After angiogenesis-activated embryonic and early postnatal vascularization, endothelial cells (ECs) in most tissues enter a quiescent state necessary for proper tissue perfusion and EC functions. Notch signaling is essential for maintaining EC quiescence, but the mechanisms of action remain elusive. Here, we show that microRNA-218 (miR-218) is a downstream effector of Notch in quiescent ECs. Notch activation upregulated, while Notch blockade downregulated, miR-218 and its host gene Slit2, likely via transactivation of the Slit2 promoter. Overexpressing miR-218 in human umbilical vein ECs (HUVECs) significantly repressed cell proliferation and sprouting in vitro. Transcriptomics showed that miR-218 overexpression attenuated the MYC proto-oncogene, bHLH transcription factor (MYC, also known as c-myc) signature. MYC overexpression rescued miR-218-mediated proliferation and sprouting defects in HUVECs. MYC was repressed by miR-218 via multiple mechanisms, including reduction of MYC mRNA, repression of MYC translation by targeting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), and promoting MYC degradation by targeting EYA3. Inhibition of miR-218 partially reversed Notch-induced repression of HUVEC proliferation and sprouting. In vivo, intravitreal injection of miR-218 reduced retinal EC proliferation accompanied by MYC repression, attenuated pathological choroidal neovascularization, and rescued retinal EC hyper-sprouting induced by Notch blockade. In summary, miR-218 mediates the effect of Notch activation of EC quiescence via MYC and is a potential treatment for angiogenesis-related diseases.

During vascular development, endothelial cells (ECs) undergo arterialization in response to genetic programs and shear stress-triggered mechanotransduction, forming a stable vasculature. Although the Notch receptor is known to sense shear stress and promote EC arterialization, its downstream mechanisms remain unclear. In this study, the Notch downstream miR-342-5p was found to respond to shear stress and promote EC arterialization. Shear stress upregulated miR-342-5p in a Notch-dependent manner in human umbilical vein endothelial cells (HUVECs). miR-342-5p overexpression upregulated the shear stress-associated transcriptomic signature. Moreover, miR-342-5p upregulated arterial markers and promoted EC arterialization in a Matrigel plug assay and retinal angiogenesis model. In contrast, miR-342-5p knockdown downregulated arterial markers, compromised retinal arterialization, and partially abrogated shear stress and Notch activation-induced arterial marker upregulation. Mechanistically, miR-342-5p overexpression suppressed MYC to repress EC proliferation and promote arterialization, achieved by promoting MYC protein degradation by targeting the EYA3. Consistently, EYA3 overexpression rescued miR-342-5p-mediated MYC downregulation and EC arterialization. In vivo, miR-342-5p expression was notably decreased in the ligated artery in a hindlimb ischemia model, and an intramuscular injection of miR-342-5p promoted EC arterialization and improved perfusion. In summary, miR-342-5p, a mechano-miR, mediates the effects of shear stress-activated Notch on EC arterialization and is a potential therapeutic target for ischemic diseases.