The mechanism of all-trans retinoic acid in the regulation of apelin expression in vascular endothelial cells

Figures

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  Figure 1. ATRA up-regulated apelin transcription and translation in HUVECs HUVECs were treated with ATRA (10 μΜ) for various times or at different doses (for 24 h). (A) Total RNA was transcribed with reverse transcriptase and amplified by PCR. GAPDH was used as an internal control. (B) Crude proteins were extracted from the treated cells and then Western blotting was performed using anti-apelin antibodies to examine the expression of apelin. β-actin was used as a loading control. (C) The mRNA levels of apelin were determined by qRT-PCR analysis. Bars represent the mean ± S.D. from three independent experiments. *P<0.05 compared with the respective control group. (D) Densitometry of (B); results were normalized to β-actin. Bars represent the mean ± S.D. from three independent experiments. *P<0.05 compared with the respective control group.

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  Figure 2. The expression of apelin in balloon-injured arteries (A) Representative Hematoxylin and Eosin-stained arterial sections from the control plus vehicle (Veh), model (M) plus vehicle, and model plus ATRA. Magnification: ×100. (B) Immunostaining of apelin in carotid arteries on day 14 after balloon injury. Magnification: ×200. (C) The ratio of I/M of carotid arteries on day 14 after balloon injury. #P<0.01 compared with the control group; *P<0.01 compared with the model group (n=6 in each group). (D) The apelin index was calculated as follows: apelin-positive cells/total intimal cells. The results are means ± S.D. for four independent experiments. #P<0.01 compared with the control group; *P<0.01 compared with model group (n=6 in each group). (E) The levels of apelin mRNA were determined by qRT-PCR analysis. #P<0.01 compared with the control group; *P<0.01 compared with the model group (n=6 in each group).

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  Figure 3. RARα was detected and Ro 41-5253 reduced the response of apelin to ATRA (A) HUVECs were treated with 10 μM of ATRA for 0, 6, 12, or 24 h. Crude proteins were extracted from the treated cells and then subjected to Western blotting with the antibodies against apelin, RARα, RARβ, and RARγ respectively. β-actin was used as a control for equal protein loading. (B) Densitometry of (A); results were normalized to β-actin. Bars represent the mean ± S.D. from three independent experiments. *,#P<0.05 compared with ATRA-free group. (C) HUVECs were pretreated with 20 μM of Ro 41-5253 for 2 h prior to exposure to ATRA (10 μM) for 24 h. Crude proteins from cell lysates were analyzed by Western blotting with the antibodies against apelin and RARα, respectively. β-actin was the loading control. (D) Densitometry of (C); results were normalized to β-actin. Bars represent the mean ± S.D. from three independent experiments. *P<0.05 compared with ATRA-free group. #P<0.05 compared with ATRA-treated group.

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  Figure 4. The effect of ATRA on the expression of apelin was detected by knockdown or overexpression of RARα (A) HUVECs were transfected with si-RARα or si-NS for 24 h and then treated with 10 μM of ATRA for 24 h. The cell lysates were analyzed by Western blotting with the antibodies against apelin and RARα, respectively. β-actin was the loading control. (B) Densitometry of (A); results were normalized to β-actin. Bars represent the mean ± S.D. from three independent experiments. *,#P<0.05 compared with si-NS group. (C) HUVECs were infected with pAd-null or pAd-GFP-RARα for 24 h prior to the exposure to ATRA (10 μM) for 24 h. Crude proteins from cell lysates were analyzed by Western blotting with antibodies against apelin or RARα. β-actin was the loading control. (D) Densitometry of (C); results were normalized to β-actin. Bars represent the mean ± S.D. from three independent experiments. *,#P<0.05 compared with pAd-null group.