Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mTOR signal and the inhibition of autophagy under hypoxia

Abstract

Apelin is highly expressed in the lungs, especially in the pulmonary vasculature, but the functional role of apelin under pathological conditions is still undefined. Hypoxic pulmonary hypertension is the most common cause of acute right heart failure, which may involve the remodeling of artery and regulation of autophagy. In this study, we determined whether treatment with apelin regulated the proliferation and migration of rat pulmonary arterial smooth muscle cells (SMCs) under hypoxia, and investigated the underlying mechanism and the relationship with autophagy. Our data showed that hypoxia activated autophagy significantly at 24 hrs. The addition of exogenous apelin decreased the level of autophagy and further inhibited pulmonary arterial SMC (PASMC) proliferation via activating downstream phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/the mammalian target of Rapamycin (mTOR) signal pathways. The inhibition of the apelin receptor (APJ) system by siRNA abolished the inhibitory effect of apelin in PASMCs under hypoxia. This study provides the evidence that exogenous apelin treatment contributes to inhibit the proliferation and migration of PASMCs by regulating the level of autophagy.

Keywords: PI3K/Akt/mTOR; apelin; autophagy; hypoxia; smooth muscle cells.

Figure 1

Hypoxia increases the proliferation and cell cycle progression of pulmonary arterial smooth muscle cells (PASMCs). (A) PASMCs were seeded at 1 × 10⁴ cells/well (0.1 ml) in 96-well flat-bottomed plates and incubated overnight at 37°C. After exposure to hypoxia (1% oxygen) and normoxia chamber, respectively, for 6, 12, 24 and 48 hrs, cell proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation. The values are mean ± SD,n = 5. (B) Cell migration of PASMCs under hypoxia condition at 24 hrs by transwell assays. Columns represent the mean of three individual experiments performed in triplicate. *P < 0.05 versus normoxia group. (C) Cell cycle analysis of PASMCs in hypoxia condition at 24 hrs by flow cytometry. The results were expressed as relative cell growth in percentage, which was compared with a 21% oxygen control group. The concentration of 21% oxygen was set as control. n = 5 for each group. *P < 0.05 versus normoxia group.

Figure 2

Activation of autophagy in pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. (A) Monodansylcadaverine (MDC) fluorescence staining of autophagic vacuoles in PASMCs treated with hypoxia condition. (B) The corresponding linear diagram of MDC staining results. (C) Representative immunofluorescence images of PASMCs stained with DAPI (blue) for nucleus and antibodies against LC3 (green) for autophagosomes; punctuated LC3 dots were considered as positive results. Images are at 1000×. (D) The corresponding linear diagram of LC3 staining. (E) The levels of LC3-II and LC3-I were measured in the PASMCs under hypoxia by western blot analysis. Similar results were observed in three independent experiments. (F) The ratio of LC3-II to LC3-I was normalized to GAPDH. The data were presented as a mean ± SD from three independent experiments. *P < 0.05 versus control group, **P < 0.01 versus control group.

Figure 3

3-MA inhibits autophagy and decreases the proliferation of pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia. PASMCs were pre-incubated with 3-MA (5 mM) for 30 min. after 24 hrs, cells were exposed to hypoxia and normoxia chamber for 24 hrs. (A) The formations of autophagic vacuoles were detected by punctated monodansylcadaverine (MDC) immunofluorescence staining. Microphotographs are shown as representative results from three independent experiments. Images are at 1000×. (B) The corresponding linear diagram of MDC staining results. (C) PASMCs were processed for LC3 immunofluorescence staining. (D) The corresponding linear diagram of LC3 staining. (E) Cell proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) assay. n = 5, mean ± SD. *P < 0.05 versus control group, ^#P < 0.05 versus hypoxia group. (F) Migration of PASMCs exposed to 3-MA under hypoxia was detected by transwell assay. n = 5, mean ± SD. *P < 0.05 versus control group, ^#P < 0.05 versus hypoxia group.

