Abstract
Pathological retinal neovascularization (NV) contributes to vision loss in diabetic retinopathy (DR) and retinopathy of prematurity, which are the leading causes of blindness in working-age adults and children, respectively. Retinal hypoxia is a key driver of pathological neovascularization that results in uncontrolled vessel sprouting and the formation of immature and leaky blood vessels. Anti-vascular endothelial growth factor and laser therapies are the standard of care to mitigate vision loss, but their limited effectiveness underlies the need to identify new therapeutic targets. The goal of the current study was to define the role of the enzyme histone deacetylase 3 (HDAC3) in the pathogenesis of experimental NV. Pathological neovascularization was induced by subjecting C57BL/6 J mouse pups to oxygen-induced retinopathy (OIR). Retinal tissues were analyzed by Western blotting and immunofluorescent labeling was conducted on mouse retinal flatmounts and human retinal sections from patients with DR. In vitro studies used cultured bovine retinal endothelial cells (REC) subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (R). Cells were treated with the HDAC3 inhibitor, RGFP966, the mitochondrial fission inhibitor, Mdivi-1 or DMSO as a control. Endpoints included assays of cell migration, untargeted proteomic analysis, Seahorse analysis of glycolysis, and mitochondrial morphology using MitoTracker dye. Using the methods described above, we found that HDAC3 expression was increased in retinal vessels of OIR mice and human DR retinal samples. HDAC3 also was upregulated in REC following OGD/R. Treatment with RGFP966 (2, 8 μM) attenuated OGD/R-induced angiogenesis as determined by cell migration. In confirmation, siRNA-mediated HDAC3 knockdown attenuated REC migration whereas HDAC3 overexpression increased it. OGD/R induced a strong upregulation of the rate-limiting glycolysis enzyme, hexokinase 2 (HK2), as determined by untargeted proteomic analysis, which correlated with increased glycolysis and mitochondrial fission. Treatment with RGFP966 or Mdivi-1 (5 μM), blocked HK2 upregulation, suppressed glycolytic flux, and reduced mitochondrial fission. Our findings indicate that HDAC3 plays a crucial role in pathological neovascularization by driving endothelial cell metabolic reprogramming toward glycolysis via the induction of mitochondrial fission and HK2 signaling. Targeting HDAC3 or its downstream metabolic pathways may offer a promising therapeutic strategy for mitigating pathological NV. Retinal endothelial cells (REC) respond to oxygen glucose deprivation/reperfusion (OGD/R) injury by increasing the expression of HDAC3 which, in turn, upregulates hexokinase 2 (HK2) and mitochondrial fission. These then go on to metabolically reprogram the REC toward a more glycolytic phenotype and promote the process of pathological angiogenesis of the retina. Inhibiting HDAC3 by RGFP966 protects against the OGD/R-induced metabolic changes. In a similar fashion, the inhibition of mitochondrial fission with Mdivi-1 mitigates the glycolytic shift and HK2 expression. These findings suggest a working model in which HDAC3-induced mitochondrial fission upregulates HK2, induces glycolysis and promotes REC pathological angiogenesis.