Abstract
Purpose :
Endothelial signal transducer and activator of transcription 3 (STAT3)-induced intravitreal neovascularization (IVNV) in oxygen-induced retinopathy (OIR) represents retinopathy of prematurity (ROP). To understand pathways involved in endothelial STAT3-induced IVNV, we performed bulk RNA-sequencing of transcripts isolated from human retinal microvascular endothelial cells (HRMECs) transfected with STAT3 siRNA or control siRNA, and treated with vascular endothelial growth factor (VEGF) or vehicle control. We found STAT3 was necessary to differentially regulate genes involved in VEGF-induced mitosis and vascular development signaling pathways. In this study, we used a data-driven approach to cluster the differentially expressed genes based on expression patterns and tested the hypothesis that VEGF-induced STAT3 mediates angiogenic signaling pathways involved in ROP.
Methods :
Hierarchical clustering with Ward's linkage method and Euclidean distance metric was performed using scaled regularized log counts of the differentially expressed genes (n=353). Pathway enrichment analysis of the differentially expressed genes within the clusters was assessed by Ingenuity Pathway Analysis (IPA) software. An adjusted p-value < 0.05 was considered statistically significant following Benjamini and Hochberg multiple testing correction.
Results :
Hierarchical clustering resulted in the visual identification of 4 clusters of genes. Regardless of VEGF or PBS treatment, knockdown of STAT3 downregulated cluster 1 (n=113) and upregulated cluster 3 (n=116) compared to control. Compared to control, knockdown of STAT3 reduced upregulation of cluster 2 (n=44) by VEGF and reduced downregulation of cluster 4 (n=80) by VEGF. IPA identified significant pathway enrichment in G2/M cell cycle checkpoint regulation, IGF-1 signaling, or JAK/STAT signaling in cluster 1, and glioblastoma multiforme signaling or signaling by Rho family GTPases in cluster 3. No significant pathway enrichment was observed in cluster 2 or 4.
Conclusions :
Our results corroborate our previous analysis that STAT3 regulates angiogenesis in HRMECs by mediating transcripts involved in mitosis and identified additional pathways implicated in angiogenesis. Further testing of the identified signaling pathways in cultured HRMECs and the OIR model might lead to a better understanding of the mechanisms involved in IVNV in ROP.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.