Department of Optometry and Vision, Faculty of Optics and Optometry, University Complutense of Madrid, C/Arcos de Jalon 118, 28037, Madrid, Spain.
Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), C/ de Melchor Fernández Almagro, 3, 28029, Madrid, Spain.
Exp Eye Res. 2022 Jun;219:109036. doi: 10.1016/j.exer.2022.109036. Epub 2022 Mar 31.
Given the implications of the problem of neovascularization on ocular health, as well as the growth in the number of cases, the purpose of the present study has been testing the efficacy of siRNAs (small interfering RNA) designed to silence Hypoxia Inducible Factor -1α (HIF-1α) and to demonstrate that their use stops neovascularization in a model of corneal burn. Corneal wounds in the limbic zone were made in the eyes of New Zealand white rabbits. Topical applications of siRNAs were done the next day to the wound for four consecutive days and eyes were examined with a slit lamp. Evaluation of neovascularization progress was done by analyzing images by ImageJTM and to determine the neovascular area in Matlab ® was used. At the same time, a rabbit corneal cell line was used for in vitro study of hypoxia exposure and Western blot analysis of the cell's extracts were done. Under normal cell culture oxygenation, the expression of HIF-1α was lower than that observed under hypoxic conditions. After 2 h of hypoxia, there was a significant increase in the HIF-1α expression, effect that was maintained up to 6 h. The increased in HIF-1α was mimicked by a cell permeable prolyl-4-hydroxylase inhibitor. Cobalt chloride showed no capacity to increase HIF-1α in vitro. The effect of three different siRNA on HIF-1α was tested after 4 h of hypoxia. siRNA#1 was able to silence 80% of HIF-1α expression, siRNA#2 and siRNA#3 reduce the expression in 45% and 40% respectively. In addition, the three siRNA were tested in a corneal model of neovascularization. scrambledsiRNA#2 was the most effective inhibitor of blood vessel production, followed by siRNA#3 and siRNA#1. Compared to the scrambled siRNA (100% of blood vessel generation), siRNA#2 blocked the presence of blood vessels by 83 ± 2%, siRNA#3 inhibited 45 ± 7% and siRNA#1 only inhibited 18 ± 5%. The necessary time to observe the 50% of effect showed values of NV50 of 10.2 ± 2.4 days for the scrambled siRNA, 9.1 ± 1.4 for siRNA#1, 6.5 ± 1.85 for siRNA#2 and 4.8 ± 1.8 days for siRNA#3. In conclusion, the topical application of siRNA towards HIF-1α seems to be an effective and reliable method to stop neovascularization.
鉴于新生血管化问题对眼部健康的影响，以及病例数量的增加，本研究的目的是测试专门设计用于沉默缺氧诱导因子-1α（HIF-1α）的 siRNA 的功效，并证明它们的使用可以阻止角膜烧伤模型中的新生血管形成。在新西兰白兔的眼limbic 区域制造角膜伤口。第二天，将 siRNA 局部应用于伤口连续 4 天，并用裂隙灯检查眼睛。通过 ImageJTM 分析图像来评估新生血管化进展，并在 Matlab ® 中确定新生血管区域。同时，使用兔角膜细胞系进行缺氧暴露的体外研究，并对细胞提取物进行 Western blot 分析。在正常细胞培养氧合下，HIF-1α 的表达低于缺氧条件下观察到的表达。缺氧 2 小时后，HIF-1α 的表达显著增加，该效应一直持续到 6 小时。细胞渗透性脯氨酰-4-羟化酶抑制剂可模拟 HIF-1α 的增加。氯化钴在体外没有增加 HIF-1α 的能力。在缺氧 4 小时后测试了三种不同的 siRNA 对 HIF-1α 的作用。siRNA#1 能够沉默 80%的 HIF-1α 表达，siRNA#2 和 siRNA#3 分别降低 45%和 40%的表达。此外，还在角膜新生血管化模型中测试了这三种 siRNA。 scrambledsiRNA#2 是血管生成最有效的抑制剂，其次是 siRNA#3 和 siRNA#1。与 scrambled siRNA（100%血管生成）相比，siRNA#2 通过 83±2%抑制血管生成，siRNA#3 通过 45±7%抑制血管生成，siRNA#1 仅通过 18±5%抑制血管生成。观察到 50%效果所需的时间显示 scrambled siRNA 的 NV50 值为 10.2±2.4 天，siRNA#1 为 9.1±1.4 天，siRNA#2 为 6.5±1.85 天，siRNA#3 为 4.8±1.8 天。总之，局部应用针对 HIF-1α 的 siRNA 似乎是一种有效且可靠的方法，可以阻止新生血管形成。
