Additive Intraocular Pressure-Lowering Effects of a Novel Selective EP2 Receptor Agonist, Omidenepag Isopropyl, Combined with Existing Antiglaucoma Agents in Conscious Ocular Normotensive Monkeys.

Purpose: To investigate the intraocular pressure (IOP)-lowering effects of omidenepag isopropyl (OMDI), a potent and highly selective prostanoid EP2 receptor agonist, as a potential first-line ocular hypotensive agent when combined with existing antiglaucoma agents in conscious ocular normotensive monkeys. Methods: Male cynomolgus monkeys were examined under conscious conditions. OMDI ophthalmic solution alone was topically applied to an eye or combined with other ophthalmic solutions at 5-min intervals. The contralateral eye was left untreated. IOP was measured before and at 2, 4, 6, and 8 h after instillation. Results: Topical application of OMDI to the eye resulted in statistically significant IOP reduction, which lasted for at least 6 h. The IOP-lowering effects of OMDI concomitantly administered with any of the tested antiglaucoma agents (timolol, brinzolamide, netarsudil, ripasudil, and brimonidine) were greater than those of OMDI alone. Furthermore, these enhanced IOP responses to their concomitant use were statistically significant compared with those of the tested antiglaucoma agents alone. Any combination of OMDI with the tested agents did not lead to serious abnormalities either systemically or locally in the eye. Conclusions: We demonstrated that OMDI has additive IOP-lowering effects when administered in combination with various antiglaucoma agents, namely, ß-adrenergic antagonist, carbonic anhydrase inhibitor, Rho-associated coiled-coil containing protein kinase inhibitors, and α2-adrenergic agonist. These results suggest that OMDI provides additional clinical benefits because of its unique mechanisms of action when combination therapy is required.

Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina.

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

Effects of the Selective EP2 Receptor Agonist Omidenepag on Adipocyte Differentiation in 3T3-L1 Cells.

Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 µmol/L, latanoprost free acid (LAT-A) at 0.1 µmol/L, or prostaglandin F2α (PGF2α), at 0.1 µmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 µmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process.

Efficacy and safety of the fixed combinations of tafluprost/timolol and latanoprost/carteolol.

In this study, we made a comparative efficacy and safety assessment of two different fixed combinations of drugs, viz., tafluprost/timolol (TAF/TIM) and latanoprost/carteolol (LAT/CAR), by determining their effects on intraocular pressure (IOP) in ocular normotensive monkeys and examining their toxic effects on ocular surface using human corneal epithelial cells. TAF/TIM was found to be more effective in lowering IOP for a longer duration compared to LAT/CAR. We found that the difference in the intensity of IOP-lowering effect was because of the differences in the strength of timolol compared with that of carteolol as a beta-adrenergic antagonist and strength of tafluprost compared with that of latanoprost as a prostaglandin analogue. In addition, TAF/TIM showed much less cytotoxic effects compared to LAT/CAR on the human corneal epithelial cells. Our findings showed that TAF/TIM is better than LAT/CAR with regard to the IOP-lowering effect in monkeys and toxicity on ocular surface.

Effects of a Novel Selective EP2 Receptor Agonist, Omidenepag Isopropyl, on Aqueous Humor Dynamics in Laser-Induced Ocular Hypertensive Monkeys.

