Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Corporation Limited, Beaverton, Oregon 97006, USA.
Invest Ophthalmol Vis Sci. 2011 Sep 1;52(10):7059-67. doi: 10.1167/iovs.11-7497.
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.
在 AMD、高眼压和糖尿病性视网膜病变中，人视网膜中的细胞死亡可能是由钙蛋白酶或半胱天冬酶蛋白水解酶的激活引起的。本研究的目的是确定在猴模型的视网膜缺氧中，钙蛋白酶和/或半胱天冬酶-3 是否参与细胞死亡。
分离猴视网膜细胞培养两周，并进行 24 小时缺氧/24 小时复氧。TUNEL 染色和 Müller 和光感受器标志物的免疫染色用于检测哪种视网膜细胞类型受到损伤。
培养分离的猴视网膜细胞两周会导致 Müller 细胞增殖，并通过波形蛋白、恢复蛋白和视蛋白免疫细胞化学染色来维持一些杆状和锥状光感受器细胞。缺氧/复氧增加了 TUNEL 染色阳性的细胞数量。免疫印迹显示，缺氧细胞中钙蛋白酶特异性的 145 kDa α-血影蛋白裂解产物（SBDP）增加，但未检测到半胱天冬酶特异性的 120 kDa α-血影蛋白裂解产物。用 10 和 100 μM 钙蛋白酶抑制剂 SNJ-1945 处理的视网膜细胞中，TUNEL 染色和蛋白水解明显减少。半胱天冬酶抑制剂 z-VAD 不能抑制缺氧/复氧引起的细胞损伤。实际上，在缺氧/复氧期间，活性钙蛋白酶将完整的 pro-caspase-3 裂解为前 29 kDa caspase-3 和 24 kDa 无活性片段，而未检测到形成活性 caspase-3 异二聚体的 17 和 12 kDa 片段。SNJ-1945 抑制钙蛋白酶诱导的半胱天冬酶裂解。
在培养的猴视网膜细胞中，参与缺氧损伤的是钙蛋白酶，而不是半胱天冬酶-3。
