Honeycomb Epithelial Edema Associated With Rho Kinase Inhibition: A Case Series and Review of the Literature.

ABSTRACT: The Rho kinase inhibitor netarsudil is a recently approved therapeutic option for the management of increased intraocular pressure in the United States. Although phase 3 clinical trials noted corneal changes related to the medication-namely, nonvisually-significant corneal verticillata-descriptions of a unique form of cystic epithelial edema began to surface as netarsudil (and its sister drug ripasudil, approved in Japan) gained widespread use. This series adds 3 new cases and reviews the current literature on this unique side effect.

NADPH oxidase-induced oxidative stress in the eyes of hypertensive rats.

Purpose: Increased reactive oxygen species (ROS) released by NADPH oxidase and inflammation are associated with arterial hypertension and eye diseases associated with high blood pressure, including glaucoma, retinopathies (e.g., age-related macular degeneration), and choroidopathies affecting ocular function; however, the mechanisms underlying these adverse outcomes remain undefined. The present study was designed to highlight the importance of oxidative stress in severe hypertension-related eye damage. Methods: Male Wistar rats (n = 7, unless otherwise specified for specific experiments) were administered an oral dose of 30 mg of Nω-nitro-L-arginine methyl ester (L-NAME) per kilogram of bodyweight and day for 3 weeks; chronic administration with L-NAME is a validated experimental approach resulting in severe hypertension secondary to nitric oxide (NO) depletion and subsequent vasoconstriction in the systemic circulation. Upon treatment completion, histomorphometric studies, NADPH oxidase activity, and ROS production were measured in eyecup homogenates and paraffin-embedded sections from control and L-NAME-treated animals. In addition, immunohistofluorescence, western blotting, and real-time PCR (RT-qPCR) analyses were performed in the eye and the retina to evaluate the expression of i) NADPH oxidase main isoforms (NOX1, NOX2, and NOX4) and subunits (p22phox and p47phox); ii) glial fibrillary acidic protein (GFAP), as a marker of microglial activation in the retina; iii) antioxidant enzymes; and iv) endothelial constitutive (eNOS) and inflammation inducible (iNOS) nitric oxide synthase isoforms, and nitrotyrosine as a versatile biomarker of oxidative stress. Results: Increased activity of NADPH oxidase and superoxide anion production, accompanied by transcriptional upregulation of this enzyme isoforms, was found in the retina and choroid of the hypertensive rats in comparison with the untreated controls. Histomorphometric analyses revealed a significant reduction in the thickness of the ganglion cell layer and the outer retinal layers in the hypertensive animals, which also showed a positive strong signal of GFAP in the retinal outer segment and plexiform layers. In addition, L-NAME-treated animals presented with upregulation of nitric oxide synthase (including inducible and endothelial isoforms) and abnormally elevated nitrotyrosine levels. Experiments on protein and mRNA expression of antioxidant enzymes revealed depletion of superoxide dismutase and glutathione peroxidase in the eyes of the hypertensive animals; however, glutathione reductase was significantly higher than in the normotensive controls. Conclusions: The present study demonstrated structural changes in the retinas of the L-NAME-treated hypertensive animals and strengthens the importance of NADPH oxidase as a major ROS-generating enzyme system in the oxidative and inflammatory processes surrounding hypertensive eye diseases. These observations might contribute to unveiling pathogenic mechanisms responsible for developing ocular disturbances in the context of severe hypertension.

Carbonic anhydrase inhibition and the management of glaucoma: a literature and patent review 2013-2019.

Introduction: Glaucoma affects more than 70 million people worldwide. One of the major therapeutic options for its management is based on the inhibition of the metalloenzyme carbonic anhydrases (CAs, EC 4.2.1.1). CA inhibitors (CAIs) diminish ocular hypertension in glaucomatous patients by reducing the rate of bicarbonate formation and thus, the secretion of the aqueous humor. Areas covered: This review is intended to cover the major contributions in terms of patent literature reports for the treatment of ophthalmic diseases by means of CAIs in a time frame spanning from 2013 to date. Expert opinion: The patent literature is dominated by innovative pharmaceutical formulations including a CAI alone or in combination with other therapeutic agents. Very few novelties within drug discovery are currently present and they mainly account for new CAI moieties and classical CAIs merged into scaffolds bearing additional chemical functionalities beneficial for the pharmacological treatment of the disease. It is reasonable to expect that in the near future the so-called 'old drugs' will achieve pharmacological performances in the management of ocular hypertension beyond any expectations and thus open a new era of drug repurposing merely based on material science advancements.

