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Single-cell RNA-sequencing Identifies Novel Molecular Targets Of Endothelial MicroRNA-200b In Ischemic Wound
Kanhaiya Singh, Edward Hernandez, Manishekar Kumar, Yashika Rustagi, Ahmed Abouhashem, Sujit Mohanty, Savita Khanna, Sashwati Roy, Chandan K. Sen.
Indiana Center for Regenerative Medicine and Engineering, Indiana University School of Medicine, Indianapolis, IN, USA.

BACKGROUND: In 2011, our laboratory reported that angiostatic miR-200b helps maintain resting cutaneous vascular homeostasis. Injury induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis but the underlying mechanisms remain unclear.
METHODS: To interrogate the transcriptional changes, single cell RNA sequencing (scRNA-seq) was performed on ~15000 human dermal microvascular endothelial cells at day 3 following treatment with miR-200b inhibitor or its mimic. The 3 scRNA-seq analysis was conducted using the Chromium single cell system (10x Genomics) and the NovaSeq 6000 sequencer (Illumina, Inc). Unsupervised clustering using CellRanger v3.0.2 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. To dissect specific 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. 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 (Perimed Inc.).
RESULTS: 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. 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).
CONCLUSIONS: This work identified miR-200b regulated endothelial cell clusters, the functionality of which are explained by SILAC proteomics data. 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.


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