Differential hypoxic response of human choroidal and retinal endothelial cells proposes tissue heterogeneity of ocular angiogenesis

Abstract

PurposeTo elaborate molecular differences between choroidal and retinal angiogenesis by generating and comparatively analyzing human primary choroidal and retinal endothelial cell (CEC and REC) lines.MethodsHuman CEC and REC were isolated by positive selection and cultured. Characterization was performed by immunostaining for endothelial cell (EC)‐specific markers. Total RNA and protein were extracted from normoxic or hypoxic CEC and REC cultures. qPCR arrays were used to comparatively analyze CEC and REC, and expression differences were calculated by ΔΔCt method. Angiogenesis‐related protein expression differences were investigated by western blot and proteome profiler, and were calculated by densitometry. Functional responses to hypoxia were analyzed by sprouting assay.ResultsPrimary human CEC and REC lines stained positively for all EC markers and demonstrated high purity with similar staining and morphology. Under normoxia, CEC showed significantly lower expression levels for cell proliferation and vessel maturation genes and higher expression levels for inflammation‐related genes when compared to REC. In response to hypoxia, CEC and REC displayed differential regulation for a multitude of angiogenesis‐related genes and proteins. Furthermore, within the vascular endothelial growth factor (VEGF) family, CEC showed preferential upregulation for vascular endothelial growth factor A (VEGFA) while REC upregulated placenta growth factor (PlGF) levels. Sprouting was observed in normoxia, and was significantly by hypoxia.ConclusionsDifferential normoxic and hypoxic regulation of angiogenesis‐related factors by CEC and REC outlines tissue heterogeneity of ocular angiogenesis and suggests that tissue specificity should be considered as a novel treatment modality for successfully overcoming choroidal and retinal angiogenic conditions in the clinic.