512-P: Single-Cell RNA-Sequencing Identifies Novel Molecular Targets of Endothelial MicroRNA-200b in the Diabetic Ischemic Wound

Abstract

Injury-induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis, but the underlying mechanisms remain unclear. Single cell RNA sequencing was performed on ~15000 human endothelial cells under high (mimic treated) miR-200b conditions. Unsupervised clustering using CellRanger identified five cell clusters, three of which were responsive to changes in miR-200b abundance. Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based proteomic analysis was employed to look for novel potential targets of endothelial miR-200b. 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 determine the potential role of these candidates in regulating vascular function, miR-200b-429fl/fl-Tie2 Cre mice were generated where endothelial miR-200b levels could be specifically depleted in vivo. The therapeutic significance of endothelial miR-200b inhibition was studied using ischemic hindlimb in these mice. The significance of miR-200b depletion was also studied in streptozotocin-induced diabetic miR-200b-429fl/fl-Tie2 Cre mice. Perfusion imaging was performed using Laser Speckle imaging (Perimed Inc.) at different time-points (d3, d7, d10, d14). Inhibition of endothelial miR-200b rescued hindlimb ischemia with improved perfusion (n=11). Such effect of miR-200b inhibition on rescuing HL-ischemia was markedly increased in diabetic animals (n =8). Such rescue was associated with increased abundance of CD31+/vWF+ vasculogenic cells (n=6). This work identified miR-200b regulated endothelial cell clusters, the functional significance of which have been experimentally validated. These findings provide insight into novel mechanisms explaining how transient inhibition of miR-200b in the endothelial compartment improves angiogenic outcomes in the diabetic ischemic limb. Disclosure K.Singh: None. M.Kumar: None. S.K.Mohanty: None. S.Khanna: None. S.Roy: None. C.K.Sen: n/a. Funding National Institutes of Health (DK128845); U.S. Department of Defense (W81XWH-22-1-0146, W81XWH-21-1-0033)