Calpain-specific breakdown fragment in human drusen.

PURPOSE: With aging and age-related macular dystrophy (AMD), proteolytic fragments are deposited in extracellular drusen located between the RPE and Bruch's membrane. Localized hypoxia may be a risk factor for AMD. Our hypothesis is that following hypoxia, activation of proteolytic enzymes called calpains may cause proteolysis/degeneration of retinal cells and RPE. No direct evidence has yet demonstrated activation of calpains in AMD. The purpose of the present study was to identify calpain-cleaved proteins in drusen. METHODS: Seventy-six (76) drusen were analyzed in human eye sections from six normal and twelve AMD human donor eyes. The sections were subjected to immunofluorescence for the calpain-specific 150 kDa breakdown product from α-spectrin, SBDP150 - a marker for calpain activation, and for recoverin - a marker for photoreceptor cells. RESULTS: Among 29 nodular drusen, 80% from normal eyes and 90% from AMD eyes stained positive for SBDP150. Among 47 soft drusen, mostly from AMD eyes, 72% stained positive for SBDP150. Thus, the majority of both soft and nodular drusen from AMD donors contained SBDP150. CONCLUSIONS: SBDP150 was detected for the first time in soft and nodular drusen from human donors. Our results suggest that calpain-induced proteolysis participates in the degeneration of photoreceptors and/or RPE cells during aging and AMD. Calpain inhibitors may ameliorate AMD progression.

GCL loss in BRAO.

PURPOSE: Our recent publication used optical coherence tomography (OCT) to follow thinning of the retinal ganglion cell layer (GCL) in central retinal artery occlusion (CRAO). Thinning of the inner layers also occurs in patients with branch retinal artery occlusion (BRAO). The mechanism for such thinning may be partially due to proteolysis by a calcium-activated protease called calpain. Calpain inhibitor SNJ-1945 ameliorated the proteolysis in a past series of model experiments. The purposes of the present retrospective study were to: 1) use segmentation analysis of OCT images to follow the loss of retinal layers in BRAO compared to CRAO patients, and 2) predict the number of patients and days of observation needed for a clinical trial of a calpain inhibitor against BRAO. METHODS: A retrospective, case control study was conducted by computer-aided search in a medical records database for BRAO (ICD10 Code H34.239) with at least one OCT procedure (CPT: 92134). Non-proliferative, co-morbid eye diseases were allowed in the patient data base, and manual correction of auto-segmentation errors was performed. GCL thickness changes were followed over time and Cohen-d/sample size statistics were used to predict minimal patients needed for drug trials. RESULTS: The thickness of the GCL layer in BRAO decreased rapidly with time as in CRAO, but in more limited quadrants. The data, as fit to a single-phase decay curve, showed that GCL thickness could be used to provide sample size statistics in a clinical trial to test a calpain inhibitor. For example, a 60-day trial with a 60% effective inhibitor would need a minimum of 29 patients. CONCLUSIONS: Using thickness changes in the GCL layer to monitor the efficacy of potential inhibitors against BRAO and CRAO is practical in human trials requiring a reasonable number of patients and relatively short trial period. TRANSLATIONAL RELEVANCE: Measurement of GCL thickness would be a useful indicator of amelioration of BRAO and CRAO progression in a clinical trial of a putative inhibitor.

A descriptive analysis of predoctoral surgical requirements in US dental schools in 2020.

OBJECTIVES: This study aims to describe surgical graduation requirements in US dental schools in 2020, including changes made due to the COVID-19 pandemic. METHODS: Representatives of Commission on Dental Accreditation-approved predoctoral dental programs in the US (n = 66) received a 13-item questionnaire about operative and observational surgical requirements. Responses were assigned values to tabulate a surgical score (zero- to eight-point scale) as a proxy for required surgical experience, and statistical analyses were performed to explore for predictors. RESULTS: Surveys were returned by 97% (64/66) of programs with complete data from 62.5% of responding institutions. In periodontics, 6.8% of programs require students to perform periodontal surgery, 63.8% to assist, and none require a competency assessment in periodontal surgery. In oral and maxillofacial surgery, 23.3% of programs have numerical requirements in performance of surgical extractions, 35% require an operating room experience, and 51.9% have a competency assessment involving a surgical procedure. Modifications to surgical and nonsurgical graduation requirements due to COVID-19 were reported by 51.6% and 52.5% of programs, respectively. The mean surgical score was 1.73 ± 1.2 (range = 0-4) of eight possible points. This was not predicted by class size or the presence of postgraduate surgical programs. The presence of postgraduate surgical programs roughly doubled the likelihood of requiring an observational experience in surgery. CONCLUSIONS: As of 2020, US dental programs require a small fraction of surgical experiences available to students. Class size is not a predictor of required surgical experience. The presence of postgraduate surgical programs increased the likelihood of required observational experiences.

