Korean J Physiol Pharmacol.  2010 Oct;14(5):299-304. 10.4196/kjpp.2010.14.5.299.

Losartan Inhibits Vascular Smooth Muscle Cell Proliferation through Activation of AMP-Activated Protein Kinase

• Kim JE
• Choi HC

• KMID: 2071697
• DOI: http://doi.org/10.4196/kjpp.2010.14.5.299

Abstract

Losartan is a selective angiotensin II (Ang II) type 1 (AT1) receptor antagonist which inhibits vascular smooth muscle cells (VSMCs) contraction and proliferation. We hypothesized that losartan may prevent cell proliferation by activating AMP-activated protein kinase (AMPK) in VSMCs. VSMCs were treated with various concentrations of losartan. AMPK activation was measured by Western blot analysis and cell proliferation was measured by MTT assay and flowcytometry. Losartan dose- and time-dependently increased the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in VSMCs. Losartan also significantly decreased the Ang II- or 15% FBS-induced VSMC proliferation by inhibiting the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. Compound C, a specific inhibitor of AMPK, or AMPK siRNA blocked the losartan-induced inhibition of cell proliferation and the G0/G1 cell cycle arrest. These data suggest that losartan-induced AMPK activation might attenuate Ang II-induced VSMC proliferation through the inhibition of cell cycle progression.

Keyword

AMP-activated protein kinase (AMPK); Angiotensin II type 1 (AT1) receptor antagonist; Losartan; Vascular smooth muscle cells (VSMCs); Proliferation

MeSH Terms

Acetyl-CoA Carboxylase
AMP-Activated Protein Kinases
Angiotensin II
Blotting, Western
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Contracts
Cyclin D
Cyclin E
Cyclins
Losartan
Muscle, Smooth, Vascular
Phosphorylation
Proteins
RNA, Small Interfering
AMP-Activated Protein Kinases
Acetyl-CoA Carboxylase
Angiotensin II
Cyclin D
Cyclin E
Cyclins
Losartan
Proteins
RNA, Small Interfering

Figure

• 

  Fig. 1. Effect of Losartan on the Phosphorylation of AMPK and Cell Proliferation in VSMCs. Cells were treated with the indicated concentration of losartan for 1 hr (A) or for the indicated periods (B). Protein expression of p-AMPK, p-ACC, and p-LKB1 were determined by western blot analysis. Representative blots from three independent experiments are shown. Cells were treated with Ang II (C) or 15% FBS (D) in the presence or absence of indicated concentration of losartan for 48 hrs. Cell proliferation was determined by the MTT assay. Data are represented as the mean±S.E.M (n=4). ∗p value<0.05 compared with control, †p<0.05 compared with Ang II, ∗∗p<0.05 compared with 15% FBS.

• 

  Fig. 2. Inhibitory Action of Compound C and AMPK siRNA on the Anti-Proliferative Effect of Losartan. (A) Cells were pretreated with compound C (CC) (1 μM) or transfected with control siRNA or AMPK siRNA in the presence of losartan, and then stimulated with Ang II for 48 hrs. Cell proliferation was determined by the MTT assay. Data are represented as the mean±S.E.M (n=4). ∗p<0.05 compared with control, †p<0.05 compared with Ang II, #p<0.05 compared with Ang II+losartan. (B) Cells were pretreated with CC (1 μM) or transfected with AMPK siRNA in the presence of losartan, and then stimulated with Ang II for 48 hrs. Protein expression of p-AMPK was determined by western blot analysis. Representative blots from three independent experiments are shown.

• 

  Fig. 3. Losartan-induced Expressions of p53 and p21. (A) Cells were treated with Ang II in the presence or absence of losartan for 48 hrs. Apoptosis was assessed by Annexin V-fluorescein isothiocyanate (FITC) staining by flow cytometric analysis and the percentage of apoptotic cells was then determined. (B) Cells were treated with losartan for the indicated periods of time. Protein expressions of p53, p21, and p27 were determined by western blot analysis. Representative blots from three independent experiments are shown.

• 

  Fig. 4. Losartan Inhibits Cell Cycle Progression via AMPK activation. Cells were pretreated with compound C (CC) (1 μM) or transfected with control siRNA or AMPK siRNA in the presence of losartan, and then stimulated with Ang II for 24 hrs. (A) Protein expressions of p-Rb, cyclin D, and cyclin E were determined by western blot analysis. Representative blots from three independent experiments are shown. Cell cycle analysis was assessed by PI staining and the percentage of cells in G0/G1 phase (B), S phase (C), and G2/M phase (D). Data are represented as the mean± S.E.M (n=4). ∗p<0.05 compared with control, †p<0.05 compared with Ang II, #p<0.05 compared with Ang II+losartan.

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