Journal: Journal of the American Heart Association

Article Title: miR‐342‐5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling

doi: 10.1161/jaha.115.003042

Figure Lengend Snippet: Figure 7. miR-342-5p participated in the regulation of VEGFR signaling. A and B, HUVECs were cultured in the presence of VEGF or PBS, and the expression of miR-342-5p and EVL was determined by using qRT-PCR (A) or Western blot (B). C, HUVECs were cultured with VEGF in the presence of Akt inhibitor or ERK inhibitor, and the expression of miR-342-5p and EVL mRNA was determined by using qRT-PCR. D, HUVECs were treated with VEGF or PBS for 48 hours. Endoglin expression was determined by using Western blot. E, HUVECs were transfected with miR-342- 5p or control. The expression of VEGFR2 and VEGFR3 was determined by using qRT-PCR. F, HUVECs were transfected with miR-342-5p or control. Cells were stimulated with VEGF, and the total and phosphorylated Akt and ERK were determined by using Western blot. Bands were quantitatively compared between the 2 groups. Bars indicate meanSD (n=6), *P<0.05, **P<0.01, ***P<0.001. Ctrl indicates control; DMSO, dimethyl sulfoxide; HUVEC, human umbilical vein endothelial cells; qRT-PCR, quantitative reverse transcription polymerase chain reaction; VEGF, vascular endothelial growth factor.

Article Snippet: The primary antibodies included rabbit antihuman EVL (1:50; Santa Cruz Biotechnology), mouse antihuman endoglin (1:800; BD Biosciences), rabbit anti–phosphorylated Smad1/5 (1:1000; Cell Signaling), rabbit anti–phosphorylated Smad2/3 (1:500; Santa Cruz Biotechnology), rabbit anti–phosphorylated Akt (Ser 473, 1:800; Cell Signaling), rabbit anti-Akt (1:800; Cell Signaling), rabbit anti–phosphorylated ERK (1:1000; Cell Signaling), rabbit anti-ERK (1:1000; Cell Signaling), mouse anti-b-actin (1:1000; Sigma-Aldrich), mouse anti-CD31 antibody (1:1000; Abcam), rabbit anti–a-smooth muscle actin antibody (1:200; Abcam), mouse anti–b-catenin antibody (1:1000; Millipore), rabbit anti-vimentin antibody (1:1000; Abcam).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: miR‐342‐5p Is a Notch Downstream Molecule and Regulates Multiple Angiogenic Pathways Including Notch, Vascular Endothelial Growth Factor and Transforming Growth Factor β Signaling

doi: 10.1161/jaha.115.003042

Figure Lengend Snippet: Figure 8. miR-342-5p inhibited vascularization in CNV. A, Healthy adult mice underwent laser-induced CNV. The choroid membranes of the mice were collected on days 3 and 7 after laser injury, and the expression of miR-342-5p and EVL mRNA was compared with the control group. B, Mice underwent laser-induced CNV and were injected intravitreally with miR-342-5p agomir. The level of miR-342-5p was determined with quantitative reverse transcription polymerase chain reaction. C, Healthy adult mice underwent laser-induced CNV. miR-342-5p agomir or control was injected intravitreally 24 hours after laser burns. Choroid membranes were collected on day 7 and stained with isolectin B4. The relative CNV areas were compared between the 2 groups. D, A model showing that miR-342-5p functions as a multieffect repressor of angiogenesis in ECs. The expression of miR-342-5p is downregulated by VEGFR2 signaling through Akt and upregulated by Notch signaling and TGF-b treatment. This miRNA represses angiogenesis by suppressing VEGFR and endoglin-mediated TGF-b receptor signaling, upregulating Dll4, and downregulating jagged 1 in Notch signaling, leading to decreased EC proliferation, migration, and probably increased endothelial–mesenchymal transition. Bars indicate meanSD (n=5), *P<0.05, **P<0.01. CNV indicates choroidal neovascularization; Ctrl, control; EC, endothelial cell; TGF-b, transforming growth factor b; VEGF, vascular endothelial growth factor.

Article Snippet: The primary antibodies included rabbit antihuman EVL (1:50; Santa Cruz Biotechnology), mouse antihuman endoglin (1:800; BD Biosciences), rabbit anti–phosphorylated Smad1/5 (1:1000; Cell Signaling), rabbit anti–phosphorylated Smad2/3 (1:500; Santa Cruz Biotechnology), rabbit anti–phosphorylated Akt (Ser 473, 1:800; Cell Signaling), rabbit anti-Akt (1:800; Cell Signaling), rabbit anti–phosphorylated ERK (1:1000; Cell Signaling), rabbit anti-ERK (1:1000; Cell Signaling), mouse anti-b-actin (1:1000; Sigma-Aldrich), mouse anti-CD31 antibody (1:1000; Abcam), rabbit anti–a-smooth muscle actin antibody (1:200; Abcam), mouse anti–b-catenin antibody (1:1000; Millipore), rabbit anti-vimentin antibody (1:1000; Abcam).

Techniques: Expressing, Control, Injection, Reverse Transcription, Polymerase Chain Reaction, Staining, Migration