Segmenting OCT for detecting drug efficacy in CRAO.

PURPOSE: Thinning of the inner layers of the retina occurs in patients with central retinal artery occlusion (CRAO). The mechanism for such thinning may be partially due to proteolysis by a calcium-activated protease called calpain. Calpain inhibitor SNJ-1945 ameliorated the proteolysis in a past series of model experiments. The purposes of the present retrospective study were to: 1) use segmentation analysis of optical coherence tomography (OCT) images to mathematically model the loss of specific retinal layers in CRAO patients, and 2) predict the number of patients and days of observation needed for clinical trials of inhibitors against CRAO. METHODS: A retrospective case control study was conducted by computer-aided search for CRAO (ICD10 H43.1) with at least one OCT procedure (CPT: 92134) in the OHSU Epic patient data base. RESULTS: After initial swelling, thinning of the inner retinal layers, especially the ganglion cell (GCL) layer followed exponential decay curves. Using sample size statistics and GCL thickness as a marker in a 30-day clinical trial, 19 eyes/group could theoretically detect a 20% beneficial effect of an inhibitor against CRAO. Other markers, such as the whole retinal thickness and combined inner layers could also be used as less-specific markers. CONCLUSIONS: Using thickness changes in the GCL layer to monitor the efficacy of potential inhibitors against CRAO is practical in human trials requiring a reasonable number of patients and relatively short trial period. TRANSLATIONAL RELEVANCE: Measurement of GCL thickness would be a useful indicator of CRAO progression in a clinical trial of putative inhibitors.

Activation of Cytosolic Calpain, Not Caspase, Is Underlying Mechanism for Hypoxic RGC Damage in Human Retinal Explants.

Purpose: Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber. Methods: Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation. Results: Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945. Conclusions: An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.

Contribution of Calpain and Caspases to Cell Death in Cultured Monkey RPE Cells.

Purpose: AMD is the leading cause of human vision loss after 65 years of age. Several mechanisms have been proposed: (1) age-related failure of the choroidal vasculature leads to loss of RPE; (2) RPE dysfunctions due to accumulation of phagocytized, but unreleased A2E (N-retinylidene-N-retinylethanolamine); (3) zinc deficiency activation of calpain and caspase proteases, leading to cell death. The purpose of the present study is to compare activation of calpain and caspase in monkey RPE cells cultured under hypoxia or with A2E. Methods: Monkey primary RPE cells were cultured under hypoxic conditions in a Gaspak pouch or cultured with synthetic A2E. Immunoblotting was used to detect activation of calpain and caspase. Calpain inhibitor, SNJ-1945, and pan-caspase inhibitor, z-VAD-fmk, were used to confirm activation of the proteases. Results: (1) Hypoxia and A2E each decreased viability of RPE cells in a time-dependent manner. (2) Incubation under hypoxia alone induced activation of calpain, but not caspases. SNJ-1945 inhibited calpain activation, but z-VAD-fmk did not. (3) Incubation with A2E alone induced activation of calpain, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation. z-VAD-fmk inhibited caspase activation, suggesting no interaction between calpain and caspases. Conclusions: Hypoxia activated the calpain pathway, while A2E activated both calpain and caspase pathways in monkey RPE cells. Such knowledge may be utilized in the treatment of AMD if inhibitor drugs against calpain and/or caspase are used to prevent RPE dysfunction caused by hypoxia or A2E.

In Silico Ocular Pharmacokinetic Modeling: Delivery of Topical FK962 to Retina.

