Abstract
Purpose :
In retinopathy of prematurity (ROP), angiogenesis not only leads to physiologic retinal vascular development (PRVD) but also pathologic intravitreal neovascularization (IVNV). Signal transducer and activator of transcription 3 (STAT3), a transcription factor, is activated by vascular endothelial growth factor (VEGF) and regulates IVNV formation without affecting PRVD in the rat oxygen-induced retinopathy model representative of ROP, but STAT3-mediated mechanisms are incompletely understood. We used RNA sequencing to test the hypothesis that VEGF-induced STAT3 activation is important for angiogenic gene expression in endothelial cells.
Methods :
Primary human retinal microvascular endothelial cells (HRMVCs, Cell Systems) were transfected with either control or STAT3 siRNA for 24 hours and then treated with VEGF (25 ng/mL) for 4 hours. Total RNA was isolated and submitted to core facilities at the University of Utah, where RNA-sequencing and differential expression calculations were performed. Pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID).
Results :
Compared to HRMVCs transfected with control siRNA, HRMVCs transfected with STAT3 siRNA had 90% STAT3 knockdown (p<0.001). Compared to VEGF-treated HRMVCs transfected with control siRNA, VEGF-treated HRMVCs transfected with STAT3 siRNA had 137 transcripts differentially downregulated (p<0.05) and 120 differentially upregulated (p<0.05). Downregulated transcripts were significantly involved in mitosis (i.e., CDK1, CCNB1, and KIF22) (p<0.05), whereas upregulated transcripts were significantly involved in vascular development (i.e., PLAT, COL18A1, CDH13, and PDGFA) (p<0.05). Using DAVID, we identified pathway enrichment in p53 signaling and cell cycle regulation for downregulated transcripts and in focal adhesion modulation, Notch signaling and oxidative phosphorylation for upregulated transcripts in VEGF-treated HRMVCs transfected with STAT3 siRNA compared to VEGF treated HRMVCs transfected with control siRNA (p<0.05).
Conclusions :
Our results suggest that STAT3 regulates angiogenesis by mediating expression of genes involved in HRMVC mitosis and vasculature development, and may involve other pathways implicated in angiogenesis. Further testing to mechanistically understand how STAT3 affects signaling cascades may lead to a better understanding of pathologic and physiologic angiogenesis in ROP.
This is a 2020 ARVO Annual Meeting abstract.