G protein-coupled receptor (GPCR) signalling, including that involving apelin (APLN) and its receptor APLNR, is known to be important in vascular development. How this ligand–receptor pair regulates the downstream signalling cascades in this context remains poorly understood. Here, we show that mice with Apln, Aplnr or endothelial-specific Aplnr deletion develop profound retinal vascular defects, which are at least in part due to dysregulated increase in endothelial CXCR4 expression. Endothelial CXCR4 is negatively regulated by miR-139-5p, whose transcription is in turn induced by laminar flow and APLN/APLNR signalling. Inhibition of miR-139-5p in vivo partially phenocopies the retinal vascular defects of APLN/APLNR deficiency. Pharmacological inhibition of CXCR4 signalling or augmentation of the miR-139-5p-CXCR4 axis can ameliorate the vascular phenotype of APLN/APLNR deficient state. Overall, we identify an important microRNA-mediated GPCR crosstalk, which plays a key role in vascular development.

Rationale: Sprouting angiogenesis is governed by the concept of tip/stalk cells that guides our understanding of the transition from vascular sprouting to maturation and ultimately quiescence. The VEGF and Notch signaling pathways have been extensively described in regulating the discrimination between these two cell populations. However, several additional tip and stalk cell specific genes have been identified. To date, unresolved questions remain, and our understanding of the mechanisms by which these signaling processes are integrated is incomplete. Objective: We set out to investigate novel mechanisms by which signaling pathways involving two G protein coupled receptors (GPCRs), expressed in a mutually exclusive fashion in the tip/stalk cell populations, are intricately linked in vascular development. Methods/Results: Using a combination of in vivo and in vitro techniques, we demonstrate the critical role of crosstalk between APLNR and CXCR4 in vascular maturation. We show robust flow induced expression of the stalk cell specific APLNR, that leads to marked suppression of CXCR4 expression, a mechanism to achieve tip cell restricted expression of the latter. Retinas from Apln (ligand), Aplnr (receptor) and endothelial specific Aplnr deleted mice show retarded vascular expansion, reduced vascularized area and fewer vascular branch points. These phenotypes are in part due to increased expression of Cxcr4 in Apln-/- and Aplnr-/- retinal vessels as Cxcr4 inhibition through a selective inhibitor can ameliorate the Aplnr phenotype. The crosstalk between the two GPCRs was found to involve a key shear responsive microRNA, miR-139-5p, which is upregulated by APLN/APLNR signaling and directly targets CXCR4 in endothelial cells. In accordance, Apln-/- and Aplnr-/- retinal endothelial cells showed depleted levels of miR-139-5p. Lastly, we demonstrate that atorvastatin, an HMG-CoA reductase inhibitor shown to enhance APLNR signaling, can induce miR-139-5p expression and rescue the vascular phenotypes associated with APLN/APLNR deficiency. Conclusions: These findings provide key mechanistic insights into a critical microRNA based crosstalk between two GPCR signaling cascades, which regulates important steps in vascular maturation.