PURPOSES: To establish the in silico ocular pharmacokinetic modeling for eye drops, and to simulate the dose regimen for FK962 in human choroid/retinal diseases. METHODS: Pharmacokinetics for FK962 in vivo was performed by a single instillation of drops containing 0.1% 14C-FK962 in rabbit eyes. Permeation of FK962 across the cornea, sclera, and choroid/retina was measured in vitro. Neurite elongation by FK962 was measured in cultured rat retinal ganglion cells. Parameters from the experimental data were used in an improved in silico model of ocular pharmacokinetics of FK962 in man. RESULTS: The mean concentration of FK962 in ocular tissues predicted by in silico modeling was consistent with in vivo results, validating the in silico model. FK962 rapidly penetrated into the anterior and posterior segments of the eye and then diffused into the vitreous body. The in silico pharmacokinetic modeling also predicted that a dose regimen of 0.0054% FK962 twice per day would produce biologically effective concentrations of FK962 in the choroid/retina, where FK962 facilitates rat neurite elongation. CONCLUSIONS: Our in silico model for ocular pharmacokinetics is useful (1) for predicting drug concentrations in specific ocular tissues after topical instillation, and (2) for suggesting the optimal dose regimens for eye drops. The pharmacodynamics for FK962 produced by this model may be useful for clinical trials against retinal neuropathy.

FK962 induces neurite outgrowth in cultured monkey trigeminal ganglion cells.

PURPOSE: Corneal sensation, cell proliferation, and wound healing all depend on adequate corneal innervation. Disruption of corneal innervation can lead to dry eye and delayed wound healing. Our studies in rats and rabbits show that the substituted fluorobenzamide drug FK962 accelerates the extension of neuronal processes and recovery of corneal sensitivity. The purpose of the present study was 1) to determine whether FK962 induces sprouting and elongation of neurites in cultured monkey trigeminal ganglion cells, and 2) to investigate the involvement of the neurotrophic peptide GDNF in FK962-induced neurite elongation. METHODS: Dissociated, cultured trigeminal ganglion cells, containing neuronal and Schwann cells were cultured for 48 h with or without FK962. Neuronal elongation was evaluated by immunostaining with a neurofilament-specific antibody. Culture with or without GDNF, or with antibody against GDNF, was used to determine the role of GDNF in FK962-induced neurite elongation. RESULTS: FK962 or GDNF were found to significantly induce neurite elongation. The GDNF antibody significantly inhibited elongation induced by FK962. CONCLUSION: GDNF was found to be a mediator of FK962-induced neurite elongation in a relevant primate model. FK962 may be a candidate drug for treatment of neurotrophic disorders in the human cornea.

Selecting Fuchs patients for drug trials involving endothelial cell proliferation.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) might be managed by drug treatment before becoming severe enough to require surgery. For a clinical trial of such a drug, we hypothesize that selecting an adequate number of patients with FECD with only moderately compromised cell densities will be challenging. Thus, the purpose of the present study was to measure the prevalence of patients with FECD exhibiting moderately decreased corneal cell densities. METHODS: A retrospective data mining study (cross-sectional study) was performed on patient charts presenting at a large US northwestern academic health center by searching for diagnosis ICD-9 code 371.57 and Fuchs corneal dystrophies, including those with prior cataract surgeries and/or existing glaucoma. Patients with prior corneal transplants were excluded. Noncontact specular photomicroscopic data (Topcon 2000) were obtained from the central region whenever possible, and individual eyes were grouped according to cell density (cells/mm2): severe (<800), moderate (800-1,500), and mild (>1,500). RESULTS: The values for 98 eyes from 61 patients with FECD were as follows (mean ± SD): corneal thickness 573 ± 59 µm, cell size 627 ± 336 µm2/cell, coefficient of variation 23 ± 7, and density 1,883 ± 703 cells/mm2. The moderate subgroup with cell density values averaging 1,184 ± 212 (26) comprised 27% of the total FECD patient pool. CONCLUSIONS: Only approximately 1 out of 4 patients with FECD will show moderately compromised corneal cell densities. A moderate level of damage may be optimal for clinical trials for testing topical drugs on endothelial cell viability. Thus, investigators will need to initially screen a fourfold excess of all patients with FECD.

Hypoxia Activates Calpains in the Nerve Fiber Layer of Monkey Retinal Explants.

