Ganesh Prasanna

Endothelin: is it a contributor to glaucoma pathophysiology?

Endothelin is a vasoactive peptide that has been shown to play an important role in vascular homeostasis. Recently, endothelin and its receptors have been found in ocular tissues where it appears to have a regulatory function. Endothelin is found in both the aqueous and vitreous humors and its concentration is elevated in glaucoma patients and in animal models of glaucoma. In the current review, the authors present information about the distribution of endothelin and endothelin receptors in the eye and the ocular actions of endothelins. Specifically, endothelin/aqueous humor dynamics, endothelin/nitric oxide interactions, endothelin and ischemia, and endothelin/optic nerve head effects. Observations concerning the potential role of endothelin in glaucoma pathophysiology is presented and discussed relative to its effects on the optic nerve head and in relation to glaucoma theories.

A novel nitric oxide releasing prostaglandin analog, NCX 125, reduces intraocular pressure in rabbit, dog, and primate models of glaucoma.

PURPOSE Nitric oxide (NO) is involved in a variety of physiological processes including ocular aqueous humor dynamics by targeting mechanisms that are complementary to those of prostaglandins. Here, we have characterized a newly synthesized compound, NCX 125, comprising latanoprost acid and NO-donating moieties. METHODS NCX 125 was synthesized and tested in vitro for its ability to release functionally active NO and then compared with core latanoprost for its intraocular pressure (IOP)-lowering effects in rabbit, dog, and nonhuman primate models of glaucoma. RESULTS NCX 125 elicited cGMP formation (EC(50) = 3.8 + or - 1.0 microM) in PC12 cells and exerted NO-dependent iNOS inhibition (IC(50) = 55 + or - 11 microM) in RAW 264.7 macrophages. NCX 125 lowered IOP to a greater extent compared with equimolar latanoprost in: (a) rabbit model of transient ocular hypertension (0.030% latanoprost, not effective; 0.039% NCX 125, Delta(max) = -10.6 + or - 2.3 mm Hg), (b) ocular hypertensive glaucomatous dogs (0.030% latanoprost, Delta(max)= -6.7 + or - 1.2 mm Hg; 0.039% NCX 125, Delta(max) = -9.1 + or - 3.1 mm Hg), and (c) laser-induced ocular hypertensive non-human primates (0.10% latanoprost, Delta(max) = -11.9 + or - 3.7 mm Hg, 0.13% NCX 125, Delta(max) = -16.7 + or - 2.2 mm Hg). In pharmacokinetic studies, NCX 125 and latanoprost resulted in similar latanoprost-free acid exposure in anterior segment ocular tissues. CONCLUSIONS NCX 125, a compound targeting 2 different mechanisms, is endowed with potent ocular hypotensive effects. This may lead to potential new perspectives in the treatment of patients at risk of glaucoma.

Role of the ETB receptor in retinal ganglion cell death in glaucoma

Recent observations suggest that the vasoactive peptide endothelin-1 (ET-1) may be an important contributor to the etiology of glaucoma. ET-1 administration has been shown to produce optic nerve axonal loss and apoptosis of retinal ganglion cells. Ocular ET-1 levels are elevated in aqueous humor in response to elevated intraocular pressure both in glaucoma patients and in animal models of glaucoma; however, the precise mechanisms by which ET-1 mediates glaucomatous optic neuropathy are not clear. Presently we report that ET-1-mediated apoptosis was markedly attenuated in ETB receptor-deficient rats, suggesting a key role for ETB receptors in apoptosis of retinal ganglion cells by ET-1 treatment. Using virally transformed rat retinal ganglion cells (RGC-5 cells), we found that ET-1 (100 nmol/L) treatment produced apoptotic changes in these cells that was determined by flow cytometric analyses, release of mitochondrial cytochrome c to the cytosol, and increased phosphorylation of c-Jun N-terminal kinase. Pretreatment with the ETB-receptor antagonist BQ788 (1 micromol/L) was able to significantly attenuate ET-1-mediated apoptosis in RGC-5 cells. ET-1-mediated apoptotic changes in RGC-5 cells were associated with ETB-receptor activation and were accompanied by a significant upregulation of ETB-receptor expression. These studies suggest that ocular ET-1 acts through ETB receptors to mediate apoptosis of retinal ganglion cells, a key event in glaucoma and related optic neuropathies.

