calpain and Retinal-Diseases

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.. 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.. 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.. 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.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Calpain; Carbamates; Caspase 3; Cells, Cultured; Child; Cytosol; DNA Fragmentation; Enzyme Activation; Humans; Hypoxia; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Macaca mulatta; Middle Aged; Retinal Diseases; Retinal Ganglion Cells

Topics: Aged; Calpain; Eye Diseases; Female; Humans; Retinal Diseases; Tissue Adhesions; Tomography, Optical Coherence; Vitreoretinopathy, Proliferative; Vitreous Body

We found that hesperidin, a plant-derived bioflavonoid, may be a candidate agent for neuroprotective treatment in the retina, after screening 41 materials for anti-oxidative properties in a primary retinal cell culture under oxidative stress. We found that the intravitreal injection of hesperidin in mice prevented reductions in markers of the retinal ganglion cells (RGCs) and RGC death after N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Hesperidin treatment also reduced calpain activation, reactive oxygen species generation and TNF-α gene expression. Finally, hesperidin treatment improved electrophysiological function, measured with visual evoked potential, and visual function, measured with optomotry. Thus, we found that hesperidin suppressed a number of cytotoxic factors associated with NMDA-induced cell death signaling, such as oxidative stress, over-activation of calpain, and inflammation, thereby protecting the RGCs in mice. Therefore, hesperidin may have potential as a therapeutic supplement for protecting the retina against the damage associated with excitotoxic injury, such as occurs in glaucoma and diabetic retinopathy.

Topics: Animals; Apoptosis; Calpain; Cells, Cultured; Disease Models, Animal; Evoked Potentials, Visual; Hesperidin; Male; Mice; N-Methylaspartate; Neuroprotective Agents; Oxidative Stress; Retina; Retinal Diseases; Retinal Ganglion Cells; Treatment Outcome; Tumor Necrosis Factor-alpha

Retinal ischemic phenomena occur in several ocular diseases that share the degeneration and death of retinal ganglion cells (RGCs) as the final event. We tested the neuroprotective effect of azithromycin, a widely used semisynthetic macrolide antibiotic endowed with anti-inflammatory and immunomodulatory properties, in a model of retinal ischemic injury induced by transient elevation of intraocular pressure in the rat.. Retinal ischemia was induced in adult rats with transient elevation of intraocular pressure. RGCs were retrogradely labeled with Fluoro-Gold, and survival was assessed following a single dose of azithromycin given systemically at the end of the ischemia. The expression of death-associated proteins and extracellular signal-regulated kinase (ERK) activation was studied with western blotting. Expression and activity of matrix metalloproteinase-2 (MMP-2) and -9 were analyzed with gelatin zymography.. Acute post-injury administration of azithromycin significantly prevented RGC death. This effect was accompanied by reduced calpain activity and prevention of Bcl-2-associated death promoter (Bad) upregulation. The observed neuroprotection was associated with a significant inhibition of MMP-2/-9 gelatinolytic activity and ERK1/2 phosphorylation.. Azithromycin provides neuroprotection by modifying the inflammatory state of the retina following ischemia/reperfusion injury suggesting potential for repurposing as a drug capable of limiting or preventing retinal neuronal damage.

Topics: Animals; Anti-Bacterial Agents; Apoptosis; Azithromycin; bcl-Associated Death Protein; Blotting, Western; Calpain; Cell Survival; Cytoprotection; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neuroprotective Agents; Phosphorylation; Rats; Rats, Wistar; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells

Vasomotor responses of retinal arterioles to luminal flow/shear stress and VEGF have a critical role in governing retinal blood flow possibly via nitric oxide synthase (NOS) activation. However, the cellular mechanism for flow-sensitive vasomotor activity in relation to VEGF signaling in retinal arterioles has not been characterized. We used an isolated vessel approach to specifically address this issue.. Porcine retinal arterioles were isolated, cannulated, and pressurized to 55 cm H2O luminal pressure by two independent reservoir systems. Luminal flow was increased stepwise by creating hydrostatic pressure gradients across two reservoirs. Diameter changes and associated signaling mechanisms corresponding to increased flow and VEGF receptor 2 (VEGFR2) activation were assessed using videomicroscopic, pharmacological, and molecular tools.. Retinal arterioles developed basal tone under zero-flow condition and dilated concentration-dependently to VEGF165. Stepwise increases in flow produced graded vasodilation. Vasodilations to VEGF165 and increased flow were abolished by endothelial removal, and inhibited by pharmacological blockade of VEGFR2, NOS, phosphoinositide 3-kinase (PI3K), calpains, or sirtuin-1 (SIRT1) deacetylase. A VEGF165 antibody blocked vasodilation to VEGF165 but not flow. Immunostaining indicated that VEGFR2 was expressed in the endothelial and smooth muscle layers of retinal arterioles.. Ligand-dependent and ligand-independent activation of VEGFR2 in the endothelium mediates NO-dependent dilations of porcine retinal arterioles in response to VEGF165 and luminal flow/shear stress, respectively. It appears that NOS stimulation via PI3K, calpain proteases, and SIRT1-dependent deacetylation downstream from VEGFR2 activation contributes to these vasodilator responses.

