snj-1945 and Hypoxia

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

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.. Calpain activation was detected by immunoblotting for calpain substrates in human and monkey retinas cultured in gas generating pouches to reduce oxygen.. 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.. 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.

Topics: Aged; Aged, 80 and over; Animals; Calpain; Carbamates; Cell Death; Cells, Cultured; Disease Models, Animal; Female; Haplorhini; Humans; Hypoxia; Immunoblotting; Male; Middle Aged; Peptide Hydrolases; Proteolysis; Retina

Cell death occurring in human retina during AMD, high IOP, and diabetic retinopathy could be caused by activation of calpain or caspase proteolytic enzymes. The purpose of the present study was to determine whether calpains and/or caspase-3 were involved in cell death during retinal hypoxia in a monkey model.. Dissociated monkey retinal cells were cultured for two weeks and subjected to 24-hour hypoxia/24-hour reoxygenation. TUNEL staining and immunostaining for Müller and photoreceptor markers were used to detect which retinal cell types were damaged.. Culturing dissociated monkey retina cells for two weeks resulted in proliferation of Müller cells and maintenance of some rod and cone photoreceptor cells, as identified by vimentin, recoverin, and rhodopsin immunocytochemical staining. Hypoxia/reoxygenation increased the number of cells staining positive for TUNEL. Immunoblotting showed that the calpain-specific 145 kDa α-spectrin breakdown product (SBDP) increased in hypoxic cells, but no caspase-specific 120 kDa α-spectrin breakdown product was detected. TUNEL staining and proteolysis were significantly reduced in the retinal cells treated with 10 and 100 μM calpain inhibitor SNJ-1945. Caspase inhibitor, z-VAD, did not inhibit cell damage from hypoxia/reoxygenation. Intact pro-caspase-3 was in fact cleaved by activated calpain during hypoxia/reoxygenation to pre 29 kDa caspase-3 and 24 kDa inactive fragments. No 17 and 12 kDa fragments, which form the active caspase-3 hetero-dimer, were detected. Calpain-induced cleavage of caspase was inhibited by SNJ-1945.. Calpain, not caspase-3, was involved in hypoxic damage in cultured monkey retinal cells.

Topics: Animals; Apoptosis; Biomarkers; Calpain; Carbamates; Caspase 3; Caspase Inhibitors; Cell Count; Cell Proliferation; Cells, Cultured; Fluorescent Antibody Technique, Indirect; Hypoxia; Immunoblotting; In Situ Nick-End Labeling; Macaca mulatta; Neuroglia; Oligopeptides; Photoreceptor Cells, Vertebrate; Recoverin; Reperfusion Injury; Rhodopsin; Spectrin; Vimentin