Departments of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, USA.
Invest Ophthalmol Vis Sci. 2013 Feb 1;54(2):939-49. doi: 10.1167/iovs.12-10536.
Mice with moderate/severe hyperhomocysteinemia due to deficiency or absence of the cbs gene encoding cystathionine-beta-synthase (CBS) have marked retinal disruption, ganglion cell loss, optic nerve mitochondrial dysfunction, and ERG defects; those with mild hyperhomocysteinemia have delayed retinal morphological/functional phenotype. Excess homocysteine is a risk factor for cardiovascular diseases; however, it is not known whether excess homocysteine alters retinal vasculature.
Cbs(+/+), cbs(+/-), and cbs(-/-) mice (age ∼3 weeks) were subjected to angiography; retinas were harvested for cryosections, flat-mount preparations, or trypsin digestion and subjected to immunofluorescence microscopy to visualize vessels using isolectin-B4, to detect angiogenesis using anti-VEGF and anti-endoglin (anti-CD105) and activated glial cells (anti-glial fibrillary acidic protein [anti-GFAP]) and to investigate the blood-retinal barrier using the tight junction markers zonula occludens-1 (ZO-1) and occludin. Expression of vegf was determined by quantitative RT-PCR (qRT-PCR) and immunoblotting. Human retinal endothelial cells (HRECs) were treated with excess homocysteine to analyze permeability.
Angiography revealed vascular leakage in cbs(-/-) mice; immunohistochemical analysis demonstrated vascular patterns consistent with ischemia; isolectin-B4 labeling revealed a capillary-free zone centrally and new vessels with capillary tufts midperipherally. This was associated with increased vegf mRNA and protein, CD105, and GFAP in cbs(-/-) retinas concomitant with a marked decrease in ZO-1 and occludin. Homocysteine-treated HRECs showed increased permeability.
Severe elevation of homocysteine in cbs(-/-) mutant mice is accompanied by alterations in retinal vasculature (ischemia, neovascularization, and incompetent blood-retinal barrier). The marked disruption of retinal structure and decreased visual function reported in cbs(-/-) mice may reflect vasculopathy as well as neuropathy.
由于 CBS 基因（编码胱硫醚-β-合酶）缺乏或缺失导致的中重度高同型半胱氨酸血症的小鼠，其视网膜出现明显破坏、神经节细胞丢失、视神经线粒体功能障碍和 ERG 缺陷；轻度高同型半胱氨酸血症的小鼠则出现视网膜形态/功能表型延迟。高同型半胱氨酸是心血管疾病的一个危险因素；然而，高同型半胱氨酸是否改变视网膜血管尚不清楚。
对 Cbs(+/+)、cbs(+/-)和 cbs(-/-) 小鼠（年龄约 3 周）进行血管造影；取视网膜进行冰冻切片、平片制备或胰蛋白酶消化，并用异硫氰酸荧光素-B4 进行免疫荧光显微镜观察以显示血管，用抗血管内皮生长因子（抗-VEGF）和抗内皮糖蛋白（抗-CD105）和激活的神经胶质细胞（抗胶质纤维酸性蛋白 [抗-GFAP]）检测血管生成，并通过紧密连接标记物紧密连接蛋白-1（ZO-1）和闭锁蛋白检测血视网膜屏障。通过定量 RT-PCR（qRT-PCR）和免疫印迹法检测 vegf 的表达。用过量同型半胱氨酸处理人视网膜内皮细胞（HRECs）以分析通透性。
血管造影显示 cbs(-/-) 小鼠存在血管渗漏；免疫组织化学分析显示血管模式符合缺血；异硫氰酸荧光素-B4 标记显示中央无毛细血管区和周边中部有毛细血管丛的新血管。这与 cbs(-/-) 视网膜中 vegf mRNA 和蛋白、CD105 和 GFAP 的增加以及 ZO-1 和闭锁蛋白的显著减少有关。同型半胱氨酸处理的 HRECs 显示通透性增加。
cbs(-/-) 突变小鼠中同型半胱氨酸的严重升高伴随着视网膜血管的改变（缺血、新生血管形成和血视网膜屏障功能障碍）。cbs(-/-) 小鼠报告的视网膜结构明显破坏和视力下降可能反映了血管病变和神经病变。
