Single-Cell Transcriptome Analysis Reveals Embryonic Endothelial Heterogeneity at Spatiotemporal Level and Multifunctions of MicroRNA-126 in Mice

Abstract

Background: Endothelial cells (ECs) play a critical role in angiogenesis and vascular remodeling. The heterogeneity of ECs has been reported at adult stages, yet it has not been fully investigated. This study aims to assess the transcriptional heterogeneity of developmental ECs at spatiotemporal level and to reveal the changes of embryonic ECs clustering when endothelium-enriched microRNA-126 (miR-126) was specifically knocked out. Methods: C57BL/6J mice embryos at day 14.5 were harvested and digested, followed by fluorescence-activated cell sorting to enrich ECs. Then, single-cell RNA sequencing was applied to enriched embryonic ECs. Tie2 (Tek receptor tyrosine kinase)-cre–mediated ECs-specific miR-126 knockout mice were constructed, and ECs from Tie2-cre–mediated ECs-specific miR-126 knockout embryos were subjected to single-cell RNA sequencing. Results: Embryonic ECs were clustered into 11 groups corresponding to anatomic characteristics. The vascular bed (arteries, capillaries, veins, lymphatics) exhibited transcriptomic similarity across the developmental stage. Embryonic ECs had higher proliferative potential than adult ECs. Integrating analysis showed that 3 ECs populations (hepatic, mesenchymal transition, and pulmonary ECs) were apparently disorganized after miR-126 being knocked out. Gene ontology analysis revealed that disrupted ECs were mainly related to hypoxia, glycometabolism, and vascular calcification. Additionally, in vivo experiment showed that Tie2-cre–mediated ECs-specific miR-126 knockout mice exhibited excessive intussusceptive angiogenesis; reductive glucose and pyruvate tolerance; and excessive accumulation of calcium. Agonist miR-126-3p agomir significantly rescued the phenotype of glucose metabolic dysfunction in Tie2-cre–mediated ECs-specific miR-126 knockout mice. Conclusions: The heterogeneity of ECs is established as early as the embryonic stage. The deficiency of miR-126 disrupts the differentiation and diversification of embryonic ECs, suggesting that miR-126 plays an essential role in the maintenance of ECs heterogeneity.

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“…Of note, miR-205 expression itself appears to be regulated by HIF-mediated signaling [40]. This trait highlights the need for a better understanding of miR networks, as other miRs like miR-126, which also serves as a biomarker candidate in ccRCC [41], were shown to regulate HIF1a in turn [42]. Apart from counteracting angiogenic signaling, other MF effects such as inhibition of mitochondrial function could also contribute to the clinically observed added value of MF in TKI trialsas recent findings suggest that mitochondrial signaling could also play a crucial role in ccRCC [43].…”

Exercise Training-Induced MicroRNA Alterations with Protective Effects in Cardiovascular Diseases

“…Robust evidence has elucidated that Propofol can ameliorate IRI in patients by increasing the expression of miR-30b-5p and targeting Beclin-1, thereby inhibiting excessive autophagy and apoptosis [ 111 ]. Several studies showed thatmiR-126-3p is important for maintaining vascular integrity [ 21 , 112 , 113 , 114 ]. miR-126 deficiency was found to impair the differentiation and diversification of embryonic ECs, suggesting its essential role in maintaining EC heterogeneity [ 112 ].…”

“…Several studies showed thatmiR-126-3p is important for maintaining vascular integrity [ 21 , 112 , 113 , 114 ]. miR-126 deficiency was found to impair the differentiation and diversification of embryonic ECs, suggesting its essential role in maintaining EC heterogeneity [ 112 ]. miR-126-5p could maintain the integrity of endothelial cells under high shear stress and autophagy by exerting non-canonical posttranslational functions.…”