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Purpose To assess the clinical safety, tolerability, and efficacy of topically administered MGV354, a soluble guanylate cyclase (sGC) activator, in patients with ocular hypertension (OH) or glaucoma. Design Double-masked, randomized, and vehicle-controlled study. Methods Parts 1 and 2 evaluated safety and tolerability to identify the maximum tolerated dose (MTD) of once-daily MGV354 in 32 healthy volunteers (Part 1) and 16 patients with OH or glaucoma (Part 2) at a single clinical site. Part 3 was a multisite trial that evaluated intraocular pressure (IOP)-lowering efficacy of the MTD administered nightly for 1 week in 50 patients with minimum IOP of 24 mm Hg at 8 AM, with a main outcome measure of mean diurnal IOP at day 8 compared to baseline ( ClinicalTrials.gov NCT02743780 ). Results There was no difference in favor of MGV354 for IOP lowering; change from baseline to day 8 in mean diurnal IOP was -0.6 mm Hg for MGV354-treated patients and -1.1 mm Hg for vehicle-treated patients in Part 3, with a confidence interval of -0.7 to 1.7. The most common adverse events reported after MGV354 administration were conjunctival and ocular hyperemia. Conclusions Overall, MGV354 0.1% demonstrated no statistically significant effect compared to vehicle in lowering IOP based on the study's main outcome measure. MGV354 produced ocular hyperemia consistent with its pharmacology.

The use of marijuana is now legal for medical purposes in 39 of the 50 United States. Eleven of these 39 states have also legalized marijuana for non-medical usage. Marijuana impairs the motor skills of users, making Driving Under the Influence of Marijuana (DUIM) a growing public health concern. There are currently few accessible and accurate methods to assess the impairment levels of drivers who have used marijuana. Current assessment methods include self-reports and testing urine, oral fluid, and blood. However, self-reports are often biased and biological tests are cumbersome to perform in situ. In this paper, we investigate whether dose-dependent changes in participants gait (walk) can be detected using data gathered from their smartphone motion sensors (accelerometer and gyroscope). We envision WeedGait, a smartphone sensing system that will assess the gait of marijuana users passively and warn them when they are too impaired to drive safely. To the best of our knowledge, this is the first study on using smartphones to assess marijuana-induced gait impairment. Gait data was collected from 10 subjects and pre-processing steps included low pass filtering, step cycle detection and segmentation, and normalization. We present a novel gait analysis approach that analyzes normalized, single-step segments to achieve higher accuracy than prior approaches. We compared the classification results of various machine and deep learning models, and found that Long Short Time Memory (LSTM) and Support Vector Machines performed best, discriminating the gait of subjects after smoking either marijuana with 3% or 7.2% THC versus smoking a placebo marijuana cigarette with an accuracy of 92.1%. These results suggest that smartphone-based marijuana testing is more accurate than urine-based tests but slightly less accurate than oral fluid based testing. Moreover, smartphone sensing of marijuana is completely passive and hence more convenient, which facilitates pervasive testing in natural settings and could have massive impact due to the near-ubiquity of smartphones.

Intoxicated driving causes 10,000 deaths annually. Smartphone sensing of user gait (walk) to identify intoxicated users in order to prevent drunk driving, have recently emerged. Such systems gather motion sensor (accelerometer and gyroscope) data from the users' smartphone as they walk and classify them using machine or deep learning. Standard Field Sobriety Tests (SFSTs) involve various types of walks designed to cause an intoxicated person to lose their balance. However, SFSTs were designed to make intoxication apparent to a trained law enforcement officer who manually proctors them. No prior work has explored which types of walk yields the most accurate results when assessed autonomously by a smartphone intoxicated gait assessment system. In this paper, we compare how accurately Long Short Term Memory (LSTM), Convolution Neural Network (CNN), Random Forest, Gradient Boosted Machines (GBM) and neural network classifiers are able to detect intoxication levels of drunk subjects who performed normal, walk-and-turn and standing on one foot SFST walks. We also compared the accuracy of intoxication detection on the ascending (increasing intoxication) vs descending (decreasing intoxication) limbs of drinking sessions (bi-phasic). We found smartphone intoxication sensing more accurate on the descending limb of the drinking episode and that intoxication detection on the normal walks of subjects were just as accurate as the SFSTs.

