P382MicroRNA-24 regulates angiogenesis acting on the notch pathway

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Abstract

Purpose

MicroRNAs (miRs) inhibit the expression of their target genes. The effect of miR-24 manipulation in post-ischemic recovery is still controversial. The Notch system regulates angiogenesis. It is composed by 4 receptors (Notch-1 to -4) and several Delta-like (Dll) and Jagged ligands. Dll4 is a validated miR-24 target gene, while Notch-1 and Dll1 are predicted targets (bioinformatics). We have investigated: 1) the impact of miR-24 on endothelial cell (EC) functions; 2) the Notch pathway as miR-24 target in ECs; 3) the effect of miR-24 inhibition on blood flow (BF) recovery and angiogenesis in a limb ischemia (LI) model.

Methods

Human umbilical vein ECs (HUVECs) were transfected with pre-miR-24, anti-miR-24 or negative control or infected with an adenovirus carrying a miR-24 decoy (Ad.decoymiR-24, 250 MOI) or control Ad.LacZ and prompted in proliferation (BrdU incorporation), apoptosis (caspase-3 activity), migration (EC layer scratch) and Matrigel assays. HUVEC mRNA and protein expression of Notch-1, Dll-1, Dll-4 and Hes-1 (Notch effector) were measured. 3'UTR luciferase assays investigated Notch-1 and Dll-1 as direct miR-24 targets. Left LI was induced by femoral artery occlusion in mice and Ad.decoymiR-24 (10^9 pfu) or Ad.Null control were injected in the ischemic adductor. Post-ischemic BF recovery (color laser Doppler) was sequentially measured for up to 21 days. Then, mice were sacrificed and muscle whole mounts prepared to investigate the blood vasculature (confocal microscopy followed by morphometry)

Results

miR-24 inhibition by anti-miR24 or miR-24-decoy enhanced HUVEC survival, proliferation, migration and networking on Matrigel, while pre-miR-24 overexpression inhibited these processes. We validated Notch-1 and Dll-1 as directed targets of miR-24. miR-24 expression levels were inversely correlated with Notch1, Dll1, Dll4, and Hes-1 levels in HUVECs. Moreover, the gamma-secretase/Notch inhibitor DAPT prevented anti-miR-24-induced Hes-1 upregulation. Furthermore, transfection with a Notch intracellular domain restored in vitro angiogenesis (Matrigel) from HUVECs overexpressing pre-miR-24. Ad.decoymiR-24 severely compromised post-LI BF recovery (P < 0.05 vs Ad.Null at 21 days). Ad.decoymiR-24-treated ischemic muscles presented a denser (vascular area per field: 2.5X10^4 ± 0.6X10^4 um2 vs 5.1X10^4 ± 0.8X10^4 um2 in Ad.Null, P < 0.02), but possibly disorganized microvessel network.

Conclusions

miR-24 promotes angiogenesis. The Notch pathway is targeted by miR-24 in ECs. miR-24 inhibition impairs post-LI BF recovery, possibly due to dysfunctional angiogenesis.

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