Topics: Animals; Arterioles; Blotting, Western; Calpain; Disease Models, Animal; Female; Gene Expression Regulation; Immunohistochemistry; Male; Muscle, Smooth, Vascular; Phosphatidylinositol 3-Kinases; Retinal Diseases; Retinal Vessels; RNA; Sirtuin 1; Stress, Mechanical; Swine; Vascular Endothelial Growth Factor Receptor-2; Vasodilation

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.. 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, β-III tubulin, and γ-synuclein were performed with specific antibodies. Cell death was assessed by TUNEL staining.. 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.. 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.

Topics: Animals; Calpain; Cell Death; Cells, Cultured; Disease Models, Animal; Hypoxia; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Macaca mulatta; Nerve Fibers; Retinal Diseases; Retinal Ganglion Cells

In ischemic retinopathies, underlying hypoxia drives abnormal neovascularization that damages retina and causes blindness. The abnormal neovasculature is tortuous and leaky and fails to alleviate hypoxia, resulting in more pathological neovascularization and retinal damage. With an established model of ischemic retinopathy we found that calpain inhibitors, when administered in moderation, reduced architectural abnormalities, reduced vascular leakage, and most importantly reduced retinal hypoxia. Mechanistically, these calpain inhibitors improved stability and organization of the actin cytoskeleton in retinal endothelial cells undergoing capillary morphogenesis in vitro, and they similarly improved organization of actin cables within new blood vessels in vivo. Hypoxia induced calpain activity in retinal endothelial cells and severely disrupted the actin cytoskeleton, whereas calpain inhibitors preserved actin cables under hypoxic conditions. Collectively, these findings support the hypothesis that hyper-activation of calpains by hypoxia contributes to disruption of the retinal endothelial cell cytoskeleton, resulting in formation of neovessels that are defective both architecturally and functionally. Modest suppression of calpain activity with calpain inhibitors restores cytoskeletal architecture and promotes formation of a functional neovasculature, thereby reducing underlying hypoxia. In sharp contrast to "anti-angiogenesis" strategies that cannot restore normoxia and may aggravate hypoxia, the therapeutic strategy described here does not inhibit neovascularization. Instead, by improving the function of neovascularization to reduce underlying hypoxia, moderate calpain inhibition offers a method for alleviating retinal ischemia, thereby suggesting a new treatment paradigm based on improvement rather than inhibition of new blood vessel growth.

Topics: Actins; Animals; Calpain; Catalytic Domain; Cell Line; Cysteine Proteinase Inhibitors; Cytoskeleton; Endothelial Cells; Glycoproteins; Humans; Hypoxia; Mice; Mice, Inbred C57BL; Retina; Retinal Diseases; Retinal Neovascularization; Retinal Vessels

After an acute ischemia/reperfusion of the rat retina, the activation of cytotoxic proteases, including calpain, results in necrosis and apoptosis of retinal ganglion cells resulting in their degeneration. Using a systemically administered calpain inhibitor that crosses the blood-retinal barrier would provide for novel systemic intervention that protects the retina from acute injury and loss of function. Herein, we study a novel calpain peptide inhibitor, cysteic-leucyl-argininal (CYLA), in an in-vivo rat model of retinal ischemia to determine functional protection using electroretinography. The CYLA prodrug was administered intraperitoneally before and/or after ischemia-reperfusion at concentrations of 20-40 mg/kg. We found that administering 20 mg/kg of CYLA only after ischemia provides significant preservation of retinal function.

Topics: Animals; Calpain; Ischemia; Leupeptins; Male; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Vessels

