Yu Wenzhen, Bai Yujing, Han Na, Wang Fei, Zhao Min, Huang Lvzhen, Li Xiaoxin
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China.
Mol Vis. 2013 Jun 27;19:1397-405. Print 2013.
Pathological retinal angiogenesis is a major cause of vision loss. Semaphorin 3A (Sema3A), a chemorepellent guidance protein, plays crucial roles in neural and vascular patterning. To identify its role in retinal neovascularization, we investigated its antiangiogenic effects.
Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study, and an oxygen-induced retinopathy (OIR) mouse model was used for the in vivo study. The HUVECs were incubated with Sema3A, and cell proliferation, migration, apoptosis, cell cycle, tube formation, and c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38 MAPK) signaling pathways were measured using Cell Counting Kit-8, Transwell, flow cytometry, Matrigel assays, and western blot. C57BL/6J mouse pups were exposed to 75% oxygen for 5 days and then brought to room air and injected with Sema3A intravitreously. At postnatal day 18, the retinal nonperfused areas were measured. The in vitro and in vivo vascular endothelial growth factor-165 (VEGF165) secretion was measured using enzyme-linked immunosorbent assay.
Sema3A not only inhibited VEGF165-induced proliferation, but also induced cell cycle arrest in a dose-dependent manner. Furthermore, Sema3A inhibited migration and tube formation, both in general and in VEGF165-containing culture medium. Using an enzyme-linked immunosorbent assay, we showed that Sema3A did not affect VEGF165 secretion, but it did impede VEGF165 function. Additionally, Sema3A significantly inhibited the phosphorylation of the JNK and p38MAPK signaling pathways. When administered intravitreously, Sema3A reduced the pathological vascular changes seen in the retinal neovascularization OIR model.
These results suggest that the administration of Sema3A could be a useful therapeutic strategy for preventing hypoxia/ischemic-induced retinal neovascularization.
病理性视网膜血管生成是视力丧失的主要原因。信号素3A（Sema3A）是一种化学排斥导向蛋白，在神经和血管模式形成中起关键作用。为确定其在视网膜新生血管形成中的作用，我们研究了其抗血管生成作用。
体外研究采用人脐静脉内皮细胞（HUVECs），体内研究采用氧诱导视网膜病变（OIR）小鼠模型。将HUVECs与Sema3A共同孵育，使用细胞计数试剂盒-8、Transwell、流式细胞术、基质胶实验和蛋白质印迹法检测细胞增殖、迁移、凋亡、细胞周期、管腔形成以及c-Jun氨基末端激酶（JNK）和p38丝裂原活化蛋白激酶（p38 MAPK）信号通路。将C57BL/6J幼鼠置于75%氧气环境中5天，然后置于室温空气中，并经玻璃体腔内注射Sema3A。在出生后第18天，测量视网膜无灌注区域。使用酶联免疫吸附测定法检测体外和体内血管内皮生长因子-165（VEGF165）的分泌。
Sema3A不仅抑制VEGF165诱导的增殖，还以剂量依赖的方式诱导细胞周期停滞。此外，Sema3A抑制迁移和管腔形成，无论是在普通培养基还是含VEGF165的培养基中。通过酶联免疫吸附测定法，我们发现Sema3A不影响VEGF165的分泌，但确实阻碍了VEGF165的功能。此外，Sema3A显著抑制JNK和p38MAPK信号通路的磷酸化。经玻璃体腔内给药时，Sema3A减少了视网膜新生血管形成OIR模型中出现的病理性血管变化。
这些结果表明，给予Sema3A可能是预防缺氧/缺血诱导的视网膜新生血管形成的一种有效治疗策略。
