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1.
Figure 2

Figure 2. miR-16 and miR-424 modulate VEGFR2, FGFR1 and VEGF expression in ECs. From: MicroRNA-16 and MicroRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting VEGFR2 and FGFR1.

(A–C) HUVECs were transfected for 12 hours with miR-16, miR-424 or CM or (B–D) I-miR-16, I-miR-424 or CI. In both cases, cells were harvested 36 hours post-transfection. (A–B) Protein levels of VEGR2, FGFR1 and VEGF were detected by western blot. HSP-90 was used as a loading control. In the quantification (right graphs) data are expressed as relative total protein levels and correspond to mean ± SEM of three experiments. (C–D) mRNA expression of VEGFR2, FGFR1 and VEGF was quantified by qRT-PCR. Data are expressed as relative expression and correspond to mean ± SEM of three experiments. *Significantly different from cells transfected with CM or CI, p≤0.05.

Aránzazu Chamorro-Jorganes, et al. Arterioscler Thromb Vasc Biol. ;31(11):2595-2606.
2.
Figure 5

Figure 5. miR-16 modulates VEGF signaling. From: MicroRNA-16 and MicroRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting VEGFR2 and FGFR1.

BAECs were transfected for 12 hours with miR-16 or CM. 36 hours post-transfection, cells were starved (0.1% BSA) for 12 hours and then treated with (A) VEGF (50 ng/mL) (B) bFGF (25 ng/mL) or (C) S1P (400 nM) for the indicated times. Phospho-AKT (Ser-473) and phospho-ERK (Thr202/Tyr204) protein levels were detected by western blot. Blots for VEGFR2 and FGFR1 are shown as control of miR-16 action. HSP-90 was used as loading control. In the quantification (right graphs) data are expressed as fold activation over control and correspond to mean ± SEM of three experiments. *Significantly different from cells without VEGF or bFGF treatment, p≤0.05.

Aránzazu Chamorro-Jorganes, et al. Arterioscler Thromb Vasc Biol. ;31(11):2595-2606.
3.
Figure 1

Figure 1. miR-16 and miR-424 regulate VEGFR2, FGFR1 and VEGF expression by targeting the VEGFR2, FGFR1 and VEGF 3′UTR. From: MicroRNA-16 and MicroRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting VEGFR2 and FGFR1.

(A) Predicted target sites (indicated in blue) for miR-16 and miR-424 in the 3′UTR regions of human VEGFR2, FGFR1 and VEGF 3′UTR. “PM” (indicated in red) refers to mutation of the predicted miR-16/-424 seed region in the 3′UTR regions of human VEGFR2, FGFR1 and VEGF 3′UTR. (B–C and D) COS-7 cells were co-transfected with the indicated constructs (containing or not the above mentioned PM) and with 30nM of miR-16, miR-424 or CM. Data are expressed as relative luciferase activity to control samples co-transfected with an equal concentration of CM and correspond to the mean ± SEM of three experiments performed in duplicate. *Significantly different from cells co-transfected with CM or # from cells co-transfected with miR-16 or miR-424 and control 3′UTR, p≤0.05.

Aránzazu Chamorro-Jorganes, et al. Arterioscler Thromb Vasc Biol. ;31(11):2595-2606.
4.
Figure 3

Figure 3. VEGF and bFGF regulate miR-16 and miR-424 expression in ECs. From: MicroRNA-16 and MicroRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting VEGFR2 and FGFR1.

(A–B) HUVECs were starved (0.1% BSA) for 12 hours and then treated with VEGF (50 ng/mL) or bFGF (25 ng/mL) for the indicated times. (A) Mature miR-16 and miR-424, and (B) pri-miR-16-1/-2 and pri-miR-424 expression was quantified by qRT-PCR. Data are expressed as relative expression and correspond to mean ± SEM of three experiments. *Significantly different from cells without treatment, p≤0.05. (C) HUVECs were transfected for 6 hours with empty vector, VEGFR2 or FGFR1 3′UTR construct, 12 hours post-transfection cells were starved (0.1% BSA) for 12 hours and then treated with VEGF for the indicated time. (D) HUVECs were transfected as above in the presence or not of 60 nM of I-miR16, I-miR-424 or control inhibitor (CI). 36 hours post-transfection cells were starved (0.1% BSA) for 12 hours and then treated with VEGF for the indicated time. Data are expressed as relative luciferase activity to control samples co-transfected with an equal concentration CI and correspond to the mean ± SEM of three experiments performed in duplicate. *Significantly different from cells co-transfected with CI and VEGF 3′UTR or # from cells co-transfected with CI and VEGF 3′UTR plus VEGF treatment, p≤0.05.

