Wnt2 Contributes to the Development of Atherosclerosis

Figures

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  Figure 1. TGF-β2 induces EndMT in HAECs. (A) The morphological change of HAECs before and after a 3-day TGF-β2 treatment. Scale bars, 100 μm. (B) WB detection of the expression of the EndMT markers VE-cadherin, CD31, PDGFRα, and αSMA. Hsp90 was used as a loading control. (C,D) Same experiments were performed as those in (B,A) to control HAECs and TGF-β2-overexpressing HAEC cells. (E) Immunostaining of VE-cadherin and αSMA in control HAECs and HAECs treated with TGF-β2 for 3 days. Scale bars, 100 μm. Experiments were repeated three times. (F) The morphological change of HAECs 2 days post TGF-β2 removal following a 3-day TGF-β2 treatment. (G) Immunoblotting for the expression of the EndMT markers VE-cadherin, CD31, PDGFRα, and αSMA in HAECs for (F). Hsp90 was used as a loading control.

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  Figure 2. A cell invasion assay of the effect of TGF-β2 treatment of HAECs. (A) The comparison of invasive capabilities between control HAECs and TGF-β2-overexpressing HAECs. Top: The cells on the bottom of the basement membrane before the invasion (0 h). Bottom: The cells on the bottom of membrane after 48 h of invasion. (B) The quantification of cell invasion assays in (B). n = 3, **P < 0.01.

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  Figure 3. Wnt ligand expression is upregulated during TGF-β-mediated EndMT. (A) RT-PCR analysis of Wnt2, Wnt4, Wnt10B, and Wnt11 mRNA expression during TGF-β-mediated EndMT for different time courses. Each value represents the mean ± SD of three separate experiments. n = 3, *P < 0.05, **P < 0.01. (B) WB analysis of Wnt ligand protein expression during TGF-β-mediated EndMT. WCLs collected from cells treated with TGF-β2 for 0, 1, and 3 days were subjected to WB analysis. Hsp90 protein expression is used as a loading control.

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  Figure 4. Wnt2 is required the TGF-β2 mediated-EndMT in cells migration and invasion. (A) HAECs stably transfected with control scrambled shRNA and Wnt2 shRNA were treated with TGF-β2 over time as indicated in the figure. Then, the WCLs were subjected to WB analysis. VE-cadherin, CD31, PDGFRα, and α-SMA. Hsp90 expression was used as a loading control. Experiments were repeated three times. (B) The comparison of invasive capabilities between control and Wnt2 shRNA with TGF-β2 treatment HAECs in the indicated time. Each value represents the mean ± SD. n = 3, **P < 0.01 (C) The comparison of invasive capabilities between control and Wnt2 overexpression (OVE) with TGF-β2 treatment HAECs in the indicated time. Each value represents the mean ± SD. n = 3, **P < 0.01. (D) Immunoblotting analysis of the EndMT markers in Wnt4 KD, Wnt10B KD, and Wnt11 KD HAECs with or without TGF-β2 treatment for 3 days, respectively. WCLs collected from KD cells treated with TGF-β2 for 0 and 3 days were subjected to immunoblots using the indicated antibodies. Hsp90 protein level is used as a loading control. Experiments were repeated three times.

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  Figure 5. Wnt2 is involved in the atherosclerotic lesion development. Upper panels (A): Oil Red O staining of atherosclerotic lesions in the aortic root at the level of the aortic valves of LDLR−/− mice fed on a normal diet (ND) and a high-fat (HF) diet. Lower panels: Immunohistochemistry staining of Wnt2 protein. Magnification is x10, scale bars = 400 μm. (B) Immunostaining for α-SMA (Red) and Wnt2 (Green) expressions in atherosclerotic lesions derived from the aortic valves of LDLR−/− mice fed on a normal diet (ND) and a high-fat (HF) diet. (C) Quantification for the Wnt2 staining area in α-SMA-positive atherosclerotic lesions derived from the aorta. Each value represents the mean ± SD. n = 7.

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  Figure 6. TGF-β induces EndMT in atherosclerosis. The TGF-β signaling activation initiates EndMT and induces changes in the level of the endothelial markers, VE-cadherin, CD31, and the mesenchymal makers, PDGFRα and αSMA. Suppression of endothelial TGF-β receptor expression arrest EndMT development.