Wang Haibo, Han Xiaokun, Kunz Eric, Hartnett M Elizabeth
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States.
The John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States 2Department of Ophthalmology, The Fourth Affiliated Hospital of China Medical University, Shenyang, China.
Invest Ophthalmol Vis Sci. 2016 Oct 1;57(13):5525-5534. doi: 10.1167/iovs.16-19691.
This study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV).
Measurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA.
Thy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin-labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC-induced Rac1 and Src activation.
Thy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted.
本研究探讨以下假说，即与年龄相关的应激反应上调脉络膜内皮细胞（CEC）中的Thy-1，并促进CEC的活化和迁移，而这些过程在脉络膜新生血管形成（CNV）中至关重要。
采用蛋白质免疫印迹法检测CEC中Thy-1蛋白，采用实时定量聚合酶链反应检测用血管内皮生长因子（VEGF）、CC趋化因子配体11（CCL11）或磷酸盐缓冲液（PBS）处理的CEC中Thy-1信使核糖核酸（mRNA），或检测来自年轻或老年供体、或接受激光照射或未接受激光照射的小鼠的视网膜色素上皮（RPE）/脉络膜中的Thy-1 mRNA。比较年轻与老年人类供体眼、或有或无新生血管性年龄相关性黄斑变性（AMD）的眼、或激光照射与未激光照射小鼠的眼的眼组织切片中免疫标记的Thy-1。用Thy-1或对照小干扰RNA（siRNA）转染脉络膜内皮细胞，或用Thy-1阻断肽或对照预处理后，用VEGF或7-酮胆固醇（7-KC）刺激。检测脉络膜内皮细胞迁移、增殖、细胞骨架应力纤维、Rac1活化以及磷酸化的VEGF受体2（VEGFR2）、整合素β3和Src。采用方差分析进行统计学分析。
Thy-1在视网膜神经节细胞以及血管内皮钙黏蛋白标记的脉络膜中表达，并定位于人或小鼠激光诱导的CNV病变处。在用VEGF或CCL11处理的CEC中，以及在老年供体眼与年轻供体眼、或激光照射小鼠与未激光照射对照小鼠的RPE/脉络膜中，Thy-1蛋白和mRNA显著增加。在CEC中敲低或抑制Thy-1可显著降低VEGF诱导的CEC迁移和增殖、应力纤维形成以及VEGFR2、Src、整合素β3和Rac-1活化，以及7-KC诱导的Rac1和Src活化。
CEC中的Thy-1调节VEGF诱导的CEC活化和迁移，并将细胞外7-KC与细胞内信号传导联系起来。有必要开展进一步研究以阐明Thy-1在新生血管性AMD中的作用机制。