PURPOSE: To investigate the mechanism of the intraocular pressure (IOP)-lowering effect of a novel selective prostaglandin E2 receptor 2 (EP2) receptor agonist, omidenepag isopropyl (OMDI). METHODS: The effect of OMDI on IOP and aqueous humor dynamics was evaluated in cynomolgus monkeys with unilateral laser-induced ocular hypertension. In a crossover manner, the hypertensive eye of each monkey was dosed once daily with 20 µL of either 0.002% OMDI or vehicle. On day 7 of dosing, IOP was measured by pneumatonometry, aqueous humor flow and outflow facility were evaluated by fluorophotometry, and uveoscleral outflow was calculated mathematically. Treatments were compared by paired t-tests. RESULTS: OMDI at 0.002% significantly lowered IOP by 27%, 35%, and 44% at 0.5, 1.5, and 4 h after the last dosing, respectively. There was no difference in aqueous humor flow between vehicle and OMDI treatments. When comparing OMDI to the vehicle treatment, outflow facility and uveoscleral outflow were significantly (P < 0.05) increased by 71% and 176%, respectively. CONCLUSIONS: OMDI, a novel IOP-lowering compound, reduced IOP by increasing outflow facility and uveoscleral outflow in nonhuman primates.

Pharmacologic Characterization of Omidenepag Isopropyl, a Novel Selective EP2 Receptor Agonist, as an Ocular Hypotensive Agent.

Purpose: The objective of this study was to investigate the pharmacologic characteristics of omidenepag isopropyl (OMDI), a compound developed as a novel intraocular pressure (IOP)-lowering agent, with better IOP control and fewer side effects than other prostanoid receptor agonists such as prostaglandin F receptor (FP) agonists. Methods: Binding activities of OMDI and its hydrolyzed form, omidenepag (OMD), to human recombinant prostanoid receptors (DP1-2, EP1-4, FP, and IP) were evaluated. Based on these binding assays, the agonistic activities of OMDI and OMD were further evaluated using cultured cells expressing selected prostanoid receptors. The pharmacokinetics of OMDI after topical administration was assessed in rabbits by measurement of the concentrations of both OMDI and OMD in aqueous humor. The ocular hypotensive effect of OMDI was evaluated in ocular normotensive rabbits, dogs, and both ocular normotensive and hypertensive monkeys. Results: OMD was determined to be a selective EP2 receptor agonist. OMDI weakly bound to EP1; however, the agonistic activity of OMDI to this receptor was not demonstrated in the functional assay. After topical administration of OMDI, OMD was detected in aqueous humor whereas OMDI was not detectable. OMDI significantly lowered IOP in both ocular normotensive and hypertensive animals. The significant ocular hypotensive effects of OMDI were demonstrated by both single and repeated dosing, and its effective duration suggests sufficient efficacy by once-daily dosing. Conclusions: These studies demonstrated that OMDI is hydrolyzed in the eye to OMD, an EP2 receptor agonist, with a significant ocular hypotensive effect in both ocular normotensive and hypertensive animal models.

N-Glycosylation affects the stability and barrier function of the MUC16 mucin.

Transmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin N-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with N-acetyllactosamine, a preferred galectin ligand. In N-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi N-acetylglucosaminyltransferase I gene (MGAT1) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for N-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for N-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.

Association between prostaglandin E receptor 3 polymorphisms and Stevens-Johnson syndrome identified by means of a genome-wide association study.

BACKGROUND: Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucosa. They often affect the ocular surface and can result in permanent visual dysfunction. OBJECTIVES: We sought to discover genetic markers for SJS/TEN susceptibility. METHODS: We performed a genome-wide association study with 60 patients and 300 control subjects. We applied stringent filter and visual assessments for selecting single nucleotide polymorphisms (SNPs) and a high false discovery rate threshold. We fine-mapped the region where a candidate SNP was found and confirmed the results by means of sequencing. We evaluated the function of agonist-activated prostaglandin E receptor 3 (EP3), the gene for which contained several SNPs, in regulating cytokine production in human conjunctival epithelial (CE) cells. The expression levels of EP3 in the CE cells from patients and control subjects were also compared. RESULTS: We identified 3 SNPs that passed the false discovery rate threshold. One (rs17131450) was close to the EP3 gene. Therefore we analyzed the EP3 region in detail and identified 5 other SNPs. We confirmed the association between SJS/TEN and all 6 SNPs. Activated EP3 was expressed in control CE cells, and it suppressed polyI:C-stimulated cytokine production, suggesting that EP3 might help prevent ocular surface inflammation. Concordantly, the EP3 levels were much lower in the CE cells of the patients than in those of the control subjects. CONCLUSION: We demonstrated, using both genetic and functional analyses, that EP3 could be a key player in the pathogenesis of SJS/TEN accompanied by ocular complications.