Correlation of In Vitro and In Vivo Kinetics of Nitric Oxide Donors in Ocular Tissues

In the eye, nitric oxide (NO) is involved in the regulation of intraocular pressure (IOP) and ocular blood flow. The main purpose of this study was to measure the kinetics of NO release from NO donors in ocular cells and tissues using in vivo and in vitro models and demonstrate the link between the kinetics of NO release with the functional effect, IOP. Nitric oxide release was measured in human ocular cells using a fluorescent dye, diaminofluorescein (DAF), following treatment with short-acting sodium nitroprusside (SNP) and longer-acting S-nitroso-N-acetylpenicillamine (SNAP) NO donors. Both SNP and SNAP were also administered topically to rabbits; IOP was measured and levels of NO and cGMP were assessed as biomarkers over a time course in the aqueous humor (AH) and iris/ciliary body (ICB). Time- and concentration-dependent increases in NO level were produced by SNP and SNAP in human ocular cells. Both NO and cGMP levels appeared to be elevated following treatment with the aforementioned NO donors in rabbit ocular tissues. Transient IOP lowering was accompanied with these biochemical estimations in rabbits, with time of maximal effect being shifted to the right for longer-acting SNAP as compared with short-acting SNP. In vitro and in vivo NO/cGMP assay results displayed a correlation between short- and longer-acting NO donors, discriminating their respective temporal actions in the eye. Due to their translatability, the in vitro DAF assay and in vivo NO fluorometric assay can therefore be potentially useful in screening novel NO donors with different temporal/kinetic profiles.

Parallel signaling pathways in endothelin-1-induced proliferation of U373MG astrocytoma cells.

Endothelin-1 (ET-1) is a potent mitogen for many cells, especially when its levels are elevated under pathological conditions, as seen in tumor cell progression and astroglial activation in neuropathies. While ET-1 is known to cause astroglial proliferation, in the present study, multiple signaling pathways involved in ET-1–mediated astrocyte proliferation were characterized. Treatment with PD98059 and U0126 (MEK inhibitors) inhibited not only ET-1–induced cell proliferation but also ET-1–activated phosphorylation of extracellular signal–regulated protein kinase 1/2 (ERK1/2) in U373MG astrocytoma cells. Whereas the nonselective protein kinase C (PKC) inhibitor chelerythrine attenuated ET-1–induced cell proliferation, it was unable to block ET-1–induced ERK phosphorylation. However, ET-1 did not activate conventional or novel PKCs and did not elevate intracellular calcium. In addition, U73122 (a selective phospholipase C inhibitor), FTI-277 (an H-Ras inhibitor), as well as protein tyrosine kinase inhibitors also did not abolish ET-1–induced ERK1/2 phosphorylation. ET-1 treatment increased the activity of total Ras but not H-Ras. The phosphoinositide 3-kinase (PI3K) pathway appeared to be involved in signal transduction induced by ET-1, but it did not appear to participate in cross talk with the mitogen-activated protein kinase (MAPK) pathway. Activated ET receptors did not propagate signals either through protein tyrosine kinases or transactivation of EGF receptor tyrosine kinases, which typically trigger Ras-Raf-MAPK pathways. The results indicate that ET-1 stimulates cell proliferation by the activation of MAPK-, PKC-, and PI3K-dependent pathways that appear to function in a parallel manner. There is no apparent, direct “cross talk” between these pathways in U373MG cells, but rather, they might act on the independent but necessary components of the mitogenic effects of ET-1.

