阻断 Wnt/β-连环蛋白信号通路的人源化抗体的抗血管生成作用。
Anti-angiogenic effect of a humanized antibody blocking the Wnt/β-catenin signaling pathway.
机构信息
Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Charlesson LLC, Oklahoma, OK 73104, USA.
出版信息
Microvasc Res. 2018 Sep;119:29-37. doi: 10.1016/j.mvr.2018.03.011. Epub 2018 Apr 7.
PURPOSE
Our previous study demonstrated that Mab2F1, a murine monoclonal antibody blocking the Wnt/β-catenin signaling pathway, has beneficial effects on experimental diabetic retinopathy and choroidal neovascularization (NV). The aforementioned antibody has been humanized. This study evaluated effects of the humanized antibody, H1L1, on NV.
METHODS
H1L1 was evaluated in the alkali burn-induced corneal NV rat model. Rats with corneal NV were injected subconjunctivally with Mab2F1 or H1L1 using non-specific mouse or human IgG as controls. Corneal NV and opacity were evaluated using corneal NV area and inflammatory index. Expression of angiogenic and inflammatory factors and components of the Wnt/β-catenin pathway in both the corneas of the animal model and human corneal epithelial (HCE) cells exposed to Wnt3a conditioned medium (WCM) were determined by Western blotting and a luciferase-based promoter assay. Cytotoxicities of these antibodies were evaluated by MTT assay.
RESULTS
H1L1 reduced the area of corneal NV and opacity, similar to Mab2F1. Both Mab2F1 and H1L1 down-regulated the overexpression of angiogenic and inflammatory factors including VEGF, TNF-α and ICAM-1, and blocked the aberrant activation of the Wnt/β-catenin pathway as shown by down-regulation of phosphorylated LRP6, total LRP6 and non-phosphorylated β-catenin in the cornea of the NV model and cultured HCE cells exposed to WCM. Both antibodies also inhibited the transcriptional activity of β-catenin induced by WCM in HCE cells. No toxic effects of the antibodies were observed in cultured HCE cells.
CONCLUSIONS
H1L1 exhibits anti-angiogenic activities through blocking the Wnt/β-catenin pathway.
目的
我们之前的研究表明，Mab2F1，一种阻断 Wnt/β-catenin 信号通路的鼠单克隆抗体，对实验性糖尿病性视网膜病变和脉络膜新生血管化（NV）有有益作用。上述抗体已人源化。本研究评估了人源化抗体 H1L1 对 NV 的作用。
方法
在碱性烧伤诱导的角膜 NV 大鼠模型中评估 H1L1。用 Mab2F1 或 H1L1 对角膜 NV 大鼠进行结膜下注射，以非特异性鼠或人 IgG 作为对照。用角膜 NV 面积和炎症指数评估角膜 NV 及混浊度。通过 Western blot 和基于荧光素酶的启动子测定法，测定动物模型和暴露于 Wnt3a 条件培养基（WCM）的人角膜上皮（HCE）细胞中血管生成和炎症因子的表达以及 Wnt/β-catenin 通路的组成部分。通过 MTT 测定法评估这些抗体的细胞毒性。
结果
H1L1 减少了角膜 NV 和混浊的面积，与 Mab2F1 相似。Mab2F1 和 H1L1 均下调了血管生成和炎症因子的过度表达，包括 VEGF、TNF-α 和 ICAM-1，并阻断了 Wnt/β-catenin 通路的异常激活，如 NV 模型角膜和暴露于 WCM 的培养 HCE 细胞中磷酸化 LRP6、总 LRP6 和非磷酸化 β-catenin 的下调所示。两种抗体还抑制了 WCM 诱导的 HCE 细胞中β-catenin 的转录活性。在培养的 HCE 细胞中未观察到抗体的毒性作用。
结论
H1L1 通过阻断 Wnt/β-catenin 通路发挥抗血管生成作用。
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