Three susceptible loci associated with primary open-angle glaucoma identified by genome-wide association study in a Japanese population.

Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel-Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.

Molecular imaging reveals unique degenerative changes in experimental glaucoma.

Experimentally induced changes in the central visual pathway were studied by using positron emission tomography in monkeys with unilateral hypertension glaucoma. In 2-[18F]fluoro-2-deoxy-glucose studies, monocular visual stimulation of the affected eye yielded significantly reduced neural responses in the occipital visuocortical areas. The response reduction was limited to the visual cortex ipsilateral to the affected eye, indicating the unique vulnerability of ipsilateral visual cortex in experimental unilateral glaucoma. In addition, in [11C]PK11195 positron emission tomography and immunohistochemical studies, selective accumulation of activated microglia, a sign of neural degeneration, was found bilaterally in lateral geniculate nuclei. The present findings establish the usefulness of noninvasive molecular imaging for early diagnosis of glaucoma by providing a sharper surrogate end point for an early phase of glaucoma.

LOXL1 genetic polymorphisms are associated with exfoliation glaucoma in the Japanese population.

PURPOSE: We performed genetic association studies using a native Japanese population to examine the reproducibility of results of lysyl oxidase-like 1 (LOXL1) genetic association studies for exfoliation glaucoma (XFG) beyond the differences of ethnicity. We also quantified LOXL1 mRNA expression in the human lens capsule to examine the possible correlation between LOXL1 expression and XFG pathogenesis. METHODS: We performed a case-control study using 95 Japanese XFG patients and 190 controls. Real-time polymerase chain reaction (PCR) analysis was performed using lens capsules obtained during surgery. RESULTS: The TT genotype in the single nucleotide polymorphism (SNP) rs1048661 and the GG genotype in the SNP rs3825942 in exon 1 of LOXL1 were significantly associated with an increased risk of XFG under recessive models (chi(2) test, p=5.34 x 10(-34) and p=2.1 x 10(-8), respectively). Quantification of LOXL1 mRNA expression demonstrated no significant difference between XFG and senile cataract samples. CONCLUSIONS: Although the functional effects of the LOXL1 SNP appear to be qualitative rather than quantitative, the amino acid substitution (R141L) caused by SNP rs1048661 is not a simple decisive factor for XFG due to the inverted allele frequency between Japanese XFG and Caucasian XFG patients. Further genetic and functional studies are essential for clarifying XFG pathogenesis.

Intravitreal injection of endothelin-1 caused optic nerve damage following to ocular hypoperfusion in rabbits.

The purpose of this study was to investigate the time course of the ocular hypoperfusion, retinal damage, and optic nerve damage induced by intravitreal injection of endothelin-1 (ET-1) in rabbits. ET-1, at 5 pmol (20 microL, twice a week for 2 or 4 weeks), was injected from the pars plana into the posterior vitreous of the right eye. Optic nerve head (ONH) blood flow and retinal artery diameter, together with the neurofilament light chain (NF-L) content, retinal morphology, and axon density of the optic nerve, were evaluated at 2, 4, and 8 weeks after the first injection of ET-1 (n=7 or 8). Tissue blood velocity in ONH was measured using a laser speckle method, and the diameter of major retinal arteries on the rim of the ONH was calculated from fundus photographs by a masked observer. Histological analysis and immunoblot evaluation of NF-L in the optic nerve were performed to evaluate optic nerve damage. At 2 weeks after the first ET-1 injection, tissue blood velocity was decreased by approximately 20% (versus the contralateral eye), and the diameter of retinal arteries had decreased by approximately 40%. These changes were sustained at the same level until 8 weeks after the first ET-1 injection. At 4 and 8 weeks after the first ET-1 injection, the amount of NF-L in the optic nerve was significantly less in the ET-1 treated eyes than in the contralateral eyes. At 8 weeks after the first ET-1 injection, a loss of myelinated axons and increases in gliosis and connective tissue were noted in the optic nerve of the treated eye, and the optic nerve-axon number had decreased significantly (each, versus the untreated eye). Retinal ganglion cells in the retina were not observed any damage at 2, 4, and 8 weeks after ET-1 injection. In conclusion, intravitreal injection of ET-1 induced chronic hypoperfusion in the ONH and retina, which presumably caused decreases in NF-L content and axon number in the optic nerve noted in the later part of the observation period.