Regulation of Na,K-ATPase Expression by Endothelin-1 in Transformed Human Ciliary Non-Pigmented Epithelial (HNPE) Cells

Endothelin-1 (ET-1) (1-100 nM) decreases the activity of Na,K-ATPase, a key enzyme responsible for aqueous humor formation, in transformed human non-pigmented ciliary epithelial (HNPE) cells. The present study sought to determine if ET-1 alters the expression of the catalytically active alpha subunit of Na,K-ATPase in HNPE cells and identify mechanisms underlying these effects. We report that acute (15 and 30 min) treatment with ET-1 results in an increase in mRNA expression of the alpha 1 subunit of Na,K-ATPase. Similar to ET-1s effects, ouabain (100 microM), a selective inhibitor of Na,K-ATPase, and monensin (10 microM), a sodium ionophore, also increased Na,K-ATPase expression in HNPE cells. The increase in Na,K-ATPase expression by short-term treatment with ouabain and monensin was dependent on their ability to elevate intracellular sodium concentrations. However, acute ET-1 treatment mediated increase in Na,K-ATPase expression was independent of changes in intracellular sodium. A prolonged (24 hr) ET-1 treatment results in an increase in both mRNA and protein levels of the alpha 1 subunit of Na,K-ATPase. These observations suggest that ET-1 could play a homeostatic role in modulating aqueous humor formation by having differential effects on the activity and expression of Na,K-ATPase by the ciliary epithelium in the eye.

Effects of Rho Kinase Inhibitors on Intraocular Pressure and Aqueous Humor Dynamics in Nonhuman Primates and Rabbits

PURPOSE This study examines the effects of 2 Rho kinase inhibitors on intraocular pressure (IOP) and aqueous humor dynamics. METHODS IOPs of New Zealand albino rabbits with ocular normotension and cynomolgus macaques (nonhuman primate, NHP) with chronic unilateral laser-induced glaucoma were measured at baseline and periodically after a 9 a.m. dose of H-1152, Y-27632, or vehicle. In a separate group of NHPs, aqueous flow, outflow facility, uveoscleral outflow, and IOP were determined after treatment with Y-27632 or vehicle control. RESULTS Decreases in IOP were found in rabbits (n = 5) at 6 h after one dose of 2% Y-27632 (29%, P = 0.0002) or 1% H-1152 (35%, P = 0.0001), and in hypertensive eyes of NHPs (n = 7-9) at 3 h after one dose of 2% Y-27632 (35%, P = 0.005) or 1% H-1152 (51%, P = 0.0003). With 2 doses of 1% Y-27632 or vehicle in NHP hypertensive eyes (n = 12), significant drug effects were IOP reduction of 28% (P = 0.05) at 2.5 h after the second dose and increases in aqueous flow (36%; P = 0.013), uveoscleral outflow (59%, P = 0.008), and outflow facility (40%; P = 0.01). In normotensive eyes of the same animals, aqueous flow increased by 21% (P = 0.03). No significant change was found in any of the other parameters. CONCLUSIONS Y-27632 and H-1152 lower IOP in rabbits and hypertensive eyes of NHPs for at least 6 h after single doses. The Y-27632 effect on IOP in hypertensive NHP eyes is caused by increases in outflow facility and uveoscleral outflow. An increase in aqueous humor formation attenuates but does not prevent an IOP decrease.

In Vivo Evaluation of 11β-Hydroxysteroid Dehydrogenase Activity in the Rabbit Eye