Netarsudil ophthalmic solution 0.02% for the treatment of patients with open-angle glaucoma or ocular hypertension.

Once-daily (p.m.) netarsudil ophthalmic solution 0.02% (Rhopressa) is approved in the United States for lowering elevated intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension. Netarsudil, a Rho kinase (ROCK) inhibitor that lowers IOP primarily by increasing trabecular outflow, produces statistically and clinically significant reductions in mean IOP from baseline, with comparable effects on nocturnal and diurnal IOP. In three phase III trials of patients with elevated IOP, the ocular hypotensive efficacy of once-daily netarsudil 0.02% met the criteria for noninferiority to twice-daily timolol 0.5% at all time points over 3 months in patients with baseline IOP less than 25 mmHg. The most frequent adverse event (AE) was generally mild conjunctival hyperemia, the severity of which did not increase with continued dosing. Netarsudil was associated with minimal treatment-related serious or systemic AEs, likely due to the lack of systemic exposure. This report summarizes the available preclinical and clinical data on netarsudil.

Jnk2 deficiency increases the rate of glaucomatous neurodegeneration in ocular hypertensive DBA/2J mice.

The cJun N-terminal kinases (JNKs; JNK1, JNK2, and JNK3) promote degenerative processes after neuronal injury and in disease. JNK2 and JNK3 have been shown to promote retinal ganglion cell (RGC) death after optic nerve injury. In their absence, long-term survival of RGC somas is significantly increased after mechanical optic nerve injury. In glaucoma, because optic nerve damage is thought to be a major cause of RGC death, JNKs are an important potential target for therapeutic intervention. To assess the role of JNK2 and JNK3 in an ocular hypertensive model of glaucoma, null alleles of Jnk2 and Jnk3 were backcrossed into the DBA/2J (D2) mouse. JNK activation occurred in RGCs following increased intraocular pressure in D2 mice. However, deficiency of both Jnk2 and Jnk3 together did not lessen optic nerve damage or RGC death. These results differentiate the molecular pathways controlling cell death in ocular hypertensive glaucoma compared with mechanical optic nerve injury. It is further shown that JUN, a pro-death component of the JNK pathway in RGCs, can be activated in glaucoma in the absence of JNK2 and JNK3. This implicates JNK1 in glaucomatous RGC death. Unexpectedly, at younger ages, Jnk2-deficient mice were more likely to develop features of glaucomatous neurodegeneration than D2 mice expressing Jnk2. This appears to be due to a neuroprotective effect of JNK2 and not due to a change in intraocular pressure. The Jnk2-deficient context also unmasked a lesser role for Jnk3 in glaucoma. Jnk2 and Jnk3 double knockout mice had a modestly increased risk of neurodegeneration compared with mice only deficient in Jnk2. Overall, these findings are consistent with pleiotropic effects of JNK isoforms in glaucoma and suggest caution is warranted when using JNK inhibitors to treat chronic neurodegenerative conditions.

Knockout of tissue transglutaminase ameliorates TGFß2-induced ocular hypertension: A novel therapeutic target for glaucoma?

Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFß2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFß2 transduced eyes. Overall, TGM2 KO rescued the TGFß2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.

Topical trans-resveratrol ameliorates steroid-induced anterior and posterior segment changes in rats.

Steroid-induced hypertension and glaucoma is associated with increased extracellular meshwork (ECM) deposition in trabecular meshwork (TM). Previous studies have shown that single drop application of trans-resveratrol lowers IOP in steroid-induced ocular hypertensive (SIOH) rats. This IOP lowering is attributed to activation of adenosine A1 receptors, which may lead to increased matrix metalloproteinase (MMP)-2 activity. This study evaluated the effect of repeated topical application of trans-resveratrol for 21 days in SIOH animals on IOP, changes in MMP-2 level in aqueous humor, trabecular meshwork and retinal morphology and retinal redox status. We observed that treatment with trans-resveratrol results in significant and sustained IOP reduction in SIOH rats. This IOP reduction is associated with significantly higher aqueous humor total MMP-2 level; significantly reduced TM thickness and increased number of TM cells. Treatment with trans-resveratrol also significantly increased ganglion cell layer (GCL) thickness, the linear cell density in the GCL and inner retina thickness; and significantly reduced retinal oxidative stress compared to the SIOH vehicle-treated group. In conclusion, repeated dose topical application of trans-resveratrol produces sustained IOP lowering effect, which is associated with increased level of aqueous humor MMP-2, normalization of TM and retinal morphology and restoration of retinal redox status.