Retinal excitotoxicity is associated with retinal ischemia, and with glaucomatous and traumatic optic neuropathy. The present study investigates the role of c-Jun N-terminal kinase (JNK) activation in NMDA-mediated retinal excitotoxicity and determines whether neuroprotection can be obtained with the JNK pathway inhibitor, D-form of JNK-inhibitor 1 (D-JNKI-1). Young adult rats received intravitreal injections of 20 nmol NMDA, which caused extensive neuronal death in the inner nuclear and ganglion cell layers. This excitotoxicity was associated with strong activation of calpain, as revealed by fodrin cleavage, and of JNK. The cell-permeable peptide D-JNKI-1 was used to inhibit JNK. Within 40 min of its intravitreal injection, FITC-labeled D-JNKI-1 spread through the retinal ganglion cell layer into the inner nuclear layer and interfered with the NMDA-induced phosphorylation of JNK. Injections of unlabeled D-JNKI-1 gave unprecedentedly strong neuroprotection against cell death in both layers, lasting for at least 10 days. The NMDA-induced calpain-specific fodrin cleavage was likewise strongly inhibited by D-JNKI-1. Moreover the electroretinogram was partially preserved by D-JNKI-1. Thus, the JNK pathway is involved in NMDA-mediated retinal excitotoxicity and JNK inhibition by D-JNKI-1 provides strong neuroprotection as shown morphologically, biochemically and physiologically.

Topics: Adaptation, Ocular; Animals; Blotting, Western; Calpain; Carrier Proteins; Cell Count; Cell Death; Electroretinography; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Immunohistochemistry; Injections; JNK Mitogen-Activated Protein Kinases; Male; Microfilament Proteins; N-Methylaspartate; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases; Signal Transduction; Vitreous Body

We previously reported a potent calpain inhibitor 1 (SJA6017, N-(4-fluorophenyl)-l-valyl-l-leucinal), which displayed relatively low oral bioavailability (BA). Replacing the metabolically labile aldehyde moiety of 1with more chemically stable warheads, such as a cyclic hemiacetal, hydrazone, and alpha-ketoamide, provided the inhibitors with improved in vitro metabolic stability. Cyclic hemiacetal 2 was the most stable of these compounds. The optimization of 2 led to hemiacetal 8 (SNJ-1715) which exhibited high potency, good aqueous solubility, excellent oral BA, and prolonged plasma half-life in rats. Furthermore, 8 showed neuroprotective efficacy via oral administration in a rat retinal ischemia model.

Topics: Administration, Oral; Animals; Calpain; Cell Line; Dipeptides; Drug Stability; Half-Life; Humans; Ischemia; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; Solubility; Stereoisomerism; Structure-Activity Relationship; Thiourea

Calpain-mediated proteolysis has been implicated as a major process in neuronal cell death in both acute insults and the chronic neurodegenerative disorders in the central nerves system. However, activation of calpain also plays a protective function in the early phase of excitotoxic neuronal death. The exact role of calpains in neuronal death and recovery after exposure to N-methyl-D-aspartate (NMDA) is not clearly known. The purpose of present study was to examine the involvement of mu- and m-calpain in NMDA-induced excitotoxicity in the adult rat retina. Increased immunoreactivity of mu-calpain was noted in RGC layer cells and in the inner nuclear layer with maximal expression at 12 h after NMDA injection. This was further confirmed with Western blotting. TdT-mediated biotin-dUTP nick end labeling (TUNEL) positive cells in the inner retina co-localized with moderate or intense mu-calpain immunoreactivity. In contrast, there was no remarkable change in m-calpain immunoreactivity at any time point after NMDA injection. Simultaneous injection of 2 nmol of a calpain inhibitor (calpain inhibitor II) significantly reduced the number of TUNEL-positive cells in the inner retina at 18 h after NMDA injection and preserved RGC-like cells counted at 7 days after injection. The results of this study showed that mu-calpain may be involved in mediating NMDA-induced excitotoxicity in the rat retina and calpain inhibitors may play a therapeutic role in NMDA related disease.

Topics: Animals; Apoptosis; Blotting, Western; Calpain; DNA Fragmentation; Immunohistochemistry; In Situ Nick-End Labeling; Isoenzymes; Male; N-Methylaspartate; Neurons; Neurotoxins; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases

Samples of the vitreous were analysed in order to identify changes of soluble proteins in vitreo-retinal disease. The soluble proteins of the vitreous were separated on an anion exchange column (Mono-Q). The degree of neutral proteolytic activity in vitreous body was also measured. The vitreous from cataract cases without vitreoretinal disease was characterized by its low content of soluble proteins equivalent to about 1% of that of serum. Albumin and transferrin were the major identified components and their concentrations were approximately 0.85 and 0.03 g/l, respectively. In cases with vitreoretinal disease the vitreous showed changes of total soluble protein and the appearance of additional protein peaks. In patients with PVR the albumin concentration in the vitreous was found to be three times higher as compared to the control group consisting of patients with cataract. Neutral proteolytic activity in the vitreous was relatively low in both normal and pathological vitreous.

Topics: Aged; Albumins; Calpain; Cataract; Chromatography, High Pressure Liquid; Eye Diseases; Eye Proteins; Humans; Middle Aged; Retinal Diseases; Solubility; Transferrin; Uveitis; Vitrectomy; Vitreous Body