PURPOSE: The vascular ischemic hypothesis attributes nerve damage in the retina to decreased blood flow in the ophthalmic artery, reduced oxygenation, and impaired axonal transport. Activation of calpain enzymes contributes to retinal cell death during hypoxia. However, we still do not know in which specific retinal layers calpains are activated. Thus, the purpose of the present study was to investigate where and when calpains are activated in an improved culture model of hypoxic monkey retina. METHODS: Monkey retinal explants were cultured on microporous membranes with the retinal ganglion cell (RGC) side facing up. Explants were incubated under hypoxic conditions, with or without additional reoxygenation. When it was used, the calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting assays for α-spectrin, calpains 1 and 2, calpastatin, ß-III tubulin, and γ-synuclein were performed with specific antibodies. Cell death was assessed by TUNEL staining. RESULTS: Under normoxic conditions, TUNEL-positive cells were minimal in our improved culture conditions. As early as 8 hours after hypoxia, the 150-kDa calpain-specific α-spectrin breakdown product appeared in the nerve fiber layer (NFL), where calpains 1 and 2 were localized. TUNEL-positive RGCs then increased at later time periods. The calpain inhibitor SNJ-1945 ameliorated changes induced by hypoxia or hypoxia/reoxygenation. CONCLUSIONS: During hypoxia/reoxygenation in an improved, relevant monkey model, calpains were first activated in the NFL, followed by death of the parent RGCs. This observation suggest that calpain-induced degeneration of retinal nerve fibers may be an underlying mechanism for RGC death in hypoxic retinal neuropathies.

Galectin-3 enhances extracellular matrix associations and wound healing in monkey corneal epithelium.

Poor healing of epithelial wounds in cornea is a major clinical problem, leading to persistent epithelial defects and ulceration. The primary cause is poor cell migration over the wound. Carbohydrate-binding protein galectin-3 binds to extracellular matrixes (ECMs) and promotes lamellipodia formation by cross-linking to α3 integrin. Recombinant galectin-3 also facilitates wound healing in the rodent cornea. The purposes of the present experiments were to: (1) establish epithelial wound healing models in monkey corneal explant culture, the models more relevant to human, (2) evaluate the healing effect of galectin-3 in our models, and (3) determine if galectin-3 enhances cell adhesion by interacting with ECMs on corneal surface and their ligand integrins. Monkey corneas with central wounds produced by sodium hydroxide (NaOH) or n-heptanol were incubated with or without recombinant galectin-3. The defected area was stained with sodium fluorescein. Primary isolated corneal epithelial cells from monkey were cultured with or without galectin-3 on plates coated with ECMs or integrins, and the number of adhering cells was counted. Galectin-3 expression in various eye tissues was visualized by immunoblotting. NaOH caused loss of epithelial cells and basement membrane. n-Heptanol removed epithelial cells, but the basement membrane was retained. These corneal defects spontaneously became smaller in a time-dependent manner. Exogenous galectin-3 enhanced wound healing in both NaOH and n-heptanol models. Galectin-3 also enhanced cell adhesion onto the major ECMs found in the basement and Bowman's membranes and onto integrins. Relatively high levels of galectin-3 were detected in corneal and conjunctival epithelium, but tear fluid contained negligible galactin-3. These results suggested that the enhanced binding of epithelial cells to ECMs and integrins caused by galectin-3 might promote cell migration over wounded corneal surfaces. Since tear fluid contained relatively low levels of galectin-3, exogenous galectin-3 may be a beneficial drug to enhance re-epithelialization in human corneal diseases.

Serum antibodies against ßH-crystallins in the American Cocker Spaniel.