We sought to refine understanding about associations identified in prior studies between angiotensin-II receptor blockers, metformin, selective serotonin reuptake inhibitors, fibric-acid derivatives, or calcium channel blockers and progression to glaucoma filtration surgery for open-angle glaucoma (OAG). We used new-initiator, active-comparator cohort designs to investigate these drugs in two data sources. We adjusted for confounders using stabilized inverse-probability-of-treatment weights and evaluated results using "intention-to-treat" and "as-treated" follow-up approaches. In both data sources, Kaplan-Meier curves showed trends for more rapid progression to glaucoma filtration surgery in patients taking calcium channel blockers compared with thiazides with as-treated (MarketScan P = 0.15; Medicare P = 0.03) and intention-to-treat follow-up (MarketScan P < 0.01; Medicare P = 0.10). There was suggestion of delayed progression for selective serotonin reuptake inhibitor compared with tricyclic antidepressants in Medicare, which was not observed in MarketScan. Our study provided support for a relationship between calcium channel blockers and OAG progression but not for other investigated drugs.

The future of next generation therapeutics for glaucoma is strong. The recent approval of two novel intraocular pressure (IOP)-lowering drugs with distinct mechanisms of action is the first in over 20 years. However, these are still being administered as topical drops. Efforts are underway to increase patient compliance and greater therapeutic benefits with the development of sustained delivery technologies. Furthermore, innovations from biologics- and gene therapy-based therapeutics are being developed in the context of disease modification, which are expected to lead to more permanent therapies for patients. Neuroprotection, including the preservation of retinal ganglion cells (RGCs) and optic nerve is another area that is actively being explored for therapeutic options. With improvements in imaging technologies and determination of new surrogate clinical endpoints, the therapeutic potential for translation of neuroprotectants is coming close to clinical realization. This review summarizes the aforementioned topics and other related aspects.

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 Schlemm's 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.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid in vivo crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.

This study examines the effects of 2 Rho kinase inhibitors on intraocular pressure (IOP) and aqueous humor dynamics.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.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.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.

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.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.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.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.

Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.

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.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.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).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.

PURPOSE The prostaglandin F2alpha (PGF2α) analogue bimatoprost lowers intraocular pressure (IOP) by increasing uveoscleral outflow at doses shown to elicit redness of the eye. With the aim to enhance the IOP-lowering effect of bimatoprost we studied NCX 470 [(S,E)-1-((1R,2R,3S,5R)-2-((Z)-7-(ethylamino)-7-oxohept-2-enyl)-3,5-dihydroxycyclopentyl)-5-phenylpent-1-en-3-yl 6-(nitrooxy)hexanoate], a dual-acting compound combining bimatoprost with nitric oxide (NO) known to mainly act via relaxation of trabecular meshwork and Schlemm's canal. METHODS New Zealand white rabbits with transient hypertonic saline-induced IOP elevation (tOHT-rabbits), cynomolgus monkeys with laser-induced ocular hypertension (OHT-monkeys), and normotensive dogs (ONT-dogs) were used. The levels of NCX 470, bimatoprost, and bimatoprost acid were determined in aqueous humor (AH), cornea (CR), and iris/ciliary body (ICB) by liquid chromatography-mass spectrometry/mass (LC-MS/MS), while cGMP in AH and ICB was monitored using an enzyme immunoassay (EIA) kit in pigmented Dutch Belted rabbits. RESULTS NCX 470 (0.14%, 30 μL) lowered IOP in tOHT-rabbits with an E(max) of -7.2 ± 2.8 mm Hg at 90 minutes. Bimatoprost at equimolar dose (0.1%, 30 μL) was noneffective in this model. NCX 470 (0.042%, 30 μL) was more effective than equimolar (0.03%, 30 μL) bimatoprost in ONT-dogs (IOP change, -5.4 ± 0.7 and -3.4 ± 0.7 mm Hg, respectively, P < 0.05) and in OHT-monkeys (IOP change, -7.7 ± 1.4 and -4.8 ± 1.7 mm Hg, respectively, P < 0.05) at 18 hours post dosing. NCX 470 (0.042%, 30 μL) or bimatoprost (0.03%, 30 μL) resulted in similar bimatoprost acid exposure in AH, CR, and ICB while cGMP was significantly increased in AH and ICB at 18 and 24 hours after NCX 470 dosing. CONCLUSIONS NCX 470 lowers IOP more than equimolar bimatoprost in three animal models of glaucoma by activating PGF2α and NO/cGMP signaling pathways.

