Injury induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis but the underlying mechanisms remain unclear. To interrogate the transcriptional changes, single-cell RNA sequencing was performed on ∼15000 human endothelial cells following treatment with miR-200b inhibitor or its mimic. The 3’ scRNA-seq analysis was conducted using the Chromium single cell system (10x Genomics). Unsupervised clustering using CellRanger identified 13 cell clusters, few of which were miR-200b responsive. To verify the novel potential targets of miR-200b identified in endothelial cells, we used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based proteomic analysis using mass spectrometry. A total of 3818 proteins were detected which were then filtered using statistical cut-offs (p value < 0.05; % change > 10%) identifying 319 proteins targeted by miR-200b. To dissect mechanisms involved in vascular function post-miR-200b silencing in vivo, we generated miR-200b-429fl/fl-Tie2 Cre mice where endothelial miR-200b levels was depleted. The therapeutic significance of miR-200b inhibition was studied using ischemic hind limb in these mice. Perfusion imaging was performed using Laser Speckle Perfusion imaging. Color-coded perfusion maps were acquired at different time-points post-surgery (d3, d7, d10, d14) and average perfusion was calculated using PimSoft v1.4 software. Inhibition of endothelial miR-200b rescued hindlimb ischemia with improved perfusion (n=5). This rescue was associated with increased abundance of CD31+/vWF+ vasculogenic cells at the site of injury (n=5). This work identified miR-200b regulated endothelial cell clusters, the functionality of which are explained by SILAC proteomics. These findings provide insight into novel mechanisms explaining how transient inhibition of endothelial miR-200b helps switch vascular homeostasis into an angiogenic fate in response to injury.
K. Singh: None. E. Hernandez: None. M. Kumar: None. Y. Rustagi: None. S.K. Mohanty: None. A.S. Abouhashem: None. S. Khanna: None. S. Roy: None. C.K. Sen: None.
National Institutes of Health (GM108014); Eli Lilly and Company; Indiana Collaborative Initiative for Talent Enrichment (to C.K.S., S.R.)