Aránzazu Chamorro-Jorganes, et al. Arterioscler Thromb Vasc Biol. ;31(11):2595-2606.
5.
Figure 6

Figure 6. miR-16 reduces microvessel formation in vivo. From: MicroRNA-16 and MicroRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting VEGFR2 and FGFR1.

(A) HUVECs were transduced with miR-16 or scr-miR as indicated in Material and Methods section. GFP expression (FL1-H) was analyzed by flow cytometry prior gel implantation. (B, C and D) HUVECs that were transduced with either miR-16 and scr-miR as above indicated were kept in culture 7 days after implantation. (B) Representative fluorescence micrographs of GFP detection are shown. Original magnification x20. (C) Mature miR-16 and (D) mRNA expression of VEGFR2, FGFR1 and VEGF was quantified by qRT-PCR. Data are expressed as relative expression and correspond to mean ± SEM. *Significantly different from cells transduced with scr-miRNA, p≤0.05. Grafts from miR-16 or scr-miR-transduced HUVECs were isolated 14 days after implantation. (E) Representative micrographs of the gel implants and H&E staining are shown. Original magnification x0.5 and x10 respectively. (F) Grafts from miR-16 or scramble miRNA (scr-miR)-transduced HUVECs were isolated 14 days after implantation. Representative PECAM-1 and UEA-1 staining is shown. Scale bars represent 100 μM. In the quantification data are expressed as PECAM-1 or UEA-1 positive capillary structures of two to four images from five sections per animal and correspond to mean ± SEM (n=5 animals per group). *Significantly different from cells transduced with scr-miRNA, p≤0.05.

Aránzazu Chamorro-Jorganes, et al. Arterioscler Thromb Vasc Biol. ;31(11):2595-2606.
6.
Figure 4

Figure 4. miR-16 and miR-424 reduce proliferation, migration and impair endothelial cell cord formation. From: MicroRNA-16 and MicroRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting VEGFR2 and FGFR1.

(A) HUVECs were transfected for 12 hours with miR-16, miR-424 or CM. Cells were harvested and counted 36 hours post-transfection. Data are expressed as relative number of cells and correspond to mean ± SEM of three experiments performed in duplicate. (B) HUVECs were transfected as indicated in A. 36 hours post-transfection, cells were starved (0.1% BSA) for 16 hours and then treated or not with VEGF for an additional 9 hours. Cells were washed and stained by crystal violet as indicated in the Material and Methods section. Data are expressed as cell survival and correspond to mean ± SEM of three experiments. (C) BAECs were transfected as indicated in A. 36 hours post-transfection, migration in response to VEGF or bFGF was quantified as described in Material and Methods section. The average number of cells from five randomly chosen fields on the lower side of the membrane of each well was counted. Data are expressed as migrated cells and correspond to mean ± SEM of three experiments performed in duplicate. (D) HUVECs were transfected as indicated in A. 36 hours post-transfection, cells were counted and seeded on a Growth Factor Reduced Matrigel in the presence of 0.1% FBS (basal; left panels), 0.1% FBS + VEGF (VEGF stimulated; middle panels) and 0.1% FBS + bFGF (bFGF stimulated; right panels). Cumulative sprout length of capillary-like structures was measured by light microscopy after 12 hours. Representative micrographs and quantification are shown. Scale bars represent 100 μM. Data are expressed as cumulative tube lengths and correspond to mean ± SEM of three experiments performed in duplicate. * Significantly different from cells transfected with CM # and from basal conditions, p≤0.05.

Aránzazu Chamorro-Jorganes, et al. Arterioscler Thromb Vasc Biol. ;31(11):2595-2606.

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