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bioRxiv · 10.1101/2023.10.18.563021

Identification of new markers of angiogenic sprouting using transcriptomics: New role for RND3

Abbey, C. A.·Duran, C. L.·Chen, Z.·Chen, Y.·Roy, S.·Coffell, A.·Chakraborty, S.·Wells, G. B.·Chang, J.·Bayless, K.
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Abstract

BackgroundNew blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. Approach and ResultsUsing a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from non-invading cells. We used this model to perform multiple transcriptomics analyses from multiple donors to monitor endothelial gene expression changes. Single-cell Population Analyses, single-cell Cluster Analyses, and bulk RNA sequencing were used to delineate transcriptomic changes in invading cells. The results revealed a 39 gene signature that was consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis, and include multiple tip cell markers. Upregulation of SNAI1, PTGS2, and JUNB protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated Rounding 3 (RND3), also known as RhoE, which has not yet been implicated in angiogenesis. Silencing RND3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for RND3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared to wild type littermates. ConclusionValidation of multiple genes, including RND3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes that are associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation. HIGHLIGHTSO_LITranscriptomic analyses identified 39 candidate genes that were upregulated at the onset of endothelial sprouting C_LIO_LIThe gene signature includes signal transduction, morphogenesis, and immune responses C_LIO_LINewly-identified RND3 is associated with filopodial extension and pathfinding C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/563021v1_ufig1.gif" ALT="Figure 1"> View larger version (92K): org.highwire.dtl.DTLVardef@1045439org.highwire.dtl.DTLVardef@1357703org.highwire.dtl.DTLVardef@1189cf2org.highwire.dtl.DTLVardef@e7d574_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Abbey, C. A., Duran, C. L., Chen, Z., Chen, Y., Roy, S., Coffell, A., Chakraborty, S., Wells, G. B., Chang, J., Bayless, K.. 2023-10-19. Identification of new markers of angiogenic sprouting using transcriptomics: New role for RND3. https://doi.org/10.1101/2023.10.18.563021

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