OBJECTIVE: To detect antibodies for lens ßH-crystallins in the serum from the American Cocker Spaniel (ACS) presenting with and without cataracts and with and without uveitis. ANIMAL STUDIED: Seventy-three American Cocker Spaniels and six normal Beagles. PROCEDURES: Sera were collected from 73 ACSs, including those with normal lenses and those with cataracts, or uveitis. Fractionated, normal Beagle lens ßH-crystallins were separated by one- or two-dimensional electrophoresis. The separated lens ßH-crystallins were used on immunoblots as sentinel substrates against which the ACS sera were tested for the presence of antibodies against ßH-crystallins. RESULTS: Sera from approximately two-thirds of study animals contained antibodies to some ßH-crystallin polypeptides, but reactivity varied among patients. Contrary to some hypotheses, serum antibodies to groups of ßH-crystallins did not relate to the stages of cataract. However, detailed analysis by two-dimensional immunoblotting and mass spectrometry showed that three spots originating from ßA1-crystallin were detected only in sera from cataract patients. CONCLUSION: Serum antibodies to ßA1-crystallin may be associated with the development of cataract.

Loss of calpastatin leads to activation of calpain in human lens epithelial cells.

PURPOSE: Activation of calpains (calpain 2 and Lp82) in rodent lenses readily causes proteolysis and cataract formation. In contrast, primate lenses are quite resistant to activation of calpains. The hypothesis is that high levels of human endogenous calpain inhibitor, calpastatin (CS), prevent calpain activation in human lenses. The purpose of the present study was to directly test if CS is a major inhibitory factor in a human lens epithelial cell line, HLE B-3. METHODS: Small interfering RNAs (siRNAs) were used to knock down expression of CS in HLE B-3. The cells then were cultured with the calcium ionophore, ionomycin, with or without a calpain inhibitor SNJ-1945. Transcripts for calpain 2 and CS were measured by quantitative PCR (qPCR). Calpain 2 activity was detected by immunoblotting for the calpain-specific, α-spectrin breakdown product and for activation-associated, fragments of calpain 2. RESULTS: Expression of CS in HLE B-3 was remarkably higher than in α-TN4 (mouse comparator cell line). Proteolysis of α-spectrin was observed in the soluble proteins from α-TN4 incubated with Ca(2+), but not in the human HLE B-3. When CS-reduced HLE B-3 cells (transfected with CS siRNA) were cultured with ionomycin, calpain 2 was activated, specific proteolysis of α-spectrin occurred, and cell death ensued; SNJ-1945 inhibited these changes. CONCLUSIONS: Our data demonstrated that the high levels of endogenous CS do, indeed, inhibit calpain activity in normal human lens epithelial cells. We speculate that age-related oxidation might cause loss of CS activity in human lens epithelial cells, allowing activation of long-dormant calpain 2, proteolysis of critical cytoskeletal proteins, and cataract formation.

Dry eye in LASIK patients.

BACKGROUND: Increasing age is a known risk factor for developing dry eye. The specific aims of the present study were to determine the prevalence of dry eye syndrome (DES) and use of post-operative dry eye medications in a relatively young population presenting for LASIK surgery at an academic ophthalmology clinic. FINDINGS: A retrospective, analysis of 948 de-identified patient charts (median age 39 years, not age stratified) was performed to extract pre-LASIK diagnoses and post-LASIK medication lists. Clinical evaluation for DES and the results of Schirmer's reflex tear flow test were used to assign LASIK patients into Normal, Pre-dry eye (Pre-DES), and Dry Eye Syndrome (DES) groups; which were then compared for use of dry eye medications.Based on pre-operative diagnoses, only 2% (CI: 1.3 - 3.1) of LASIK patients presented with overt DES. Unexpectantly, 25% (CI: 22.2 - 27.6) of LASIK patients labeled Pre-DES were not classified by the clinician as having overt DES, yet they showed poor reflex tear flow rates ≤ 5 mm before surgery, and frequently used post-operative lubricant dry eye medications. CONCLUSIONS: Although the number of patients with pre-existing eye conditions was unknown, a sizable portion of relatively young LASIK patients displays poor reflex tear flow without overt DES. Such patients could go on to develop more serious consequences of poor tear flow, such as corneal abrasion and erosion. More specific, dry eye medications may be needed for ideal treatment.

Mechanism for laser-induced neovascularization in rat choroid: accumulation of integrin α chain-positive cells and their ligands.

