MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3

Abstract

MicroRNAs (miRNAs) are small non-protein-coding RNAs that function as negative gene expression regulators. In the present study, we investigated miRNAs role in endothelial cell response to hypoxia. We found that the expression of miR-210 progressively increased upon exposure to hypoxia. miR-210 overexpression in normoxic endothelial cells stimulated the formation of capillary-like structures on Matrigel and vascular endothelial growth factor-driven cell migration. Conversely, miR-210 blockade via anti-miRNA transfection inhibited the formation of capillary-like structures stimulated by hypoxia and decreased cell migration in response to vascular endothelial growth factor. miR-210 overexpression did not affect endothelial cell growth in both normoxia and hypoxia. However, anti-miR-210 transfection inhibited cell growth and induced apoptosis, in both normoxia and hypoxia. We determined that one relevant target of miR-210 in hypoxia was Ephrin-A3 since miR-210 was necessary and sufficient to down-modulate its expression. Moreover, luciferase reporter assays showed that Ephrin-A3 was a direct target of miR-210. Ephrin-A3 modulation by miR-210 had significant functional consequences; indeed, the expression of an Ephrin-A3 allele that is not targeted by miR-210 prevented miR-210-mediated stimulation of both tubulogenesis and chemotaxis. We conclude that miR-210 up-regulation is a crucial element of endothelial cell response to hypoxia, affecting cell survival, migration, and differentiation.

FIGURE 1.

miR-210 induction by hypoxia. A, activation of miR-210 by hypoxia is fast and long lasting. HUVEC were exposed to hypoxia for the indicated time. Separated bars indicate miR-210 expression of HUVEC exposed to 1% oxygen for 24 h and then exposed to atmosphere oxygen for 8 and 24 h (^*, p < 0.001; #, p < 0.02; n = 4–11). B, Northern blotting showing time-dependent induction of miR-210 by hypoxia in HUVEC.

FIGURE 2.

miR-210 enhances the development of capillary-like structures. A and B, effect of hypoxia and miR-210 blockade on HUVEC tubulogenesis. HUVEC (4 × 10³/cm²) were transfected with anti-miR-210. Eighteen hours later, cells were exposed to hypoxia for further 24 h, and then their organization into capillary-like structures was assayed. A shows representative phase-contrast images. Size bar = 200 μm. B shows the quantitative assessment of capillary-like structures (hypoxic control versus normoxic control: ^*, p < 0.01; anti-miR-210 versus control: #, p < 0.005; n = 3). C, HUVEC were transduced with a retroviral vector bearing the miR-210 pre-miRNA sequence under the control of a constitutive promoter. These cells expressed almost 30-fold more mature miR-210 than control (^*, p < 0.004; n = 4). D and E, miR-210 stimulated HUVEC tubulogenesis. The differentiation into capillary-like structures of HUVEC overexpressing miR-210 (pSUPER-210) and control (pSUPER) was assayed. D shows representative phase-contrast images. Size bar = 200 μm. E shows the quantitative assessment of capillary-like structures (^*, p < 0.003; n = 3).

FIGURE 3.

miR-210 expression enhances VEGF-induced chemotaxis. A, representative experiment of the chemotactic responses of HUVECs transfected with either anti-miR-210 or control in response to 20 ng/ml VEGF. Size bar = 50 μm. B, miR-210 blockade decreases HUVEC migration index (^*, p < 0.015; n = 3). C, HUVECs were transduced with either a retroviral vector encoding miR-210 (pSUPER-210) or vector alone (pSUPER), and their chemotactic responses in response to 20 ng/ml VEGF was assayed. Size bar = 50 μm. D, miR-210 expression increases HUVEC migration index (^*, p < 0.001; n = 7).

FIGURE 4.

EFNA3 expression is inhibited by hypoxia and miR-210 expression. A, EFNA3 inhibition in hypoxic cells. HUVEC were exposed to hypoxia for 24 and 48 h. Then, cells were fixed and stained with both an antibody to EFNA3 (K-19, Santa Cruz Biotechnology, green) and the DNA intercalating agent Hoechst 33342 (blue). Size bar = 50 μm. EFNA3 signal was quantified using Scion Image software, and after 24 and 48 h of hypoxia, the decrease of EFNA3 fluorescence was about 80% when compared with the normoxic control (^*, p < 0.0001; n = 3). B, HUVEC were transfected with anti-miR-210 and, 18 h later, were exposed to hypoxia for 24 h. Then, cells were fixed and stained with both α-EFNA3 (green) and the DNA intercalating agent Hoechst 33342 (blue). Size bar = 50 μm. EFNA3 signal was quantified using Scion Image software (anti-miR-210 versus control: ^*, p < 0.02; hypoxia versus control: #, p < 0.001; ns, not significant; n = 3). C, miR-210 expression levels comparable with these observed during hypoxia induce EFNA3 inhibition. HUVEC infected either with a retroviral vector encoding miR-210 (pSUPER-210) or vector alone (pSUPER) were stained with both an antibody to EFNA3 (green) and the DNA intercalating agent Hoechst 33342 (Blue). Size bar = 50 μm. EFNA3 signal was quantified using Scion Image software (^*, p < 0.005; n = 3).

FIGURE 5.

miR-210 inhibits EFNA3 expression directly. A, structure of pLUC firefly luciferase reporter plasmids. miR-210 seed sequence and its complementary binding site in EFNA3 3′-UTR are highlighted. CMV, cytomegalovirus. B, U2OS were transfected with pLUC derivatives along with a plasmid encoding either miR-210 or a control sequence. Luciferase values were normalized for their mRNA levels, and the ratio of luciferase activity of each construct was calculated either in the presence or in the absence of exogenous miR-210 (B;^*, p < 0.003; n = 6) or in the presence or absence of hypoxia (C;#, p < 0.03; ^*, p < 0.005; n = 3).

FIGURE 6.

miR-210 repression of EFNA3 is necessary to stimulate capillary-like formation and cell chemotaxis. HUVEC expressing either EFNA3Δ or vector alone (4 × 10³/cm²) were transfected with mature miR-210 RNA or a scramble sequence and further cultivated for 36 h (A and C). Alternatively, HUVEC expressing either EFNA3Δ or vector alone were exposed to hypoxia or normoxia for 24 h (B and D). Then, cells were harvested, and either their organization into capillary-like structures (A and B) or their chemotactic response to 20 ng/ml VEGF (C and D) was assayed. The prevention of EFNA3 down-modulation by EFNA3Δ expression inhibited capillary-like structure increase induced both by miR-210 transfection (A; n = 4) and by hypoxia (B; n = 8). Moreover, EFNA3Δ expression completely prevented the migration index increase induced both by miR-210 transfection (C;^*, p < 0.007 versus all the other conditions; n = 3) and by hypoxia (D;^*, p < 0.005 versus all the other conditions; n = 4).