Figure 4

Apelin decreases the proliferation and migration via inhibiting autophagy in pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. (A) PASMCs were pre-incubated with different concentrations (0.1, 0.5 and 1 μM) apelin for 30 min., and then exposed to hypoxia chamber and normoxia chamber for 24 hrs; cell proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) assay. n = 5, mean ± SD. *P < 0.05 versus control group. (B) The apoptosis rate of PASMCs in hypoxia condition, which was pre-incubated with 1 μM apelin for 30 min. and then placed in 1% oxygen for 24 or 48 hrs. (C) Apelin inhibited cell migration of PASMCs in hypoxia condition. PASMCs were pre-incubated with apelin and then placed in 1% oxygen for 24 hrs; scratches were made with a pipette tip. The widths of scratched gaps were measured. *P < 0.05 versus control group, #P < 0.05 versus hypoxia group. n = 5. (D) Cell migration and representative pictures of PASMCs were taken at different conditions. (E) Effect of apelin on autophagy in PASMCs under hypoxia. PASMCs were labelled with monodansylcadaverine (MDC) and observed with a fluorescent microscope. Images are at 1000×. Microphotographs were shown as representative results from three independent experiments. (F) The corresponding linear diagram of MDC staining results. **P < 0.01 versus control group, ^#P < 0.05 versus hypoxia group. (G) Representative images of PASMCs were stained with DAPI (blue), and antibodies against LC3 (green), punctuated LC3 dots were considered as positive results. Images are at 1000×. (H) The corresponding linear diagram of LC3 staining. *P < 0.05 versus control group, ^#P < 0.05 versus hypoxia group.

Figure 5

The effect of apelin on autophagy in pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia is related to the regulation of PI3K/Akt/mTOR pathways. (A) apelin increases the phosphorylation of PI3K/Akt/mTOR signals. The protein expressions were measured by western blot analysis. (B) Densitometry was applied to quantify the protein density. Standard error represents three independent experiments. *P < 0.05 versus hypoxia group. (C) Expression of phosphorylated-PI3K/Akt/mTOR and LC3 protein in PASMCs under hypoxia with apelin and Akt inhibitor LY294002. (D) Densitometry was applied to quantify phospho-PI3K/AKT/mTOR protein density. *P < 0.05 versus hypoxia group, ^#P < 0.05 versus apelin-treated hypoxia group. (E) The ratio of normalized LC3-II to LC3-I; the data were presented as a mean ± SD from three independent experiments. *P < 0.05 versus hypoxia group, ^#P < 0.05 versus apelin-treated hypoxia group.

Figure 6

The effect of siRNA-APJ on the proliferation and activation of PI3K/Akt/mTOR signals in pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. (A) Western blot analysis of APJ receptor protein expression in PASMCs transfected with siRNA-APJ and scramble vectors as described above for 24 hrs. (B) Densitometry was applied to quantify the protein density. Data were presented as a mean ± SD from three independent experiments. ^#P < 0.01 versus scramble group. (C) PASMCs treated with siRNA-APJ and scramble siRNA vectors for 24 hrs, cell proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) assay. *P < 0.05 versus hypoxia group. ^#P < 0.05 versus apelin-treated hypoxia group. n = 5. (D) Phosphorylation of PI3K/Akt/mTOR protein in PASMCs treated with siRNA-APJ and apelin in hypoxia condition. (E) Densitometry was applied to quantify the protein density; data were presented as a mean ± SD from three independent experiments. *P < 0.05 versus apelin-treated hypoxia group.

Figure 7

Transfection of siRNA-APJ blocks the inhibitory effect of apelin on autophagy in pulmonary arterial smooth muscle cells (PASMCs) under hypoxia. PASMCs treated with apelin and transfected with siRNA-APJ in hypoxia conditions. (A) Representative images of PASMCs were stained with DAPI (blue) and antibodies against LC3 (green). Images are at 1000×. Microphotographs were shown as representative results from three independent experiments. (B) The corresponding linear diagram of LC3 staining. (C) The protein levels of ATG4B and LC3 were detected with immunoblotting. (D) The ratio of normalized LC3-II to LC3-I. Data were presented as a mean ± SD from three independent experiments. *P < 0.05 versus control group, ^#P < 0.05 versus hypoxia group, ^$P < 0.05 versus apelin-treated hypoxia group. (E) The ratio of normalized ATG4B protein. Data were presented as a mean ± SD from three independent experiments. *P < 0.05 versus control group.