Therapeutic potential of topical administration of siRNAs against HIF-1α for corneal neovascularization. (June 2022)

• Record Type: Journal Article
• Authors: Peral, Assumpta; Mateo, Jesús; Domínguez-Godínez, Carmen O.; Carracedo, Gonzalo; Gómez, Jose Antonio; Crooke, Almudena; Pintor, Jesús
• Is Part Of: Experimental eye research. Volume 219(2022)
• Publication Date: 2022-06
• Subjects: Blood vessel -- Cornea -- HIF-1α -- Hypoxia -- Neovascularization -- siRNA; Ophthalmology -- Periodicals; Eye -- Periodicals; Œil -- Périodiques; Ophthalmology; Periodicals; Electronic journals; 612.8405
• DOI: 10.1016/j.exer.2022.109036
• Languages: English
• ISSNs: 0014-4835

Abstract

Abstract: 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. Highlights: Statens Seruminstitut Rabbit Cornea cells were incubated with siRNAs-small interfering RNA, to study gene HIF-1α expression. siRNA#1 silenced 87% of HIF-1α expression, siRNA#2 and siRNA#3 silenced 55% and 60% respectively. Control experiments with a scramble siRNA, showed a wide extension vessel. Not all siRNAs depicted the same silencing effect. siRNA#2 was the most effective inhibitor followed by siRNA#3 and siRNA#1. The topical application of siRNA against gene HIF-1α seems to be an effective and reliable method to halt neovascularization.