PURPOSE: Inhibitors binding to integrins α5 and αv are antiangiogenic in models of choroidal neovascularization (CNV). However, a comprehensive understanding of the accumulation of integrin α isoform-positive cells, their ligands, and associations is limited. The purpose of the present study was to examine the localization of integrin α chain-positive cells and their extracellular matrix (ECM) ligands in the RPE/choroid after laser injury. METHODS: CNV, observed with fluorescein isothiocyanate (FITC)-labeled isolectin, was produced in Brown Norway rats with a 532 nm green laser. Localization of α5 and αv integrins and their ligands was performed with immunohistochemistry in consecutive cryosections. To test the binding specificity between the integrin α chains and ECM ligands, an in vitro cell adhesion assay was performed using retinal endothelial cells and specific antibodies. RESULTS: Angiogenesis was observed on day 7 after laser injury in choroidal flat mounts and cryosections. The number of integrin α5- and αv-positive cells markedly increased at day 3 and then gradually decreased, but was still elevated on day 14. One day after laser treatment, α integrin ligands fibronectin (FN) and vitronectin (VN) were markedly increased, and localized closely to integrins in the laser-injured regions. FN decreased on day 7, but was still retained until 14 days. In contrast, VN disappeared. Cell adhesion assays showed specific association of integrin α5 to FN, and integrin αv to VN. CONCLUSIONS: Laser-induced choroidal injury increased FN and VN, followed by accumulation of integrin α5- and αv-positive cells. The interaction between integrin α chain-positive cells and their specific ligands FN and VN may be important steps leading to CNV.

Degeneration and dysfunction of retinal neurons in acute ocular hypertensive rats: involvement of calpains.

PURPOSE: Retinal ischemic diseases primarily lead to damage of the inner retinal neurons. Electrophysiological studies also suggest impairment of the inner retinal neurons. Our recent studies with acute ocular hypertensive rats confirmed damage predominantly in the inner retinal layer along with the ganglion cell layer, changes that are ameliorated by the calpain inhibitor SNJ-1945. However, we do not know which specific neuronal cells in the inner retinal layer are damaged by calpains. Thus, the purpose of the present study was to identify specific calpain-damaged neuronal cells in the inner retina from acute ocular hypertensive rats. METHODS: Intraocular pressure was elevated to 110 mm Hg for 40 min. One hour after ocular hypertension (OH), SNJ-1945 was administrated as a single oral dose of 50 mg/kg. Retinal function was assessed by scotopic electroretinography (ERG). Histological degeneration was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL), and immunostaining in thin sections and flat mounts of the retina. Calpain activation was determined by proteolysis of the calpain substrate α-spectrin. RESULTS: OH caused calpain activation, increased TUNEL-positive staining, decreased thickness of the inner nuclear layer (INL), and decreased amplitudes of the ERG a- and b-waves and oscillatory potentials (OPs). SNJ-1945 significantly inhibited calpain activation and the decrease in ERG values. Interestingly, the changes in the b-wave and OPs amplitudes were significantly correlated to changes in the thickness of the INL. In the inner retinal layer, the numbers of rod bipolar, cone-ON bipolar, and amacrine cells were decreased after OH. SNJ-1945 suppressed the loss of cone-ON bipolar and amacrine cells, but did not inhibit the loss of rod bipolar cells. We also observed increased glial fibrillary acid protein-positive staining in the Müller cells after OH and the treatment with SNJ-1945. CONCLUSIONS: Calpains may contribute to ischemic retinal dysfunction by causing the loss of cone-ON bipolar and amacrine cells and causing the activation of Müller cells. Calpain inhibitor SNJ-1945 may be a candidate compound for treatment of retinal ischemic disease.

Calpain protease causes hypoxia-induced proteolysis in cultured human retina.

PURPOSE/AIM: Calpain proteases are known to be involved in retinal cell death in animal models. The purpose of the present study was to test for calpain activation in human retinas cultured under hypoxic conditions. MATERIALS AND METHODS: Calpain activation was detected by immunoblotting for calpain substrates in human and monkey retinas cultured in gas generating pouches to reduce oxygen. RESULTS: Hypoxia caused activation of calpains as measured by accumulation of the calpain-specific 145 kDa α-spectrin breakdown product. Opsin-1 (photoreceptor marker) and vimentin (Müller cell marker) were degraded. Calpain inhibitor SNJ-1945 ameliorated these changes. Results were similar to comparative data from cultured monkey retinas. CONCLUSIONS: In cultured human retina, hypoxia caused activation of calpain and subsequent proteolysis of critical substrates. The efficacy of SNJ-1945 in ameliorating these changes indicated that it might be useful to test as a drug for protecting against pathologic proteolysis of photoreceptor and Müller cells.