PURPOSE Steroids are used in a diverse range of conditions in clinical ophthalmology and one of the most significant complications is corticosteroid-induced glaucoma, which is characterized by an increase in intraocular pressure (IOP). 11beta-Hydroxysteroid dehydrogenase-1 (11beta-HSD1) is known to catalyze the interconversion of hormonally inactive cortisone to hormonally active cortisol and is widely expressed in the eye, particularly ciliary epithelium. Carbenoxolone (CBX), an 11beta-HSD1 inhibitor, has been shown to reduce IOP in healthy volunteers and patients with ocular hypertension (OHT). The purpose of this study was to: (1) develop an in vivo model for the assessment of cortisone to cortisol conversion in the eye, that is, 11beta-HSD1 activity and (2) assess the pharmacokinetic/pharmacodynamic relationship following topical treatment with 11beta-HSD1 inhibitors using an in vivo rabbit model. METHODS Potent and selective 11beta-HSD1 inhibitors were topically administered to the rabbit eye and exogenous cortisone to endogenous cortisol conversion in the eye was assessed in rabbits. Tissues were then evaluated for cortisone, cortisol, and 11beta-HSD1 inhibitor levels by LC/MS/MS. Concomitantly cortisol activity in ocular tissue samples was determined using a secondary mechanistic pLuc-GRE assay. RESULTS Topical treatment with potent and selective 11beta-HSD1 inhibitors resulted in complete inhibition in the conversion of cortisone to cortisol in the rabbit eye as well as decreased pLuc-GRE luciferase activity. The reduction of cortisone conversion was time- and dose-dependent as well as dependent on dosing volume (suggestive of increased spillover and washout with greater dosing volume). CONCLUSIONS In conclusion, topical delivery of 11beta-HSD1 inhibitors can reduce or inhibit the conversion of cortisone to cortisol in the eye, indicating that the rabbit eye possesses an active enzyme for glucocorticoid synthesis. Dosing concentration and volume play an important role in the pharmacokinetic and pharmacodynamic effects of topically delivering an 11beta-HSD1 inhibitor. The rabbit model is useful for mechanistically assessing the conversion of cortisone to cortisol in the eye.

Pharmacology of novel intraocular pressure-lowering targets that enhance conventional outflow facility: Pitfalls, promises and what lies ahead?

Intraocular pressure (IOP) lowering drugs that are approved for the treatment of glaucoma and ocular hypertension have limited activity on increasing aqueous humor movement through the trabecular meshwork and Schlemms canal (TM/SC). The TM/SC complex is considered the conventional outflow pathway and is a primary site of increased resistance to aqueous humor outflow in glaucoma. Novel mechanisms that enhance conventional outflow have shown promise in IOP reduction via modulation of several pathways including Rho kinase, nitric oxide/soluble guanylate cyclase/cGMP, adenosine A1, prostaglandin EP4/cAMP, and potassium channels. The clinical translatability of these pharmacological modulators based on pre-clinical efficacy models is currently being explored. In addition, identification of pathways from GWAS and other studies involving transgenic rodent models with elevated/reduced IOP phenotypes have begun to yield additional insights into IOP regulation and serve as a source for the next generation of IOP lowering targets. Lastly, improvements in drug delivery technologies to enable sustained IOP reduction are also discussed.

Thrombin-Induced Endothelin-1 Synthesis and Secretion in Retinal Pigment Epithelial Cells Is Rho Kinase Dependent

PURPOSE The retinal pigment epithelium (RPE) is a major source for endothelin-1 (ET-1), a potent vasoactive peptide, at the outer blood–retinal barrier. Factors that regulate ET-1 synthesis at this site may help identify its normal function and its role in pathologic states accompanying retinal injury. Thrombin is one such factor that might act on the RPE after injury and breakdown of the blood–retinal barrier. The present study was conducted to identify signaling intermediates in thrombin-induced ET-1 synthesis and secretion in primary human RPE (hRPE) and transformed RPE cells (ARPE-19) and a possible pharmacological strategy to block excess release of ET-1. METHODS Cultured hRPE cells were treated with different concentrations of thrombin and thrombin receptor agonists, and a time course to measure levels of preproET-1 (ppET-1) mRNA and secreted mature ET-1 was performed. Levels of secondary messengers [Ca²+]i and RhoA were measured and pharmacologically inhibited to determine how receptor-mediated thrombin activity lead to changes in ET-1 levels. RESULTS Thrombin primarily acts via the protease-activated receptor-1 (PAR-1) subtype in RPE to induce ET-1 synthesis. Thrombin and other receptor agonists increased both [Ca²+]<]i and active RhoA. PAR-1-dependent rho/Rho kinase activation led to increase in ppET-1 mRNA and mature ET-1 secretion. CONCLUSIONS Transient intracellular calcium mobilization and protein kinase C activation by thrombin play a minor role, if any, in ET-1 synthesis in RPE. Instead, rho/Rho kinase activation after PAR-1 stimulation strongly increased ppET-1 mRNA and ET-1 secretion in hRPE cells.