Endothelin-1 impairs retrograde axonal transport and leads to axonal injury in rat optic nerve.

The purpose of this study was to examine the effects of endothelin-1 (ET-1) on retrograde axonal transport in the rat optic nerve. Vehicle or ET-1 (0.2, 1, or 5 pmol/eye) were injected into the vitreous body in Sprague-Dawley rats. Retinal vessels were observed, using a fundus camera, before, and at 10 min, 3 days and 7 days after a single intravitreous injection. Two days after the injection, a neuronal tracer, fluoro gold, was administered via the superior colliculi to retrogradely label active retinal ganglion cells (RGCs). Five days after the tracer administration, retrogradely labeled RGCs were evaluated in the flat-mounted retina, and cross sections from each optic nerve were graded for injury by four independent, masked observers. ET-1 at 5 pmol/eye caused a significant constriction of retinal vessels (versus the vehicle-treated group) at 10 min after the injection. Intravitreous injection of ET-1 caused a dose-related decrease in the number of retrogradely labeled RGCs. Injection of 5 pmol/eye ET-1 led to a statistically significant decrease in the number of retrogradely labeled RGCs (versus the vehicle-treated group). ET-1 at 1 and 5 pmol/eye caused histological optic nerve damage (evaluated using a graded scale). The histological optic nerve damage correlated with the number of retrogradely labeled RGCs. In conclusion, a single intravitreous injection of ET-1 impaired retrograde axonal transport in the rat optic nerve and this impairment correlated with the histological optic nerve damage.

A new calcium channel antagonist, lomerizine, alleviates secondary retinal ganglion cell death after optic nerve injury in the rat.

PURPOSE: We investigated whether lomerizine, a new diphenylmethylpiperazine calcium channel blocker, exerted a neuroprotective effect on axonal or retinal damage induced by optic nerve injury in the rat. METHODS: A partial crush lesion was inflicted unilaterally on the optic nerve, 2 mm behind the globe, in adult Wistar albino rats. Animals were treated with the vehicle, 10 or 30 mg/kg lomerizine. Each solution was given orally twice daily for 4 weeks. One week before euthanization, Fluoro-Gold (FG) was injected into both superior colliculi to retrogradely label surviving retinal ganglion cells (RGCs). Approximately 1 month after the optic nerve injury, the retinal damage was assessed morphologically, and the optic nerve axons surrounding the initial lesion were examined histologically. RESULTS: The mean RGC density in the control group decreased to 65.9 +/- 1.32% of the contralateral eye, whereas the systemic application of 10 or 30 mg/kg of lomerizine significantly enhanced the RGC survival to 88.1 +/- 0.38% and 89.8 +/- 0.28%, respectively. Histological examination of damaged axons revealed no significant enhancement of the density or total number of axons of the retinal ganglion cells in the lomerizine-treated group. The crush force we employed caused no significant morphological differences in the retinal layers between the sham-operated animals and the animals from the experimental groups. CONCLUSIONS: Our findings suggest that lomerizine alleviates secondary degeneration of RGCs induced by an optic nerve crush injury in the rat, presumably by improving the impaired axoplasmic flow.

