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.

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.

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.

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.

Pituitary adenylate cyclase-activating peptide induces neurite outgrowth in cultured monkey trigeminal ganglion cells: involvement of receptor PAC1.

PURPOSE: Our previous studies in the rabbit trigeminal nerve (TgN) showed that pituitary adenylate cyclase-activating peptide (PACAP) accelerated the extension of neuronal processes and recovery of corneal sensitivity. The purposes of the present study were 1) develop a procedure to culture trigeminal nerve (TgN) cells from monkeys, 2) test whether PACAP induces sprouting and elongation of axons in our culture system, 3) investigate the signaling mechanisms producing axon elongation induced by PACAP, and 4) test the action of PACAP on tear protein secretion by monkey lacrimal acinar cells. METHODS: Primary cultures of TgN cells were established from rhesus monkeys. Cellular distribution of the PACAP receptor, PAC1, was determined with immunostaining. Axonal length in cultured TgN ganglion cells was evaluated with staining by antibody for neurofilament. mRNA expression was determined with quantitative real-time polymerase chain reaction (qPCR). Secretion of tear protein from cultured acinar cells was measured with immunoblotting. RESULTS: Our results showed that dissociated, cultured TgN cells contained neuronal ganglion and Schwann cells, and the PAC1 receptor was expressed in both cell types. PACAP-27 significantly induced neurite outgrowth, which was inhibited by PACAP 6-27. Inhibitors for adenylate cyclase and phospholipase C also inhibited neurite outgrowth. Follistatin was upregulated by PACAP-27 during the culture period. PACAP enhanced secretion of tear proteins. CONCLUSIONS: Our data suggested PAC1 activation is involved in TgN neurite outgrowth.

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).

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.

PACAP induces neurite outgrowth in cultured trigeminal ganglion cells and recovery of corneal sensitivity after flap surgery in rabbits.

PURPOSE: To evaluate the ability of pituitary adenylate cyclase-activating polypeptide (PACAP) to induce growth of neuronal processes in cultured trigeminal ganglion cells, and to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of a corneal flap in a rabbit model of laser-assisted in situ keratomileusis (LASIK) surgery. DESIGN: Animal study. METHODS: The cDNA of rabbit PACAP was sequenced, and the expression of PACAP receptors in the trigeminal ganglia from rabbits was quantified by quantitative real-time polymerase chain reaction. Trigeminal ganglion cells were isolated from rabbits and cultured for 48 hours with or without PACAP27 (bioactive N-terminal peptide from PACAP). Cells were stained with antibody against neurofilaments, and neurite outgrowth was quantified by cell counting. In the rabbit LASIK model, a corneal flap with a planned thickness of 130 microm and 8.5 mm diameter was created with a microkeratome. The rabbits then received eyedrops containing PACAP27 four times a day for eight weeks, and corneal sensitivity was measured. Neurite outgrowth was assessed by staining histologic sections of the flap area for cholinesterase. RESULTS: The deduced amino acid sequence of PACAP in rabbit was identical to that of human. PACAP receptor, PAC1, was highly expressed in trigeminal ganglia from newborn and adult rabbits. PACAP27 at 1 microM induced growth of neuronal processes in cultured primary trigeminal ganglion cells. In the LASIK model, extensions of neuronal processes from amputated nerve trunks in cornea were observed after administration of eyedrops containing 1 or 10 microM PACAP27. The 10 microM PACAP27 treatment also greatly accelerated recovery of corneal sensitivity. CONCLUSIONS: PACAP may be a candidate drug for ameliorating dry eye after LASIK surgery.

Contribution of calpain to cellular damage in human retinal pigment epithelial cells cultured with zinc chelator.