Aqueous Humor Outflow Physiology in NOS3 Knockout Mice.

PURPOSE: To investigate the role of endothelial nitric oxide synthase (eNOS) on conventional outflow function using NOS3 knockout (KO) mice. METHODS: Intraocular pressure was measured in both NOS3 KO and wild type (WT) by rebound tonometry. Outflow facility was measured by perfusing enucleated mouse eyes at multiple pressure steps. A subset of eyes was sectioned and stained for histology. Mock aqueous humor ± the nitric oxide (NO) donors nitroprusside dihydrate (SNP) or S-Nitroso-N-Acetyl-D,L-Penicillamine (SNAP) was perfused into enucleated eyes. SNP and SNAP was administered topically at 0, 1, 2, and 3 hours while the contralateral eyes served as vehicle controls. Intraocular pressure was measured in both eyes before and after the last drug treatment. RESULTS: Intraocular pressure was higher in KO mice (18.2 ± 0.7 mm Hg vs. 13.9 ± 0.5 mm Hg, mean ± SEM, n = 30, P < 0.05), and pressure-dependent conventional drainage was significantly lower (0.0058 ± 0.0005 µL/min/mm Hg, mean ± SEM, n = 21) compared with WT mice (0.0082 ± 0.0013 µL/min/mm Hg, n = 23, P < 0.05). No obvious morphological differences in iridiocorneal angle tissues were observed in hematoxylin and eosin (H&E)-stained sections. SNP and SNAP significantly increased pressure-dependent drainage in KO animals (n = 12, P < 0.05). In WT mice, SNP and SNAP caused a significant increase in pressure dependent drainage (n = 12, P < 0.05) to a similar degree as in KO mice. Topical application of SNP significantly reduced IOP in WT and KO mice (n = 12, P < 0.05), but SNAP did not change IOP significantly (n = 19). CONCLUSIONS: NOS3 KO mice have elevated IOP, which is likely the result of reduced pressure-dependent drainage. These findings are consistent with human data showing polymorphisms in the NOS3 gene associate with ocular hypertension and the development of glaucoma.

RhoA GTPase-induced ocular hypertension in a rodent model is associated with increased fibrogenic activity in the trabecular meshwork.

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.

Digoxin derivatives with enhanced selectivity for the α2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits.

In the ciliary epithelium of the eye, the pigmented cells express the α1ß1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2ß3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1ß1, α2ß1, and α3ß1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH2 substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.

Inhibition of cyclophilin D by cyclosporin A promotes retinal ganglion cell survival by preventing mitochondrial alteration in ischemic injury.

Cyclosporin A (CsA) inhibits the opening of the mitochondrial permeability transition pore (MPTP) by interacting with cyclophilin D (CypD) and ameliorates neuronal cell death in the central nervous system against ischemic injury. However, the molecular mechanisms underlying CypD/MPTP opening-mediated cell death in ischemic retinal injury induced by acute intraocular pressure (IOP) elevation remain unknown. We observed the first direct evidence that acute IOP elevation significantly upregulated CypD protein expression in ischemic retina at 12 h. However, CsA prevented the upregulation of CypD protein expression and promoted retinal ganglion cell (RGC) survival against ischemic injury. Moreover, CsA blocked apoptotic cell death by decreasing cleaved caspase-3 protein expression in ischemic retina. Of interest, although the expression level of Bcl-xL protein did not show a significant change in ischemic retina treated with vehicle or CsA at 12 h, ischemic damage induced the reduction of Bcl-xL immunoreactivity in RGCs. More importantly, CsA preserved Bcl-xL immunoreactivity in RGCs of ischemic retina. In parallel, acute IOP elevation significantly increased phosphorylated Bad (pBad) at Ser112 protein expression in ischemic retina at 12 h. However, CsA significantly preserved pBad protein expression in ischemic retina. Finally, acute IOP elevation significantly increased mitochondrial transcription factor A (Tfam) protein expression in ischemic retina at 12 h. However, CsA significantly preserved Tfam protein expression in ischemic retina. Studies on mitochondrial DNA (mtDNA) content in ischemic retina showed that there were no statistically significant differences in mtDNA content among control and ischemic groups treated with vehicle or CsA. Therefore, these results provide evidence that the activation of CypD-mediated MPTP opening is associated with the apoptotic pathway and the mitochondrial alteration in RGC death of ischemic retinal injury. On the basis of these observations, our findings suggest that CsA-mediated CypD inhibition may provide a promising therapeutic potential for protecting RGCs against ischemic injury-mediated mitochondrial dysfunction.

VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.

Vascular endothelial growth factor A (VEGF-A) is a validated therapeutic target in several angiogenic- and vascular permeability-related pathological conditions, including certain cancers and potentially blinding diseases, such as age-related macular degeneration and diabetic retinopathy. We and others have shown that VEGF-A also plays an important role in neuronal development and neuroprotection, including in the neural retina. Antagonism of VEGF-A function might therefore present a risk to neuronal survival as a significant adverse effect. Herein, we demonstrate that VEGF-A acts directly on retinal ganglion cells (RGCs) to promote survival. VEGF receptor-2 signaling via the phosphoinositide-3-kinase/Akt pathway was required for the survival response in isolated RGCs. These results were confirmed in animal models of staurosporine-induced RGC death and experimental hypertensive glaucoma. Importantly, we observed that VEGF-A blockade significantly exacerbated neuronal cell death in the hypertensive glaucoma model. Our findings highlight the need to better define the risks associated with use of VEGF-A antagonists in the ocular setting.

Lamotrigine monotherapy does not provide protection against the loss of optic nerve axons in a rat model of ocular hypertension.

Sodium channel blocking agents such as lamotrigine are potent agents for neuroprotection in several animal models of neurodegenerative and neuroinflammatory disease. We therefore explored whether lamotrigine therapy was neuroprotective in a rat model of ocular hypertension characterized by axonal injury and selective loss of retinal ganglion cells. Twenty-seven male Wistar rats were injected subcutaneously twice daily with either lamotrigine (14 mg/kg/day) or vehicle. Two weeks after the first injection, experimental ocular hypertension was induced in one eye by 532 nm trabecular laser treatment. Intraocular pressure (IOP) was monitored by rebound tonometry and four weeks after the elevation of IOP the loss of optic nerve axons was quantified relative to eyes without either IOP elevation or lamotrigine exposure. In other animals with ocular hypertension, the optic nerves were examined by immunohistochemistry for the expression of the inducible form of nitric oxide synthase (iNOS) at 7 and 28 days. Four weeks after initiation of IOP elevation, no significant difference in axonal loss was observed between rats treated with lamotrigine (30.8% ± 10.5%) or vehicle (17.8% ± 5.7%) (P = 0.19, T-test). There was no significant difference in mean IOP, peak IOP and integral IOP exposure. Furthermore, optic nerve axon counts per unit integral IOP exposure were similar in both groups (P = 0.44). The optic nerves were not positive for the expression of iNOS. In conclusion, this study provides no evidence that lamotrigine is neuroprotective for RGC axons after four weeks of experimental ocular hypertension in the rat, in a model where axonal degeneration occurs in the absence of iNOS expression.

Steady-state levels of retinal 24S-hydroxycholesterol are maintained by glial cells intervention after elevation of intraocular pressure in the rat.