Morphometric evaluation of changes with time in optic disc structure and thickness of retinal nerve fibre layer in chronic ocular hypertensive monkeys.

We examined the time course of changes in optic disc structure by means of a scanning laser ophthalmoscope (Heidelberg Retina Tomograph, HRT) in ocular hypertensive (experimental glaucoma) monkeys, and clarified the relationships between the histological RNFL thickness and HRT parameters. Further, the time course of changes in retinal nerve fiber layer (RNFL) thickness in individual eyes was measured using a scanning laser polarimeter with fixed corneal polarization compensator (GDx FCC). In the present study, two separate experiments were carried out. A chronic intraocular pressure (IOP) elevation was induced by laser trabeculoplasty in the left eye in 11 cynomolgus monkeys. In Experiment 1, the HRT and GDx parameters were measured 12 weeks after the laser treatment in 10 eyes in five monkeys. In Experiment 2, the time course of changes in the HRT and GDx parameters was examined before and 1, 3, 4, 5, 6, 8, 10, 12, 14, and 16 weeks after the laser treatment in 12 eyes in six monkeys. The retardation values (thickness parameters) obtained from the GDx were used to derive thickness and ratio parameters in the superior, inferior, nasal and temporal quadrants. Ratio parameters were expressed as a ratio of superior and inferior quadrant to nasal quadrant. After the last measurements, each eye was enucleated, and retinal cross sections were prepared for histological analysis. In the left (hypertensive) eyes, IOP was persistently elevated throughout the observation periods in both Experiments 1 and 2. In the HRT measurements in Experiment 1, seven out of eight global topographic parameters (exception, disc area) were statistically different between the hypertensive and control eyes 12 weeks after the laser treatment. In Experiment 2, the HRT parameters changed in a time-dependent manner, but each of them almost plateaued at about 4 weeks after the laser treatment. Significant correlations were seen between the histological mean RNFL thickness at 1.5 disc diameters from the optic disc margin and the HRT parameters in 21 eyes from 11 monkeys in Experiments 1 and 2. Especially good correlations with histological mean RNFL thickness were seen for the rim volume and cup volume. In Experiment 1, good correlations were found between GDx ratio parameters and histological RNFL thickness in individual right control eyes (n=5). In individual left experimental glaucoma eyes of Experiment 2 (n=6), GDx ratio parameters declined in a time-dependent manner alongside the IOP elevation. In conclusion, alongside the IOP elevation, time-related changes in optic disc topography and RNFL thickness were demonstrated in monkey eyes using HRT and GDx. HRT (rim and cup) parameters showed good correlations with histological RNFL thickness, and significant interrelations.

Nerve fiber layer measurement using scanning laser polarimetry with fixed corneal compensator in normal cynomolgus monkey eyes.

PURPOSE: The purpose of this study was to examine retinal nerve fiber layer thickness in normal cynomolgus monkeys using a scanning laser polarimeter with a fixed corneal compensator (GDx FCC), and to clarify the reproducibility and symmetries (right-left differences) between both eyes for the GDx parameters. METHODS: GDx parameters were measured in 36 normal eyes of 18 cynomolgus monkeys aged 4.0-5.5 years. The retardation values (thickness parameters) at peripapillary and macular areas obtained from the GDx FCC were measured and calculated thickness, ratio, and modulation parameters in the superior and inferior quadrants. Mean and standard deviation (SD), coefficient of variation (CV), and binocular differences were obtained for each parameter from three independent measurements made during a 1-week period. Correlation between both eyes in macular retardation and baseline values, which indicated the combined minimum retardation values for the nasal and temporal quadrants, and between macular retardation and baseline values were analyzed. RESULTS: The intraocular pressure values (mean +/- SD, n = 18) obtained for the right and left eyes were 20.7 +/- 3.8 and 20.0 +/- 3.2 mm Hg, respectively (no significant differences in both eyes). No significant differences between right and left eyes were detected for any GDx parameters. All parameters showed small right-left differences. The CVs (SD/mean x 100) for all parameters were less than 10%. Highly significant correlations were seen between bilateral eyes for macular retardation (r = 0.936, p < 0.0001) or baseline values (r = 0.946, p < 0.0001). A significant correlation (r = 0.883, p < 0.0001) was also seen between macular retardation and baseline values. CONCLUSIONS: Considering individual differences in corneal birefringence, GDx parameters obtained from a GDx FCC may be useful for the objective evaluation of time-related changes in individual eyes or for binocular comparisons in cynomolgus monkeys.

