Fangfang Qiu1, Younghwa Shin2, Danyang Chen3, Rui Cheng4, Qian Chen5, Kelu Zhou6, James W Larrick7, Andrew R Mendelson7, Jian-Xing Ma8. 1. Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. Electronic address: Fangfang-Qiu@ouhsc.edu. 2. Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. Electronic address: YoungHwa-Shin@ouhsc.edu. 3. Charlesson LLC, Oklahoma, OK 73104, USA. Electronic address: dchen@charlessonllc.com. 4. Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. Electronic address: rui-cheng@ouhsc.edu. 5. Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. 6. Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. Electronic address: Kelu-Zhou@ouhsc.edu. 7. WntGen, LLC, Sunnyvale, CA 94089, USA. 8. Department of Physiology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA. Electronic address: Jian-Xing-Ma@ouhsc.edu.
Abstract
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.
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 NVrat model. Rats with corneal NV were injected subconjunctivally with Mab2F1 or H1L1 using non-specific mouse or humanIgG 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 humancorneal 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.