Tawfik Amany, Markand Shanu, Al-Shabrawey Mohamed, Mayo Jamie N, Reynolds Jason, Bearden Shawn E, Ganapathy Vadivel, Smith Sylvia B
Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, Georgia; James and Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia.
James and Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia; Department of Oral Biology and Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, Georgia; Department of Ophthalmology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia.
Am J Pathol. 2014 Sep;184(9):2573-85. doi: 10.1016/j.ajpath.2014.05.018. Epub 2014 Jul 10.
Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-β-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.
轻度至中度高同型半胱氨酸血症在人类中普遍存在，并与神经血管疾病有关，最近还与某些视网膜疾病有关。在此，我们使用缺乏编码胱硫醚-β-合酶（Cbs(+/-)）的Cbs基因的高同型半胱氨酸血症小鼠来评估视网膜血管完整性。对Cbs(+/+)（野生型）和Cbs(+/-)（杂合子）小鼠（16至52周龄）进行荧光素血管造影和光学相干断层扫描，以评估体内血管系统。收获用于冷冻切片或平铺标本制备的视网膜，进行免疫荧光显微镜检查，以检测血管（异凝集素-B4）、血管生成[抗血管内皮生长因子（VEGF）和抗CD105]、胶质增生[抗胶质纤维酸性蛋白（GFAP）]、周细胞（抗神经/胶质抗原2）、血视网膜屏障[抗闭合蛋白1（ZO-1）和抗闭合蛋白]以及缺氧[抗盐酸匹莫硝唑（Hypoxyprobe-1）]。通过免疫印迹法测定VEGF、GFAP、ZO-1和闭合蛋白的水平。这些分析结果显示年轻小鼠存在轻度血管表型，且随年龄增长而进展。荧光素血管造影显示Cbs(+/-)小鼠出现进行性新生血管形成和血管渗漏；光学相干断层扫描证实到1岁时玻璃体中有新血管。免疫荧光显微镜检查显示血管模式与缺血一致，包括老年小鼠中央无毛细血管区和周边有毛细血管丛的新血管。这与Cbs(+/-)视网膜中VEGF、CD105和GFAP增加以及ZO-1/闭合蛋白水平降低有关。在一些Cbs(+/-)小鼠视网膜中观察到视网膜静脉阻塞。我们得出结论，Cbs(+/-)小鼠中同型半胱氨酸轻度至中度升高伴随着视网膜血管系统的进行性改变，其特征为缺血、新生血管形成、血视网膜屏障功能不全和血管阻塞。
