Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis

Abstract

MicroRNAs are negative regulators of gene expression that play a key role in cell-type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR-126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC-specific expression of miR-126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR-126 in arteriogenesis and angiogenesis. Using miR-reporter constructs, we show that miR-126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR-126. To study the consequences of miR-126 silencing on vascular regeneration, mice were injected with a single dose of antagomir-126 or a control 'scramblemir' and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR-126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir-126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir-126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR-126 in angiogenesis. Our data demonstrate a facilitary function for miR-126 in ischemia-induced angiogenesis and show the efficacy and specificity of antagomir-induced silencing of EC-specific microRNAs in vivo.

Fig. 1

MiR-126 is specifically expressed in EC. (A) Total RNA was harvested from different cell types and analyzed by qRT-PCR on miR-126. Obtained values were normalized by qRT-PCR on U6 snRNA. (B) Representative microscopic images of immunohistochemistry of CD31 on human renal sections and in situ hybridization of miR-126 with locked nucleic acid (LNA)-probe or a control LNA-probe. Upper two pictures represent consecutive sections.

Fig. 2

MiR-126 is functionally active in vitro and can be specifically repressed by antagomir-126 in vitro and in vivo. (A) Relative firefly luciferase expression of HEK-293T and EC-RF24 transfected with a luciferase reporter plasmid harbouring 0 (pMIR), 1 (pMIR-126 M) or 4 (pMIR-126Q) perfect match target sites for miR-126 in the 3’UTR of the luciferase transcript (**=P< 0.01). (B) Relative firefly luciferase expression of transfected EC-RF24 incubated for 16 hrs with 5 μg/ml scram-blemir or antagomir-126 (*=P< 0.05 and **=P< 0.01).

Fig. 3

Repression of miR-126 in vitro does not influence the formation of capillary structures, or EC proliferation and migration. (A) Quantification of area and length of capillary structures after 8 hrs of culture on matrigel (data expressed in arbitrary units). (B) Representative microscopic images of the formation of capillary-like structures by HUVEC. (C) Quantification of the degree of EC regrowth in a scratch/wound assay (expressed in μm from scratch border). (D) Representative microscopic images of HUVEC incubated with antagomir-126 or scramblemir before and 24 hrs after the scratch/wound assay.

Fig. 4

MiR-126 silencing has no effect on collateral formation, but miR-126 facilitates ischemia-induced angiogenesis. (A and B) Total RNA was harvested from lungs of mice after 11 day treatment with high and low doses of antagomir-126 or scramblemir (HD or LD). Samples were analyzed by qRT-PCR for A, miR-126 and (B) miR-423 expression. Obtained values were normalized by qRT-PCR on U6 snRNA (*=P< 0.05 and ***=P< 0.001). (C) Quantitative evaluation of blood flow recovery measured before and after induction of ischemia by laser Doppler perfusion imaging and expressed as a ratio of the left (ischemic) and right (non-ischemic) limb. (D) Representative microscopic images of CD31 staining in ischemic murine gastrocnemius muscles. (E) Quantification of the total area of capillaries in sections of the gastrocnemius muscle 10 days after induction of ischemia (*=P< 0.05, **=P< 0.01 and ***=P< 0.001).

Fig. 5

Repression of miR-126 impairs endothelial outgrowth or aortic explants. (A) Quantification of total surface (in μm²) covered by endothelial outgrowth of aortic explants. (B) Representative microscopic images of endothelial outgrowth from aortic explants.