USP39 promotes retinal pathological angiogenesis in retinopathy of prematurity by stabilizing SIRT2 expression through deubiquitination.

Abstract

Background: Retinopathy of prematurity (ROP) is a major cause of childhood blindness worldwide, highlighted by retinal neovascularization. Ubiquitin is present throughout the retina. The deubiquitinating enzyme ubiquitin-specific protease 39 (USP39) has been reported to be involved in angiogenesis. Here, this study aimed to investigate the effects of USP39 on ROP and its associated mechanism.

Methods: Hypoxia-induced human retinal microvascular endothelial cells (hRMECs) were adopted for functional analyses. Detection of mRNA and protein was conducted using quantitative real-time PCR and western blotting. Cell migration, invasion and angiogenesis were evaluated using transwell and tube formation assays. Protein interaction was determined by immunoprecipitation assay. Oxygen-induced retinopathy (OIR) mouse models were used for in vivo analysis.

Results: USP39 level was higher in hypoxia-induced hRMECs, functionally, USP39 silencing reversed hypoxia-induced migration, invasion and angiogenesis in hRMECs. In further mechanism analysis, we found that USP39 stabilized SIRT2 protein expression in hRMECs by inducing SIRT2 deubiquitination. Moreover, SIRT2 up-regulation abated hypoxia-evoked migration, invasion and angiogenesis in hRMECs. Besides that, the inhibitory effects of USP39 silencing on hypoxia-induced metastatic and angiogenic behaviors were abolished after SIRT2 overexpression. In addition, USP39 silencing blocked the activation of phosphoinositide 3-kinase (PI3K)/protein kinase B pathway (AKT) by regulating SIRT2. In vivo assay showed that levels of USP39, SIRT2, matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9 and Vascular endothelial growth factor A (VEGFA) were increased in the retinas of OIR mice, while intravitreal injection of USP39 short hairpin RNA (shRNA) could reduce their expression.

Conclusion: USP39 stabilized SIRT2 expression by deubiquitination and promoted hypoxia-induced metastatic and angiogenic behaviors of RMECs in vitro, as well as retinal angiogenesis in vivo.