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Mitsuyoshi Azuma, Katherine B. Hammond, Emi Nakajima, Thomas R. Shearer; Calpain Protease Causes Hypoxia-Induced Proteolysis in Cultured Human Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4279.
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Decreased blood flow in the choroid or retinal arteries leads to rapid cell death and blindness in age-related macular degeneration, glaucoma from high intraocular pressure, and diabetic retinopathy. Calpain and caspase proteases are known to be involved in retinal cell death in a number of animal models. Interestingly, in monkey retinal cells cultured under hypoxia, calpain protease caused the cell damage. This is not always the case because most tissues contain significant amounts of the highly specific, endogenous calpain inhibitor calpastatin. Thus, the clinically relevant purpose of the present study was to test for calpain activation in human retinas cultured under hypoxic conditions.
Human and monkey retinas were quartered and incubated in DMEM. Hypoxia was imposed using the GasPack pouch system. Retinal cell marker proteins, activation of calpains, and proteolysis of α-spectrin substrate were detected by immunoblotting. Calpain inhibitor SNJ-1945 was used to confirm activation of calpain.
In cultured human retina, hypoxia caused autolysis of calpain (indicative of calpain activation) and accumulation of the 145 kDa calpain-specific, α-spectrin breakdown product. Opsin (photoreceptor marker) and vimentin (Müller cell marker) were also degraded. SNJ-1945 inhibited calpain activation and degradation of opsin and vimentin in a dose dependent manner. Retinas cultured under normoxic conditions showed a lower level of calpain activation and substrate proteolysis. Similar results were observed in monkey retina.
The most important finding was that human retina shows calpain activation when cultured under hypoxia. Activated calpain proteolyzes critical substrates such as opsin, vimentin and α-spectrin; leading to cell death. Synthetic calpain inhibitor SNJ-1945 may be a useful drug to protect against proteolysis in photoreceptor and Müller cells from patients with ischemic retinal diseases.Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are employees of, Senju Pharmaceutical Co, Ltd.
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