■角膜新生血管形成(CNV)是一种威胁视力的疾病,需要进行表观遗传控制。赖氨酸特异性脱甲基酶1(LSD1)在CNV中的作用尚不清楚,尽管它在肿瘤血管生成调节中具有重要意义。
■在体内使用碱烧伤诱导的CNV小鼠模型。通过裂隙灯检查LSD1抑制剂tranylcypromine盐酸盐(TCP)的作用,组织学染色,和免疫荧光。丙二醛(MDA)的表达,超氧化物歧化酶(SOD),评估角膜组织中的谷胱甘肽(GSH)水平。使用4-HNE测定CNV期间的氧化应激和亚铁离子表达,GPX4和铁橙染色。体外,使用人脐静脉内皮细胞(HUVECs)建立缺氧-复氧(H/R)模型,以研究LSD1或缺氧诱导因子(HIF-1α)敲低和HIF-1α的慢病毒过表达。对HUVEC迁移的影响,入侵,通过细胞划痕试验评估血管生成,transwell迁移测定和试管形成测定。使用ROS染色研究了铁死亡的作用,铁橙染色,和关键的铁凋亡蛋白。Further,通过结膜下注射AG490的体内实验探索JAK2/STAT3通路参与CNV调节。
■结果显示角膜损伤与LSD1水平之间存在实质性相关性。此外,碱烧伤后HIF-1α表达也升高,结膜下注射TCP可减少角膜炎症和新生血管形成。角膜碱烧伤增加了ROS水平,降低了抗氧化应激指标,伴随着亚铁离子水平升高,通过TCP注射逆转。体外,TCP或siRNA通过影响特定蛋白表达和MDA抑制H/R诱导的HUVECs铁凋亡和血管生成,SOD,和GSH水平。HIF-1α水平,与ROS生产相关,铁性凋亡,和血管生成,在H/R期间增加,但通过TCP或siRNA给药逆转。HIF-1α过表达抵消了LSD1抑制作用。此外,AG490注射可有效降低CNV模型中HIF-1α和VEGFA的表达。
■这些研究结果表明,通过HIF-1α驱动途径抑制LSD1可防止血管生成,氧化应激,角膜碱烧伤诱导的CNV的铁性凋亡,强调LSD1是一个潜在的治疗靶点。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.