A significant loss and remodeling of the lamina cribrosa tissue leading to the excavation of the optic nerve is seen in glaucoma. Elevated endothelin-1 (ET-1) levels are detected in the aqueous humor of patients of open-angle glaucoma and in the plasma of patients with normal- tension glaucoma. Optic nerve damage, including axonal loss, can be mimicked by ET-1 injection near the optic nerve. ET-1 is produced from its precursor Big ET-1 (38 amino acids) by endothelin-converting enzyme (ECE). Although ET-1 and its receptors have been identified in the retina, little is known of the distribution of ECE at the optic nerve. Presently, ET-1 receptors and Big ET-1 converting activities were characterized in bovine optic nerve and the retina. The ET(B) receptor was detected in both the optic nerve and retina by immunoblotting and cross-linking, using 125I-ET-1. However, the ET(A) receptor was detected only in the retina. Big ET-1 conversion activities were detected in the plasma membrane (PM) of bovine retina, but not in the PM of the optic nerve. The retinal PM Big ET-1 converting activity was inhibited by phosphoramidon, thiorphan, and acidification. Furthermore, ECE cytosolic activities were detected in both the optic nerve and retina. Unlike the PM-ECE, cytosolic Big ET-1 converting activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2-like activity and/or cathepsin activity. Pepstatin, a potent inhibitor of cathepsins, inhibited the optic nerve (ON) cytosolic conversion of Big ET-1 peptide by 50%, and the combination of pepstatin and phosphoramidon, a potent inhibitor of ECE, inhibited the ON cytosolic activity by 86%. By contrast, the combination of both inhibitors weakly inhibited the cytosolic retinal Big ET-1 converting activity. Western blotting revealed the presence of ECE-1 at the PM of the retina not the ON. ECE-2 and cathpesins B, D, and L were detected only in the cytosol of both the retina and ON. In summary, it appears that ET-1 could be produced in the retina and optic nerve by at least two ECE subtypes and, perhaps, cathepsins. Big ET-1 converting activity may be an important target in preventing ET-1-induced optic nerve pathology.

Elevated endothelin-1 (ET-1) levels are detected in patients with glaucoma. ET-1 is produced from its precursor, Big ET-1, by endothelin-converting enzyme (ECE). Characterization of ET- 1 secretion and ECE activity was performed in ARPE-19 cells, a human retinal pigmented epithelial cell-line. The ET(B) receptor but not the ET(A) receptor was detected by immunoblotting and cross-linking using 125I-ET-1 at the plasma membrane (PM). Tumor necrosis factor-alpha (10 nmol/L) induced a 700% increase in ET-1 levels and such an effect was further potentiated by BQ788, an ET(B) receptor antagonist, suggesting the involvement of ET(B) receptor in ET-1 clearance. Big ET-1-converting activities were detected in both the PM and cytosol. Phosphoramidon, thiorphan, acidification, and phenanthroline inhibited PM ECE activity; the cytosolic ECE activity was not affected by phenanthroline but was inhibited by the others. In contrast, ECE cytosolic activities were activated by acidification (pH 6.4), suggesting the involvement of ECE-2 or cathepsin-like activity. Pepstatin, a potent inhibitor of cathepsins, and phosphoramidon, a potent inhibitor of ECE, inhibited the cytosolic conversion of Big ET-1 peptide by 46% and 35%, respectively, whereas the combination of both inhibited the cytosolic activity by 93%. Based on immunoblotting, ECE-1 was detected only at the PM, whereas ECE-2 and cathpesins B and D were detected in the cytosol. In summary, ET-1 production in RPE is regulated by at least two isoforms of ECE, (cytosolic and PM) as well as cathepsins.

