J Korean Med Sci.  2020 Sep;35(35):e289. 10.3346/jkms.2020.35.e289.

Activation of AMPK by Telmisartan Decreases Basal and PDGF-stimulated VSMC Proliferation via Inhibiting the mTOR/p70S6K Signaling Axis

• Hwang YJ
• Park JH
• Cho DH

• KMID: 2506085
• DOI: http://doi.org/10.3346/jkms.2020.35.e289

Abstract

Background
Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is widely used to treat hypertension by blocking the renin-angiotensin-aldosterone system. Although abnormal proliferation of vascular smooth muscle cells (VSMCs) is a well-established contributor to the development of various vascular diseases, such as atherosclerosis, the effect of telmisartan on VSMC proliferation and its mechanism of action have not been fully revealed. Herein, we investigated the molecular mechanism whereby telmisartan inhibits rat VSMC proliferation.
Methods
We measured VSMC proliferation by MTT assay, and performed inhibitor studies and western blot analyses using basal and platelet-derived growth factor (PDGF)-stimulated rat VSMCs. To elucidate the role of AMP-activated protein kinase (AMPK), we introduced dominant-negative (dn)-AMPKα1 gene into VSMCs.
Results
Telmisartan decreased VSMC proliferation, which was accompanied by decreased phosphorylations of mammalian target of rapamycin (mTOR) at Ser2448 (p-mTOR-Ser²⁴⁴⁸ ) and p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr³⁸⁹ ) in dose- and time-dependent manners. Telmisartan dose- and time-dependently increased phosphorylation of AMPK at Thr172 (p-AMPK-Thr¹⁷² ). Co-treatment with compound C, a specific AMPK inhibitor, or ectopic expression of the dn-AMPKα1 gene, significantly reversed telmisartan-inhibited VSMC proliferation, p-mTOR-Ser²⁴⁴⁸ and p-p70S6K-Thr³⁸⁹ levels. Among the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr¹⁷² and decreased p-mTOR-Ser²⁴⁴⁸ , p-p70S6K-Thr³⁸⁹ , and VSMC proliferation. Furthermore, GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, did not affect any of the telmisartan-induced changes. Finally, telmisartan also exhibited inhibitory effects on VSMC proliferation by increasing p-AMPK-Thr¹⁷² and decreasing p-mTOR-Ser²⁴⁴⁸ and p-p70S6K-Thr³⁸⁹ in a PDGF-induced in vitro atherosclerosis model.
Conclusion
These results demonstrated that telmisartan-activated AMPK inhibited basal and PDGF-stimulated VSMC proliferation, at least in part, by downregulating the mTOR/p70S6K signaling axis in a PPARγ-independent manner. These observations suggest that telmisartan could be used to treat arterial narrowing diseases such as atherosclerosis and restenosis.

Keyword

Telmisartan; Vascular Smooth Muscle Cells; Proliferation; Adenosine Monophosphate-activated Protein Kinase; Mammalian Target of Rapamycin; p70 S6 Kinase

Figure

• 

  Fig. 1 Telmisartan decreases rat VSMC proliferation by inhibiting p-mTOR-Ser2448 and p-p70S6K-Thr389. (A) VSMC proliferation was measured by MTT assay as described in the METHODS. VSMCs were treated with various doses of telmisartan (0, 10, 20, or 40 μM) for 24 hours. (B) Cells were treated as described above, and then p-mTOR-Ser2448 and p-p70S6K-Thr389 levels were detected using western blot analyses. Nitrocellulose membranes were re-probed with antibodies against mTOR, p70S6K, or β-actin to assess equal sample loading. (C) VSMCs were treated with 40 µM telmisartan or vehicle (DMSO) for the indicated times (0, 1, 3, 6, 12, or 24 hours), and the subsequent p-mTOR-Ser2448 and p-p70S6K-Thr389 levels were detected using western blot analyses as described in Fig. 1B. (B, C) Using western blot results, densitometry was performed to quantitate p-mTOR-Ser2448 and p-p70S6K-Thr389 levels relative to those of total mTOR and p70S6K, respectively. All experiments were performed at least four times independently, and the blots shown are representative of at least four experiments (n = 4). Bar graphs depict mean fold alterations below the control level (± standard deviation).Differences were considered statistically significant at *P < 0.05, #P < 0.05, **P < 0.01, and ##P < 0.01.