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells.

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).

Involvement of NFκB in the production of chemokines by rat and human conjunctival cells cultured under allergenic conditions.

PURPOSE: The purpose of present studies was to determine the involvement of NFκB and STAT6 transcription factors in the production of cytokines by the fibroblasts and epithelial cells in conjunctiva. METHODS: An in vitro model of allergic conjunctivitis was developed by sensitizing and challenging rat mast cells with anti-dinitrophenyl (DNP) IgE and DNP-BSA, and then using the conditioned medium to stimulate rat conjunctival fibroblasts. Chemokines (eotaxin-1, IL-8, and RANTES -- Regulated and Normal T cell Expressed and Secreted) released from cells into the medium was determined by ELISA. Human conjunctival fibroblasts and epithelial cells were also directly stimulated with exogenous cytokines tumor necrosis factor (TNF)-α or IL-4. Degradation of IκB-α and phosphorylation of STAT6 were assessed by immunoblotting. For inhibition of NFκB or STAT6 activation, upstream regulators IκB kinase and Janus protein tyrosine kinases (JAK) were inhibited by use of BMS-345541 and JAK inhibitor 1. An in vivo model of conjunctivitis was also produced in rats by intraperitoneal injection of ovalbumin (OA) with aluminum hydroxide and challenge at 21 d with OA eye drops. RESULTS: Stimulated rat mast cells released TNF-α and IL-4. TNF-α induced NFκB activation in rat and human conjunctival fibroblasts and epithelial cells, and caused production and release of cytokines IL-8 and RANTES. IL-4 activation of STAT6 did not cause release of these cytokines. Only fibroblasts produced the eosinophil-recruiting cytokine, eotaxin-1, after treatment with TNF-α- plus IL-4. As observed in the cultured cells, allergic stimulation in the in vivo model caused degradation of IκB-α in conjunctiva, and infiltration of eosinophils and other inflammatory cells. CONCLUSION: Activated NFκB was found to be a major transcription factor for the release of cytokines from conjunctival cells and intensification of the allergic response. Inhibition of the NFκB pathway by therapeutic drugs may be an important objective for the treatment of human allergic conjunctivitis.

Lacritin-induced secretion of tear proteins from cultured monkey lacrimal acinar cells.

PURPOSE: During inflammation of the ocular surface, increased proinflammatory cytokines depress tear protein secretion, suggesting that a decline in lacrimal cell function contributes to dry eye. Lacritin, a glycoprotein secreted from lacrimal acinar cells, may function as an autocrine factor to stimulate tear protein secretion. The purpose of the present experiment was to investigate lacritin-induced protein secretion in normal and cytokine-pretreated (inflammation model) monkey acinar cells. METHODS: Acinar cells from monkey lacrimal glands were cultured with or without tumor necrosis factor alpha (TNF-α) plus interferon gamma (IFN-γ). Protein secretion was induced by lacritin or carbachol (Cch, positive control). Proteins were detected and identified by immunoblotting and immunocytochemistry. Intracellular Ca(2+) was measured with the fluorophore Calcium-4, and cell damage was determined by LDH leakage into the culture medium. RESULTS: In cultured monkey acinar cells, lacritin stimulated tear protein secretion in normal cells without elevating intracellular Ca(2+). In contrast, Cch elevated intracellular Ca(2+) and release of tear proteins. This contrast suggested an alternate, calcium-independent mechanism for lacritin-induced protein secretion. TNF-α plus IFN-γ caused LDH leakage from sensitive human corneal epithelial cells, but even higher doses of TNF-α plus IFN-γ did not cause LDH leakage from monkey acinar cells, suggesting a higher tolerance against these cytokines. In cytokine-treated acinar cells, lacritin stimulated protein secretion as much as that in normal cells. In contrast, Cch-induced elevation of Ca(2+) and release of proteins were depressed by cytokines. CONCLUSIONS: Lacritin might be a useful biotechnology-based treatment agent against ocular surface diseases where endogenous lacritin is inadequate.