State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China.
Diabetes Obes Metab. 2013 Mar;15(3):224-33. doi: 10.1111/dom.12008. Epub 2012 Sep 30.
Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are upregulated in many ocular neovascular diseases such as diabetic retinopathy (DR). KH902 is a recombinant fusion protein with its binding ligand taken from the domains of VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2) and can bind all VEGF-A isoforms and PlGF. The aim of this study was to investigate the underlying mechanisms of anti-angiogenic effects of KH902.
The toxic effect of KH902 on cultured human retinal endothelial cells (HRECs) was measured by Annexin V/PI staining and MTT assay. The concentrations of secreted VEGF and PlGF were measured by ELISA. The migration of HRECs was assessed by scratch wound and transwell assay. The sprouting of HRECs was determined by tube formation assay. The protein levels of Src, p-Src, PI3K, Akt1, p-Akt1, Erk1/2 and p-Erk1/2 were measured by Western blot.
KH902 at the concentrations from 100 ng/ml to 100 µg/ml had no cytotoxicity to cultured HRECs. KH902 bound not only VEGF165, but also PlGF that were secreted by HRECs under high glucose condition. A 500 ng/ml of KH902 significantly suppressed high glucose-induced migration and sprouting of HRECs through downregulating the expression of PI3K and inhibiting the activation of Src, Akt1 and Erk1/2.
Our study indicates that KH902 suppresses high glucose-induced migration and sprouting of HRECs through not only binding VEGF, but also PlGF to inhibit the activation of Src-Akt1-Erk1/2 pathway. KH902 is a drug that potentially inhibits angiogenic pathways involving in DR or other ocular neovascular diseases.
血管内皮生长因子（VEGF）和胎盘生长因子（PlGF）在许多眼部新生血管疾病中上调，如糖尿病视网膜病变（DR）。KH902 是一种重组融合蛋白，其结合配体取自 VEGF 受体-1（VEGFR-1）和 VEGF 受体-2（VEGFR-2）的结构域，可与所有 VEGF-A 同工型和 PlGF 结合。本研究旨在探讨 KH902 抗血管生成作用的潜在机制。
通过 Annexin V/PI 染色和 MTT 测定法测量 KH902 对培养的人视网膜内皮细胞（HRECs）的毒性作用。通过 ELISA 测量分泌的 VEGF 和 PlGF 的浓度。通过划痕伤口和 Transwell 测定法评估 HRECs 的迁移。通过管形成测定法确定 HRECs 的发芽。通过 Western blot 测定法测量 Src、p-Src、PI3K、Akt1、p-Akt1、Erk1/2 和 p-Erk1/2 的蛋白水平。
浓度为 100ng/ml 至 100μg/ml 的 KH902 对培养的 HRECs 无细胞毒性。KH902 不仅结合 VEGF165，还结合高糖条件下 HRECs 分泌的 PlGF。500ng/ml 的 KH902 通过下调 PI3K 的表达并抑制 Src、Akt1 和 Erk1/2 的激活，显著抑制高糖诱导的 HRECs 迁移和发芽。
我们的研究表明，KH902 通过结合 VEGF 和 PlGF 抑制 Src-Akt1-Erk1/2 通路的激活，抑制高糖诱导的 HRECs 迁移和发芽。KH902 是一种潜在的抑制涉及 DR 或其他眼部新生血管疾病的血管生成途径的药物。
