Chen Wei-Sheng, Cao Zhiyi, Leffler Hakon, Nilsson Ulf J, Panjwani Noorjahan
Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, United States.
New England Eye Center/Department of Ophthalmology, Tufts University, Boston, Massachusetts, United States.
Invest Ophthalmol Vis Sci. 2017 Jan 1;58(1):9-20. doi: 10.1167/iovs.16-20009.
Corneal neovascularization and scarring commonly lead to significant vision loss. This study was designed to determine whether a small-molecule inhibitor of galectin-3 can inhibit both corneal angiogenesis and fibrosis in experimental mouse models.
Animal models of silver nitrate cautery and alkaline burn were used to induce mouse corneal angiogenesis and fibrosis, respectively. Corneas were treated with the galectin-3 inhibitor, 33DFTG, or vehicle alone and were processed for whole-mount immunofluorescence staining and Western blot analysis to quantify the density of blood vessels and markers of fibrosis. In addition, human umbilical vein endothelial cells (HUVECs) and primary human corneal fibroblasts were used to analyze the role of galectin-3 in the process of angiogenesis and fibrosis in vitro.
Robust angiogenesis was observed in silver nitrate-cauterized corneas on day 5 post injury, and markedly increased corneal opacification was demonstrated in alkaline burn-injured corneas on days 7 and 14 post injury. Treatment with the inhibitor substantially reduced corneal angiogenesis and opacification with a concomitant decrease in α-smooth muscle actin (α-SMA) expression and distribution. In vitro studies revealed that 33DFTG inhibited VEGF-A-induced HUVEC migration and sprouting without cytotoxic effects. The addition of exogenous galectin-3 to corneal fibroblasts in culture induced the expression of fibrosis-related proteins, including α-SMA and connective tissue growth factor.
Our data provide proof of concept that targeting galectin-3 by the novel, small-molecule inhibitor, 33DFTG, ameliorates pathological corneal angiogenesis as well as fibrosis. These findings suggest a potential new therapeutic strategy for treating ocular disorders related to pathological angiogenesis and fibrosis.
角膜新生血管化和瘢痕形成通常会导致严重的视力丧失。本研究旨在确定半乳糖凝集素-3的小分子抑制剂是否能在实验小鼠模型中抑制角膜血管生成和纤维化。
分别使用硝酸银烧灼和碱烧伤动物模型诱导小鼠角膜血管生成和纤维化。角膜分别用半乳糖凝集素-3抑制剂33DFTG或单独的赋形剂处理，并进行全层免疫荧光染色和蛋白质印迹分析，以量化血管密度和纤维化标志物。此外，使用人脐静脉内皮细胞（HUVEC）和原代人角膜成纤维细胞在体外分析半乳糖凝集素-3在血管生成和纤维化过程中的作用。
在损伤后第5天，硝酸银烧灼的角膜中观察到强烈的血管生成，在损伤后第7天和第14天，碱烧伤的角膜中角膜混浊明显增加。用抑制剂治疗可显著减少角膜血管生成和混浊，同时α-平滑肌肌动蛋白（α-SMA）的表达和分布也随之减少。体外研究表明，33DFTG可抑制VEGF-A诱导的HUVEC迁移和芽生，且无细胞毒性作用。向培养的角膜成纤维细胞中添加外源性半乳糖凝集素-3可诱导纤维化相关蛋白的表达，包括α-SMA和结缔组织生长因子。
我们的数据提供了概念验证，即新型小分子抑制剂33DFTG靶向半乳糖凝集素-3可改善病理性角膜血管生成和纤维化。这些发现提示了一种潜在的新治疗策略，用于治疗与病理性血管生成和纤维化相关的眼部疾病。
