可溶性EphB4-HSA对CNV的抑制及机理研究

Choroidal neovascularization (CNV) is a leading cause of central vision loss complicated with age-related macular degeneration. Although intravitreal injection of anti-VEGF is widely used in the treatment of CNV, optimal treatment regimens for the disease are still under investigation. EphrinB2 and EphB4 can regulate angiogenesis, and interruption of signaling of EphB4/EphrinB2 has been demonstrated to inhibit angiogenesis. Our preliminary study showed that EphB4 and EphrinB2 were expressed in human choroidal vascular endothelial cells (CEC). EphB4 and EphrinB2 were highly expressed in surgically removed human CNV membrane, and double labeled with CD31 (vascular endothelial cell marker), suggesting that the involvement of EphB4 and EphrinB2 in the pathogenesis of CNV; furthermore, the expressions of EphB4 and EphrinB2 were stimulated by VEGF in CEC cells, implying that EphB4 and EphrinB2 are downstream genes of VEGF. Importantly, laser-induced rat CNV is significantly inhibited by intravitreal injection of soluble EphB4-HSA as demonstrated by FFA, CNV volume measurement and histological analysis. The expressions of phosphorylated VEGF-receptor2 and fibronectin in the CNV lesions were reduced compared with control rats demonstrated by Western blot and immunofluorescent staining. Our hypotheses is that laser can induce the expression of VEGF in the retina of rats, VEGF in turn stimulates the expression of EphB4 and EprinB2, and the interaction between EphB4 and EphrinB2 activates forward and reverse signaling and PI3K, MAPK pathway, which lead to the choroidal endothelial cell proliferation, migration, tube formation and the new vessels growth. Therefore, VEGF-EphB4/EphrinB2-PI3K-MAPK are essential in the development of CNV and suppression of VEGF-R2 phosphorylation and EphB4/EphrinB2 bidirectional signaling by application of soluble EphB4-HSA will inhibit CNV. In this study, the effects of soluble EphB4-HSA on angiogenesis will be evaluated in human CEC cells using cell and molecular biological methods in vitro and a laser induced rats CNV model in vivo.

脉络膜新生血管（choroidal neovascularization, CNV）是湿性年龄相关性黄斑变性的主要特征。VEGF是新生血管的主要诱导者，最近的研究表明EphB4/EphrinB2双向信号系统活化可激活PI3K及MAPK并促使CNV的发生。我们的预实验结果表明人CNV膜有EphB4与EphrinB2的高度表达，且VEGF能上调二者的表达，可溶性EphB4-HSA能抑制大鼠CNV及VEGF-R2的磷酸化。因此我们假设VEGF-EphB4/EphrinB2-PI3K/MAPK是形成CNV的关键环节，应用可溶性EphB4-HSA可抑制VEGF-R2的磷酸化、EphB4和EprinB2的上调及其所诱导的双向信号系统活化，从而达到抑制CNV产生的目的。本研究体外将用人CEC细胞，体内应用激光诱导的大鼠CNV模型来证实我们的假设，为CNV的药物治疗提供新的理论依据。

年龄相关性黄斑变性（age-related macular degeneration，AMD )， 尤其是AMD并发脉络膜新生血管（choroidal neovascularization，CNV）可严重损害视力，其发病机理仍然在研究中。虽然抗VEGF疗法已成为治疗CNV的主要方法，然而抗VEGF治疗的副作用也不断出现，究其原因，与EphB4的升高有关，所以本研究聚焦于可溶性EphB4对CNV 的抑制及机理研究。我们通过体内体外的研究获得了以下的结果：1. 玻璃体腔注射可溶性EphB4-HSA能显著抑制实验性脉络膜新生血管（通过CNV的体积的测定，视网膜荧光血管造影，大鼠眼球切片组织观察）我们发现这种抑制作用与可溶性EphB4-HSA对FN及VEGF受体的磷酸化的抑制密切相关，为sEphB4-HSA抑制新生血管提供了新的依据。2. 体外研究进一步阐明了EphB4-HSA抑制CNV的机制，这包括：A. EphB4-HSA能抑制VEGF对EphB4及EphrinB2的影响而抑制新生血管；B.用siRNA敲减脉络膜血管内皮细胞（CEC）EphrinB2可抑制VEGF诱导CEC细胞增殖、迁移及管腔形成；C. 敲减EphrinB2能抑制VEGF诱导的VEGFR2、PI3K、MAPK、EphB4的磷酸化及FN、EphB4、EphrinB2的表达。D. 用EphB4质粒、EphrinB2质粒转染CEC细胞可促进CEC迁移，而敲减EphrinB2可抑制VEGF诱导的CEC管腔形成；E. EphB4-Fc、EphrinB2-Fc 能诱导CEC细胞PI3K、MAPK活化，而抑制MAKP及PI3k活化可分别抑制CEC 细胞的迁移与管腔形成。F. sEphB4-HSA体外能抑制VEGF-R2的磷酸化。总之本研究证实了sEphB4-HSA能抑制实验性脉络膜新生血管，通过体外及体内的实验揭示了sEphB4-HSA抑制新生血管的机制，我们的研究结果为探讨CNV的发病机制提供了新的理论依据，并为CNV的治疗提供了新的途径。