PURPOSE: We previously showed involvement of calpains in neural retina degeneration induced by hypoxia and ischemia-reperfusion. Age-related macular degeneration (AMD) is one of the leading causes for loss of vision. AMD showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (RPE). RPE performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. The purpose of the current study was to determine the contribution of calpain-induced proteolysis to damage in cultured human RPE cells. Zinc chelator TPEN was used to induce cellular damage as zinc deficiency is a suspected risk factor for AMD. METHODS: In RPE/choroid preparations from normal and AMD patients, calpain mRNAs were measured by qPCR, and calpain activity was assessed by casein zymography. Third- to fifth-passage cells from human RPE cells were cultured with TPEN. Cell damage was morphologically assessed under the phase-contrast microscope, and TUNEL staining was performed to detect apoptosis. Leakage of lactate dehydrogenase (LDH) into the medium was measured as a marker of RPE cell damage. Activation of calpains and proteolysis of the known calpain substrate alpha -spectrin were assessed by immunoblotting. To further confirm calpain-induced proteolysis, calpain in homogenized RPE was also activated directly by addition of calcium. RESULTS: RPE/choroid from normal patients expressed mRNAs for calpain 1, calpain 2, and calpastatin moderately, and calpain 2 activity tended to be lower in AMD patients. TPEN caused RPE cell damage with positive TUNEL staining. TPEN also caused leakage of LDH into the medium from RPE cells, and calpain inhibitor SJA6017 inhibited the leakage. Caspase-3 inhibitors z-VAD and z-DEVD also showed inhibitory effects. Immunoblotting for calpain and alpha -spectrin showed activation of calpain in RPE cells cultured with TPEN. Proteolysis by activated calpain was confirmed by addition of calcium to homogenized RPE. CONCLUSIONS: These results suggested that activation of calpain contributed to cellular damage induced by TPEN in cultured human RPE cells.

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.

Calpain-specific proteolysis in primate retina: Contribution of calpains in cell death.

PURPOSE: One of the leading causes of blindness is retinal damage caused by the high intraocular pressure (IOP) in glaucoma. Previous studies in rats have suggested that the proteolytic enzyme calpain (EC 3.4.22.17) is involved in retinal cell death during ischemia and in acute high IOP. Ubiquitous, calcium-activated calpain-1 and -2 from monkey retina are highly homologous to rat calpains, although expression patterns in variants of tissue-specific calpain-3 are different between monkey and rodent retinas. Thus, the purpose of the present study was to investigate the involvement of calpain-induced proteolysis in retinal cell death in primates. METHODS: Calpain involvement in a simulated pathologic condition was examined by incubating monkey retinas in hypoxic conditions (95% N2 and 5% CO2) in RPMI medium without glucose. Endogenous tissue calpains were also directly activated in monkey and human retinal soluble proteins by incubating with 2.5 mM calcium. The resultant proteolysis of monkey retinal proteins was assessed by 2D electrophoresis (2-DE). RESULTS: In hypoxic retina, leakage of lactate dehydrogenase (LDH) from retinas into the medium increased, indicating cell death. LDH leakage was partially inhibited by the calpain inhibitor SJA6017. Calpain autolysis was observed, and the calpain-preferred substrate alpha-spectrin was proteolyzed. In retinal soluble proteins incubated with calcium, a total of 15 spots from 2-DE of retinal soluble proteins were identified by mass spectrometry. Proteolysis of major proteins, vimentin, beta-tubulin, alpha-enolase, and Hsp70 were confirmed by immunoblot analysis. Activation of calpains and proteolysis of these substrates were inhibited by the calpain-specific inhibitor SJA6017. CONCLUSIONS: Taken together, these results suggested that calpain activation in primate retinas could play an important role in cell death during hypoxia caused by elevated IOP from glaucoma.

Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic beta-cells.

