Corneal neovascularization (CoNV) can cause abnormal blood vessels to grow in the transparent cornea, leading to various sight-threatening eye diseases. MicroRNAs are known to play essential roles in the regulation of numerous biological functions. We try to clarify the role of a specific microRNA, miR‑497, which has been shown to regulate the growth of tumor cells and angiogenesis on the basis of available data. However, the association between miR-497 and vascularized cornea remains unclear. Therefore, it is urgently needed to understand the molecular mechanism of miR497 in the progress of corneal neovascularization. Animal model of CoNV was established in wildtype (WT) C57BL/6 mice, CRISPR/Cas9 mediated miR-497 knockout (KO) and overexpressed (TG) C57BL/6 mice. MiR-497, expressed in corneas, was actively involved in alkali burn-induced corneal neovascularization via targeting STAT3 and negatively regulating its expression, attenuating macrophage infiltration and M2 polarization. Knockdown of miR-497 enhanced the formation of corneal angiogenesis through targeting STAT3 and facilitating its expression, promoting recruitment of macrophages, while overexpression of miR-497 restrained blood vessel sprouting via regulating downstream STAT3 and VEGFA expression, reducing macrophage activation and inhibiting M2 polarization. Moreover, miR-497 knockout-mediated damage effect can be rescued through the inhibition of STAT3 signaling. Mechanically, miR-497 might serve as a potential strategy for pathological corneal neovascularization via macrophage through the IL-6/STAT3/VEGFA signaling pathway.

Background: Corneal neovascularization (CNV) is a sight-threatening condition that necessitates epigenetic control. The role of lysine-specific demethylase 1 (LSD1) in CNV remains unclear, despite its established significance in tumor angiogenesis regulation.Methods: An alkali burn-induced CNV mouse model was used in vivo. The effects of LSD1 inhibitor tranylcypromine hydrochloride (TCP) were examined through slit lamp, histological staining, and immunofluorescence. The expression of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed in corneal tissues. Oxidative stress and ferrous ion expression during CNV were determined using 4-HNE, GPX4, and FerroOrange staining. In vitro, a hypoxia-reoxygenation (H/R) model was established using human umbilical vein endothelial cells (HUVECs) to study LSD1 or hypoxia-inducible factor (HIF-1α) knockdown and lentiviral overexpression of HIF-1α. The effects on HUVECs migration, invasion, and angiogenesis were evaluated through cell scratching assay, transwell migration assay and tube formation assay. The role of ferroptosis was investigated using ROS staining, FerroOrange staining, and key ferroptosis proteins. Further, The JAK2/STAT3 pathway’s involvement in CNV regulation was explored through in vivo experiments with subconjunctival injection of AG490.Results: The results showed a substantial correlation between corneal damage and LSD1 levels. In addition, HIF-1α expression was also elevated after alkali burns, and subconjunctival injection of TCP reduced corneal inflammation and neovascularization. Corneal alkali burns increased ROS levels and reduced antioxidative stress indicators, accompanied by elevated ferrous ion levels, which were reversed by TCP injection. In vitro, TCP or siRNAs inhibited H/R-induced ferroptosis and angiogenesis in HUVECs by affecting specific protein expressions and MDA, SOD, and GSH levels. HIF-1α levels, associated with ROS production, ferroptosis, and angiogenesis, increased during H/R, but were reversed by TCP or siRNA administration. HIF-1α overexpression counteracted the effects of LSD1 inhibition. Additionally, AG490 injection effectively reduced HIF-1α and VEGFA expression in the CNV model.Discussion: These findings suggest that LSD1 inhibition via the HIF-1α-driven pathway prevents angiogenesis, oxidative stress, and ferroptosis in corneal alkali burn-induced CNV, highlighting LSD1 as a potential therapeutic target.