Department of Ophthalmology, University Hospital, LMU Munich, Munich, Germany.
Department of Ophthalmology, University Hospital Ulm, Ulm, Germany.
PLoS One. 2022 Mar 23;17(3):e0265805. doi: 10.1371/journal.pone.0265805. eCollection 2022.
Galectin-1 and -3 are β-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro.
To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for β-catenin, galectin-1 and -3 were performed and β-catenin expression was investigated by western blot analysis.
Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/β-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively.
Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins.
半乳糖凝集素-1 和 -3 是具有不同促血管生成和凋亡作用的β-半乳糖苷结合凝集素。由于在视网膜色素上皮细胞中，高浓度的人重组半乳糖凝集素（hr-GAL）1 和 3 可抑制细胞黏附、迁移和增殖，因此我们研究了 hr-GAL1 和 3 是否对体外培养的人视网膜微血管内皮细胞（HRMEC）具有同源作用。
为了研究半乳糖凝集素-1 和 -3 对 HRMEC 的影响，通过 BrdU-ELISA、组蛋白-DNA 复合物 ELISA、死活染色和 WST-1 测定分别分析了在 30、60 和 120μg/ml hr-GAL1 或 3 孵育后 HRMEC 的增殖、凋亡和活力。此外，还在半乳糖处理的 HRMEC 上进行了细胞黏附和管形成测定。通过划痕迁移测定和延时显微镜观察研究了迁移。此外，还对 HRMEC 进行了β-连环蛋白、半乳糖凝集素-1 和 -3 的免疫组织化学染色，并通过 Western blot 分析研究了β-连环蛋白的表达。
与未处理对照组相比，hr-GAL1 或 3 孵育导致 HRMEC 的增殖、迁移、黏附和管形成减少。未检测到 hr-GAL1 和 3 对 HRMEC 的毒性作用。有趣的是，在用 hr-GAL1 或 3 处理 HRMEC 后，观察到促血管生成的 Wnt/β-连环蛋白信号通路被激活。然而，孵育 HRMEC 与 hr-GAL1 或 3 后，细胞内的半乳糖凝集素-1 和 -3 分别被排出细胞外。
外源性添加的 hr-GAL1 或 3 可抑制体外 HRMEC 的血管生成特性，这种作用可能是通过细胞内内源性半乳糖凝集素的丢失介导的。
