/ethnology , Kidney/physiopathology , Stroke/ethnology , Age Factors , Aged , Aged, 80 and over , Asian People , Brain Ischemia/diagnostic imaging , Brain Ischemia/physiopathology , Cerebral Small Vessel Diseases/diagnostic imaging , Cross-Sectional Studies , Diffusion Magnetic Resonance Imaging , Female , Hong Kong/epidemiology , Humans , Kidney Diseases/diagnosis , Kidney Diseases/physiopathology , Male , Middle Aged , Prospective Studies , Risk Factors , Severity of Illness Index , Stroke/diagnostic imaging , Stroke/physiopathologyABSTRACT
Purpose: Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. Methods: Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer. Results: Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%. Conclusions: Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.