PURPOSE: Our previous studies suggested that CYP46A1 and 24S-hydroxycholesterol (24SOH) may be associated with glaucoma. Loss of CYP46A1-expressing retinal ganglion cells is involved in the activation of glia, and therefore possibly in the disbalance of cholesterol. In this context, the purpose of our present work was to emphasize the glial and longitudinal CYP46A1 expression after an interventional glaucoma-related stress triggered by elevated intraocular pressure (IOP). METHODS: Sprague-Dawley rats were submitted to laser photocoagulation of the trabecular meshwork, limbus and episcleral veins in one eye to induce elevated IOP. Rats were euthanized at days 3, 14, 30 and 60 (n = 10 per time-point), and 24SOH was measured in plasma and retina by gas chromatography-mass spectrometry. CYP46A1 was quantified by Western blotting. Glial activation was assessed by glial fibrillary acidic protein immunoreactivity in Western blots and retinal cryosections. RESULTS: Sustained high IOP was observed in experimental eyes from day 1 to day 21. Plasma MCP-1 and ICAM-1, quantified using multiplex assay kits, were increased at day 3. Glial activation was observed bilaterally at all time-points, at lower levels in contralateral eyes than in experimental eyes. In experimental retinas, CYP46A1 expression showed a transient increase at day 3 and then returned to baseline. Plasma and retinal 24SOH peaked at day 14 and 30, respectively. CONCLUSIONS: These data show that CYP46A1 expression was induced early in response to retinal stress but remained constant at late time-points, reinforcing the constitutive role of CYP46A1 in maintaining cholesterol balance in neuronal tissues.

Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5-MCA-NAT.

We have previously demonstrated that melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduce intraocular pressure (IOP) in New Zealand rabbits. More recently, we have shown that 5-MCA-NAT can also regulate ciliary adrenoceptor gene expression. Like adrenoceptors, carbonic anhydrase (CA) enzymes are involved in aqueous humour secretion by the ocular ciliary epithelium. Moreover, CA enzymes have been reported to be regulated by melatonin. Hence, the aim of this study was to investigate whether the hypotensive effect of 5-MCA-NAT is also because of a regulation of CA genes and enzymes. Time course of 5-MCA-NAT effect on rabbit IOP was followed for 7 hr every day for up to 144 hr (6 days). 5-MCA-NAT reduced IOP, maximally by 51.30 ± 2.41% (at 3 hr), and the hypotensive effect was maintained for up to 96 hr with a single application. IOP studies with 5-MCA-NAT plus Trusopt(®) and immunohistochemical analysis confirmed that CA are molecular targets of 5-MCA-NAT. In addition, real-time quantitative PCR (qPCR) and immunocytochemical assays were performed to determine changes in CA2 (CAII) and CA12 (CAXII) expression in cultured rabbit nonpigmented ciliary epithelial cells (NPE) treated with 5-MCA-NAT. NPE cells showed a prominent decrease in both CA, at the mRNA and protein levels. These data confirm that the long-term hypotensive effect of 5-MCA-NAT is also due, to a down-regulation of CA2 (CAII) and CA12 (CAXII) expression.

eNOS, a pressure-dependent regulator of intraocular pressure.

PURPOSE: Pathology in the primary drainage pathway for aqueous humor in the eye is responsible for ocular hypertension, the only treatable risk factor in patients with glaucoma. Unfortunately, the mechanisms that regulate pressure-dependent drainage of aqueous humor and thus intraocular pressure (IOP) are unknown. To better understand one possible underlying molecular factor that regulates IOP, nitric oxide (NO), pressure-dependent drainage in transgenic mice overexpressing endothelial NO synthase (eNOS) was studied. METHODS: IOP was measured by rebound tonometry in mice, and pressure versus flow data were measured by ex vivo perfusion at multiple pressures between 8 and 45 mm Hg, using mock AH ±100 µM L-NAME. A subset of eyes was examined histologically using standard techniques or was assayed for fusion protein expression by Western blot analysis. RESULTS: IOP was lower (9.6 ± 2.7 vs. 11.4 ± 2.5 mm Hg; mean ± SD; P = 0.04) and pressure-dependent drainage was higher (0.0154 ± 0.006 vs. 0.0066 ± 0.0009 µL/min/mm Hg; P = 0.002) in the transgenic mice than in the wild-type animals; however, pressure-independent drainage was unaffected. The NOS inhibitor L-NAME normalized pressure-dependent drainage in transgenic animals. For IOP >35 mm Hg, the slope of the pressure-flow curve in wild-type mice increased to match that seen in transgenic mice. Shear stress in the pressure-dependent pathway at elevated pressures was calculated to be in a range known to affect eNOS expression and activity in vascular endothelia. CONCLUSIONS: Endothelial NOS overexpression lowers IOP by increasing pressure-dependent drainage in the mouse eye. Data are consistent with NO's having a mechanoregulatory role in aqueous humor dynamics, with eNOS induction at elevated IOPs leading to increased pressure-dependent outflow.

Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal model of glaucoma.

We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used to derive 39, which substantially reduced intraocular pressure in a monkey model of glaucoma-associated ocular hypertension.