Endothelin is an endogenous vasoactive peptide that is considered among the most potent vasoconstrictor substances known. In addition to its vascular effects, endothelins and their receptors have been shown to be present in the eye and to have a number of ocular actions that may be important for ocular homeostasis, but, in excess can be a potential contributor to ocular neuropathy in glaucoma. The current review focuses on the cellular and molecular aspects of endothelins and its receptors in the eye with an emphasis on its relationship to ocular function and its potential role in the etiology of glaucoma pathophysiology.

Previous studies utilizing inhibitors of the Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) to address the role of this enzyme in insulin secretion have produced contradictory results. In the current study, these inconsistencies have been addressed by evaluating the effect of various CaM kinase II inhibitors to decrease Ca(2+)-induced insulin secretion from permeabilized beta-cells. KN-93 (2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlo rocinnamyl)-N-methylbenzylamine) markedly inhibited both CaM kinase II activation and insulin secretion in parallel in alpha-toxin-permeabilized beta-cells. These effects were specific since they were not mimicked by the inactive analog, KN-92 (2-[N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methy lbenzylamine). In contrast, KN-62 (1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine) , while reported to be similar to KN-93 with respect to mechanism of action, did not inhibit Ca(2+)-induced activation of CaM kinase II or insulin secretion in these cell preparations. All three agents suppressed Ca(2+) influx in intact beta-cells induced by depolarization in the presence of elevated extracellular potassium although to different extents. The synthetic peptide inhibitors of CaM kinase II, [Ala(286)]CaMK 281-302 and AIP (autocamtide-2-related inhibitory peptide), strongly inhibited Ca(2+)-induced insulin secretion from electropermeabilized islets, an effect that also correlated with an equivalent inhibition of CaM kinase II activation. This re-evaluation (i) explains a lack of effect of KN-62 on insulin secretion from permeabilized cells based on its inability to inhibit CaM kinase II activation in these preparations; (ii) has revealed that CaM inhibitors, either chemical or peptide in nature, that are capable of preventing enzyme activation uniformly suppress Ca(2+)-sensitive insulin secretion; and (iii) cautions the use of KN-62/93/92 as selective inhibitors of CaM kinase II in intact cell studies. These observations reinforce the suggestion that CaM kinase II plays an important role in insulin exocytosis in the beta-cell.

To identify which endothelin receptor subtype is expressed and is functional in the human ciliary body and trabecular meshwork, tissues that regulate aqueous humor dynamics.Immunocytochemistry was used to characterize the primary culture cells of normal human ocular cells. Endothelin receptor gene expression was probed with reverse transcription of polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) mobilization was measured with video image microscopy using Fura-2AM as a fluorescent probe.Identities of primary cultures, human ciliary smooth muscle (HCSM), ciliary nonpigmented epithelial (HCE), and trabecular meshwork (HTM) cells were confirmed by immunocytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ETA) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ETB receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca2+]i of HCSM cells was increased from 57 +/- 7 nM to 328 +/- 108 nM (n = 23; mean +/- SE; P0.05) by 1 nM endothelin-1 (ET-1). In HCE cells, [Ca2+]i increased from 40 +/- 3 nM to 90 +/- 10 nM (n = 55) (P0.001) with the same concentration of ET-1. Similarly, ET-1 (1 nM) increased the [Ca2+]i from 51 +/- 6 nM to 185 +/- 47 nM (n = 19) (P0.001) in the HTM cells. The agonist for ETB, S6c, had no effect on [Ca2+]i transients in all three cell types. No ETB receptor expression was detected in these cell types under the experimental and culture conditions.ETA receptor is expressed and is possibly responsible for mediating the signal for [Ca2+]i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) by the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle thus enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regulation of endothelin-1 (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effect of tumor necrosis factor-alpha(TNF-alpha; 10 nm), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and release in SV-40 transformed human ciliary non-pigmented epithelial cells (HNPE). ET-1 and Big endothelin-1 (Big ET-1) immunoreactivity was time-dependently increased following TNF-alphatreatment. Phorbol esters (PMA), activators of PKC, also raised the immunoreactive levels of ET-1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nm), decreased ET-1 levels in TNF-alpha-stimulated cells. Pre-treatment with phosphoramidon (1 micron) an ECE-inhibitor, followed by TNF-alpha stimulation, decreased ir-ET-1 levels. Cycloheximide (9 micron), a protein synthesis inhibitor, decreased TNF-alpha-stimulated levels for ir-ET-1 and ir-Big ET-1, suggesting that TNF-alpha may be directly regulating ET-1 expression at the ET-1 gene. Our data indicates that TNF-alpha regulates ET-1 levels in HNPE cells possibly by activating PKC either to stimulate protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an important role in aqueous humor dynamics following cytokine activation.