Alterations in neurofilament light in optic nerve in rat kainate and monkey ocular hypertension models.

The purpose of this study is to evaluate the changes in neurofilament light (NF-L) protein in the optic nerve in rat kainate and monkey ocular hypertension models. In the rat model, optic nerve damage was induced by kainate injection into the vitreous body. In the monkey model, photocoagulation of the trabecular meshwork led to laser-induced ocular hypertension. NF-L in optic nerve extract was quantified by quantitative immunoblot using an imaging analyzer. The amount of NF-L in optic nerve was compared between normal and kainate-treated groups at 7 days after kainate injection. Specimens from rat optic nerves and retinas were evaluated histologically to examine the correlations between damage and amount of NF-L in the optic nerve. In monkeys, the amount of NF-L in the optic nerve was compared between control fellow and ocular hypertensive eyes. Injection of kainate induced morphological optic nerve and retinal damage in rats. The amount of NF-L in the optic nerve was significantly reduced in kainate-treated eyes (vs. normal eyes). The amount of NF-L correlated with the cell count in the ganglion cell layer and the axon number in the optic nerve at 7 days after kainate injection. Further, 6-cyano-7-nitro-quinoxaline-2,3-dione, a non N-methyl-D-aspartate receptor antagonist, suppressed the kainate-induced reduction in NF-L and retinal damage. In the monkey model, ocular hypertension and morphological optic nerve damage were shown by laser-treated eyes. The amount of NF-L in the optic nerve was reduced in laser-treated eyes. In conclusion, in rat kainate and monkey ocular hypertension models, immunoblot evaluation of NF-L shows the reduction of the amount of NF-L in the optic nerves of treated-eyes. The amount of NF-L correlated with the morphological retinal and optic nerve damage in rats. These findings indicate that immunoblot evaluation of NF-L in the optic nerve may provide a quantitative index of optic nerve damage.

A critical role for the carboxy terminal region of the proprotein convertase, PACE4A, in the regulation of its autocatalytic activation coupled with secretion.

PACE4A is a member of the mammalian subtilisin-like proprotein convertase family which is responsible for the proteolytic activation of precursors into their biologically active forms. Previously we reported that the maturation of proPACE4A occurs via a intramolecular autoactivation and cleavage of the propeptide is a rate-limiting step for the secretion of PACE4A (Nagahama et al., FEBS Lett. (1998) 434, 155-159). Although PACE4A is a putative secretory enzyme, it matures and is secreted much slower than general secretory proteins. In this study, we investigated the molecular mechanism underlying this slow maturation. The deletion of 25 amino acids at the carboxy terminus is sufficient for a marked acceleration in both the maturation and secretion of PACE4A. The carboxyl-truncated proPACE4A existed only as a monomer-sized form in the endoplasmic reticulum, whereas the wild type of proPACE4A existed in larger forms. Further, the fusion construct of yellow fluorescent protein and the carboxy-terminal sequence of PACE4A associated with the proPACE4A moiety and inhibited maturation. Thus the carboxy terminus of PACE4A functions as a potent autoinhibitor of its activation, resulting in the retention of proPACE4A in the endoplasmic reticulum. These findings indicate that PACE4A activity is highly controlled by a unique system at post-translational level.