Differential modulation of MAPKs in relation to increased intraocular pressure in the aqueous humor of rat eye injected with hyaluronic acid.

PURPOSE: To explore the presence of several mitogem-activated protein kinases in the aqueous humor and to follow changes in levels and activity along chronic elevated intraocular pressure. MATERIAL AND METHODS: Intra-anterior chamber once-a-week injections of hyaluronic acid were used as a rat model of induced elevated intraocular pressure. Saline-injected rats served as control and the counter untreated eye was used as sham group. Aqueous humor was taken at different timepoints and analyzed by Western blot analysis for Erk, JNK, AP1, and NOS in the phosphorylated active form and total protein expression. RESULTS: Phosphorylated Erk was detected in the aqueous humor of the entire tested groups. In rats exposed to elevated intraocular pressure for longer than two weeks, a significant increase in the Erk activation was found. Total Erk expression following the hyaluronic acid injections significantly increased. Higher incidence of phosphorylated 46kD JNK was found in the aqueous humor of the hyaluronic acid injection (63%) versus the saline and sham groups (43%). Rats with elevated intraocular pressure after two weeks expressed significantly higher pJNK than the short-term injected hyaluronic acid, saline, and sham groups. The amount of the two JNK isomers declined in the hyaluronic acid injected rats, reaching significance when the elevated intraocular pressure is longer than two weeks versus sham. Among the tested samples, 74% expressed the different NOS isoforms independent of the treatment. Along with the "non responders," the majority were from the sham group. There is a significant decrease in the total amount of the three isoforms in the hyaluronic acid injected rats after two weeks versus the sham and saline groups. CONCLUSIONS: We suggest that these signaling molecules might be a new target for intervention, opening new possibilities for intraocular pressure management.

Mitochondrial cytochrome c oxidase expression in the central nervous system is elevated at sites of pressure gradient elevation but not absolute pressure increase.

The paucity of suitable experimental models has made it difficult to isolate the pathogenic role of mitochondria in central nervous system diseases associated with absolute pressure elevation and increased pressure gradients. Experimental models of traumatic brain injury (TBI) and hydrocephalus have been useful for examining the mitochondrial response following pressure increase in the central nervous system; however, the presence of multiple pathogenic factors acting on the brain in these previous studies has made it difficult to determine whether the induced changes were a result of mechanical damage, intracranial pressure elevation, or other pathogenic factors. By direct monitoring and control of pressures in the intraocular, intracranial, and vascular compartments, we use the pig optic nerve, a typical central white matter tract, to compare the temporal sequence of cytochrome c oxidase (CcO) levels between regions of absolute pressure elevation and pressure gradient increase. We demonstrate that a rise in pressure gradient without traumatic injury up-regulates CcO levels across the site of the gradient, in a manner similar to what has been previously reported for hydrocephalus. We also demonstrate that CcO changes do not occur following an absolute pressure rise. These findings taken together with our recent reports suggest that mitochondria initiate an early compensatory response to axonal damage following pressure gradient increase. Extrapolation of our results also suggests that decreased CcO levels in TBI may be secondary to mechanical damage. This study emphasises the importance of pressure gradients in regulating mitochondrial function in the central nervous system.

Determination of conventional protein kinase C isoforms involved in high intraocular pressure-induced retinal ischemic preconditioning of rats.

Evidence indicates that conventional protein kinase C (cPKC) plays a pivotal role in the development of retinal ischemic preconditioning (IPC). In this study, the effect of high intraocular pressure (IOP)-induced retinal IPC on cPKC isoform-specific membrane translocation and protein expression were observed. We found that cPKCgamma membrane translocation increased significantly at the early stage (20min-1h), while the protein expression levels of cPKCalpha and gamma were markedly elevated in the delayed retinal IPC (12-168h) of rats. The increased protein expressions of cPKCalpha at 72h and cPKCgamma at 24h after IPC were further confirmed by immunofluorescence staining. In addition, we found that cPKCgamma co-localized with retinal ganglion cell (RGC)-specific marker, neurofilaments heavy chain (NF-H) by using double immunofluorescence labeling. These results suggest that increased cPKCgamma membrane translocation and up-regulated protein expressions of cPKCalpha and gamma are involved in the development of high IOP-induced rat retinal IPC.