Calpain-10 (CAPN10) is the first type 2 diabetes susceptibility gene to be identified through a genome scan, with polymorphisms being associated with altered CAPN10 expression. Functional data have been hitherto elusive, but we report here a corresponding increase between CAPN10 expression level and regulated insulin secretion. Pancreatic beta-cell secretory granule exocytosis is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor protein complex of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin 1, and vesicle-associated membrane protein 2. We report, for the first time, direct binding of a calpain-10 isoform with members of this complex. Furthermore, SNAP-25 undergoes a Ca2+-dependent partial proteolysis during exocytosis, with calpain protease inhibitor similarly suppressing both insulin secretion and SNAP-25 proteolysis. Based upon these findings, we postulate that an isoform of calpain-10 is a Ca2+-sensor that functions to trigger exocytosis in pancreatic beta-cells.

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.

Calpain may contribute to hereditary cataract formation in sheep.

PURPOSE: To determine the involvement of calpain in ovine cataractogenesis by measuring calcium, calpain activity, proteolysis, and the effect of calpain inhibition. METHODS: Sheep with genetic cataracts were examined for cataract severity. Calcium in normal and cataract lenses was measured. The presence of calpain was detected by casein zymography and immunoblotting. Calpain activity was assayed using BODIPY-casein as a substrate. Degradation of calpain substrates spectrin and vimentin was assessed by immunoblotting. The calpain inhibitor SJA6017 was applied to the left eye of cataract lambs, leaving the right eye as an untreated control. Both eyes were monitored by slit-lamp microscopy for cataract progression. RESULTS: Cortical cataracts were first observed in lambs at 1 to 2 months of age. Lens calcium concentration increased in the early stages of cataract formation and was >10-fold higher in mature cataract than normal lenses. Three calpain isoforms were detected in young lamb lenses. Calpain activity decreased as cataracts progressed. Both spectrin and vimentin were degraded with cataract maturity, which could indicate calpain proteolysis. Cataract lambs treated with SJA6017 eyedrops over a period of 4 months showed significantly smaller cataracts in the left treated eye over the right untreated eye. CONCLUSIONS: The presence of calpains and calcium elevation during cataract formation suggests that proteolysis may play a role in opacification in ovine lens. This hypothesis is supported by the delay in opacification with SJA6017 treatment. The results also suggested that the ovine hereditary cataract is a useful nonrodent model to test the role of calpains in cataractogenesis.

Proteolysis of neuronal cytoskeletal proteins by calpain contributes to rat retinal cell death induced by hypoxia.

Our previous studies in retina on the mechanism for hypoxia-induced cell death suggested activation of a class of calcium-activated proteases known as calpains. This conclusion was based on data showing proteolysis of a calpain substrate alpha-spectrin, autolysis of activated calpain, and reduction of cell damage by calpain inhibitor SJA6017. Less is known about changes in downstream pathways after calpain activation. Thus, the purpose of the present investigation was to measure proteolysis of neuronal cytoskeletal proteins and apoptotic cell signaling factors during hypoxia-induced retinal cell death. Rat retinas were incubated in RPMI medium with glucose and 95% O2/5% CO2 to supply sufficient oxygen for retinal cell survival. Hypoxia was induced with 95% N2/5% CO2 without glucose. Immunoblotting was used to detect activation of calpain and proteolysis of substrates. Amounts of mRNA for calpain 1 and 2 were determined by quantitative PCR. Twelve times more calpain 2 mRNA than calpain 1 was present in retinas. Activation of calpain 2 and production of a calpain-specific alpha-spectrin breakdown product at 150 kDa were confirmed in hypoxic retinas. Further, pro-caspase-3 at 32 kDa was proteolyzed to a fragment at 30 kDa, tau protein was lost, and p35 was proteolyzed to p25 suggesting prolonged activation of cdk5. SJA6017 partially inhibited the production of these fragments. During hypoxia in rat retinas, calpains may be major proteases causing breakdown of neuronal proteins involved in apoptotic cell death. Calpain inhibitor SJA6017 may have potential for testing as a therapeutic agent against retinal pathologies such those caused by glaucoma, although future studies such as testing in in vivo animal models are required.