Kim Jeong Hun, Kim Jin Hyoung, Kim Dong Hun, Park Woong-Yang, Kim Kyu-Won, Yu Young Suk
Fight against Angiogenesis-Related Blindness Laboratory, Department of Ophthalmology, Seoul National University College of Medicine & Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.
Korean J Ophthalmol. 2011 Jun;25(3):189-95. doi: 10.3341/kjo.2011.25.3.189. Epub 2011 May 24.
Cancer-associated retinopathy is a paraneoplastic retinal degeneration which may primarily result from auto-immune mediated apoptosis. It has been hypothesized that high titer of auto-antibodies are able to cross the blood-retinal barrier (BRB) and to enter retinal cells to activate apoptotic pathway which has been already well-established. However, it still remains to be elucidated whether auto-antibodies could cross BRB in the retina. Herein, we demonstrated that intravenously administrated anti-recoverin antibodies could not pass through BRB and not lead to retinal cell death.
Anti-recoverin antibody was intravenously injected to C57BL/6 mice, which were sacrificed 1 and 7 days to obtain eye. Vascular endothelial growth factor was intravitreally injected to induce BRB breakdown, which was confirmed by fluorescein angiography and western blotting for zonula occludens (ZO)-1, ZO-2 and occludin. To investigate the location of anti-recoverin antibody in the retina, immunofluorescein was performed. The retinal toxicity of intravenous anti-recoverin antibody was evaluated by histological examination and transferase-mediated dUTP nick-end labeling. Immunofluorescein staining for glial fibrillary acidic protein was done to address glial activation as well.
Intravenously administrated anti-recoverin antibodies were exclusively distributed on retinal vessels which were co-localized with CD31, and led to neither increase of glial fibrillary acidic protein expression, as an indicator of retinal stress, nor apoptotic retinal cell death. Moreover, even in the condition of vascular endothelial growth factor-induced BRB breakdown, anti-recoverin antibodies could not migrate across BRB and still remained on retinal vessels without retinal cytotoxicity.
Our results suggest that high titer of intravascular anti-recoverin antibodies could not penetrate into the retina by themselves, and BRB breakdown mediated by dysregulation of tight junction might not be sufficient to allow anti-recoverin antibodies to pass through BRB.
癌症相关性视网膜病变是一种副肿瘤性视网膜变性，其可能主要由自身免疫介导的细胞凋亡引起。据推测，高滴度的自身抗体能够穿过血视网膜屏障（BRB）并进入视网膜细胞，从而激活早已确立的凋亡途径。然而，自身抗体是否能够穿过视网膜中的血视网膜屏障仍有待阐明。在此，我们证明静脉注射抗恢复蛋白抗体不能穿过血视网膜屏障，也不会导致视网膜细胞死亡。
将抗恢复蛋白抗体静脉注射到C57BL/6小鼠体内，在1天和7天后处死小鼠以获取眼球。玻璃体内注射血管内皮生长因子以诱导血视网膜屏障破坏，通过荧光素血管造影以及对紧密连接蛋白（ZO）-1、ZO-2和闭合蛋白进行蛋白质印迹分析来证实。为了研究抗恢复蛋白抗体在视网膜中的定位，进行了免疫荧光检测。通过组织学检查和末端脱氧核苷酸转移酶介导的缺口末端标记法评估静脉注射抗恢复蛋白抗体的视网膜毒性。还进行了胶质纤维酸性蛋白的免疫荧光染色以研究神经胶质激活情况。
静脉注射的抗恢复蛋白抗体仅分布在与CD31共定位的视网膜血管上，既未导致作为视网膜应激指标的胶质纤维酸性蛋白表达增加，也未导致视网膜细胞凋亡死亡。此外，即使在血管内皮生长因子诱导血视网膜屏障破坏的情况下，抗恢复蛋白抗体也不能穿过血视网膜屏障，仍保留在视网膜血管上且无视网膜细胞毒性。
我们的结果表明，血管内高滴度的抗恢复蛋白抗体自身不能穿透进入视网膜，并且由紧密连接失调介导的血视网膜屏障破坏可能不足以使抗恢复蛋白抗体穿过血视网膜屏障。