Prostaglandin receptor agonists have intraocular pressure–lowering effects in humans and are of interest in the treatment of glaucoma. The prostanoid receptor agonist PF-04475270 is a potent and selective agonist of the prostaglandin E2 receptor EP4. This paper characterizes the toxicity associated with topical ocular administration of PF-04475270 in beagles. Dogs were given PF-04475270 topically to the eye on a consecutive daily dosing schedule for one or four weeks followed by a one-or four-week reversal period, respectively. Clinical observations, ophthalmic, and laboratory parameters were recorded. Necropsies were conducted at the end of the dosing and recovery phases, and histologic examinations performed. Corneal neovascularization that was considered adverse was observed at doses of ≥1.0 μg/eye and was not reversed by the end of the recovery phase. Dogs dosed with ≥0.25 μg/eye developed a dose-related conjunctival hyperemia that persisted throughout the reversal period. Corneal neovascular cells stained positive with EP4 and the endothelial biomarker Factor VIII-vWF. Other histopathology findings observed at doses of ≥1.0 μg included single-cell necrosis and neutrophils in the cornea, inflammatory cell infiltrates in the iris/ciliary body, and iridal endothelial cell hypertrophy. A resolving acute to subacute inflammation in the iris/ciliary body was observed after the four-week recovery period.

The effect of hypoxia (24 h) on TNF-alpha-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-alpha- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-alpha (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-alpha-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-alpha- and ET-1-mediated hONA proliferation. PD142893 (1 microM), an ET(A/B) receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-alpha treatment. Also, U0126 (10 microM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-alpha and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-alpha and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.

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-1's 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.

Purpose Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-alpha. Methods Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-alpha for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. Results Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-alpha caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-alpha. There was a concentration-dependent increase in [Ca2+]i in both y- and mRPE cells in response to CCh. CCh at 1 microM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. Conclusions Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-alpha and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-alpha treatment.

Endothelin-1 (ET-1) lowers intraocular pressure (IOP) in animal models by regulating aqueous humour dynamics through both inflow and outflow mechanisms. Moreover, ET's concentration is elevated in glaucoma patients and in animal models of glaucoma. Glucocorticoid therapy often can lead to increase IOP in susceptible individuals including patients with primary open angle glaucoma (POAG). In this study, we examined the effects of dexamethasone (Dex), a frequently used anti-inflammatory glucocorticoid, on the synthesis and release of endothelin-1 and on the expression of endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells, an established source for ET-1 in the anterior chamber. As measured by ET-1 immunoreactivity, ET-1 was concentration-dependently increased following 24hr Dex treatment, with a maximum concentration (100 nM) causing a threefold increase of ET-1 release. Western blot analysis of HNPE cells showed the expression of endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)) with approximate molecular weights of 40 kDa. Dex treatment decreased ET(A) receptor expression at all Dex doses, but up-regulated ET(B) receptors with 10nM Dex having the greatest effect. Quantitative PCR demonstrated that Dex also increased the mRNA of pre-pro-ET-1 (ppET-1) and ET(B) but decreased the mRNA of ET(A). RU486, a glucocorticoid receptor antagonist, was able to block Dex's actions on ET release and ET(B) receptor expression, but did not block its action on ET(A) receptor expression. Endothelin receptors were minimally expressed in HNPE cells as determined in binding experiments (B(max): ET(A) 17, ET(B) 25 fmolmg(-1) membrane protein). However Dex treatment stimulated a dramatic increase in ET(B) receptor density while decreasing ET(A) receptors (B(max): ET(A) 11, ET(B) 116 fmolmg(-1) membrane protein). The regulation of endothelin and its receptors could be a novel mechanism associated with glucocorticoid's effects on intraocular pressure. The increase in ET-1 and disproportionate regulation in ET receptor expression by Dex could promote dysregulation in ET's mechanism on both inflow and outflow, thus affecting aqueous humour dynamics in the anterior chamber of the eye.