• 

  Fig. 2 Telmisartan-activated AMPK inhibits VSMC proliferation by decreasing mTOR/p70S6K signaling pathway. (A) VSMCs were treated with various doses of telmisartan (0, 10, 20, or 40 μM) for 24 hours followed by p-AMPK-Thr172 and p-ACC-Ser79 detection using western blot analyses as described in Fig. 1B. (B) VSMCs were treated with 40 µM telmisartan or vehicle (DMSO) for the indicated times (0, 1, 3, 6, 12, or 24 hours), and the p-AMPK-Thr172 and p-ACC-Ser79 levels were detected using western blot analyses as described in Fig. 1B. (C) VSMCs were treated with 40 µM telmisartan or vehicle (DMSO) in the absence or presence of 10 µM compound C for 24 hours followed by p-mTOR-Ser2448, p-p70S6K-Thr389, and p-ACC-Ser79 detection using western blot analyses as described in Fig. 1B. (D, E) VSMCs were transfected with rat dn-AMPKα1 (D157A) gene or empty vector before treatment with 40 µM telmisartan or vehicle (DMSO) for 24 hours. The resulting p-AMPK-Thr172, p-ACC-Ser79, p-mTOR-Ser2448, and p-p70S6K-Thr389 levels were detected using western blot analyses as described in Fig. 1B. (F) Rat dn-AMPKα1 (D157A) gene- or empty vector-transfected VSMCs were treated with 40 µM telmisartan or vehicle (DMSO) for 24 hours and the subsequent cell proliferation was assessed by MTT assay as described in the METHODS. All experiments were independently performed at least four times, and the blots shown are representative of at least four experiments (n = 4). Bar graphs depict mean fold alterations above/below the control levels (± standard deviation).Differences were considered statistically significant at *P < 0.05, #P < 0.05, **P < 0.01, and ##P < 0.01.

• 

  Fig. 3 Among the tested ARBs, only telmisartan induces p-AMPK-Thr172 and inhibits mTOR/p70S6K signaling pathway and VSMC proliferation. (A, B) VSMCs were treated with various ARBs (telmisartan, losartan, or fimasartan; all at a dose of 40 μM) for 24 hours, and then p-AMPK-Thr172, p-ACC-Ser79, p-mTOR-Ser2448, and p-p70S6K-Thr389 levels were assessed using western blot analyses as described in Fig. 1B. (C) VSMCs were treated as described above, and cell proliferation was evaluated by MTT assay as described in the METHODS. All experiments were independently performed at least four times, and the blots shown are representative of at least four experiments (n = 4). Bar graphs depict mean fold alterations above/below the control level (± standard deviation).Differences were considered statistically significant at **P < 0.01 and ##P < 0.01.

• 

  Fig. 4 Telmisartan-inhibited VSMC proliferation is mediated by a PPARγ-independent pathway. (A, B) VSMCs were treated with 40 µM telmisartan or vehicle (DMSO) in the absence or presence of 5 µM GW9662 for 24 hours, and subsequent p-AMPK-Thr172, p-ACC-Ser79, p-mTOR-Ser2448, and p-p70S6K-Thr389 levels were determined using western blot analyses as described in Fig. 1B. (C) VSMCs were treated as described above, and cell proliferation was measured by MTT assay as described in the METHODS. All experiments were independently performed at least four times, and the blots shown are representative of at least four experiments (n = 4). Bar graphs depict mean fold alterations above/below the control level (± standard deviation).n.s = not significant.

• 

  Fig. 5 Telmisartan inhibits PDGF-stimulated VSMC proliferation through AMPK-mediated inhibition of the mTOR/p70S6K signaling pathway. (A) VSMCs were treated with various doses of telmisartan (0, 10, 20, or 40 μM) in the presence of 10 ng/mL PDGF for 24 hours, and subsequent cell proliferation was measured by MTT assay as described in the METHODS. (B, C) VSMCs were treated as described above, and the p-mTOR-Ser2448, p-p70S6K-Thr389, p-AMPK-Thr172, and p-ACC-Ser79 levels were determined using western blot analyses as described in Fig. 1B. (D, E) VSMCs were transfected with rat dn-AMPKα1 (D157A) gene or empty vector, and then treated with 40 µM telmisartan or vehicle (DMSO) in the presence of 10 ng/mL PDGF for 24 hours. The p-AMPK-Thr172, p-ACC-Ser79, p-mTOR-Ser2448, and p-p70S6K-Thr389 levels were detected using western blot analyses as described in Fig. 1B. (F) Rat dn-AMPKα1 (D157A) gene- or empty vector-transfected VSMCs were treated with 40 µM telmisartan or vehicle (DMSO) in the presence of 10 ng/mL PDGF for 24 hours, and subsequent cell proliferation was assessed by MTT assay as described in the METHODS. All experiments were independently performed at least four times, and the blots shown are representative of at least four experiments (n = 4). Bar graphs depict mean fold alterations above/below the control levels (± standard deviation).Differences were considered statistically significant at *P < 0.05, **P < 0.01, and ##P < 0.01.

• 

  Fig. 6 Schematic illustration of telmisartan inhibition of VSMC proliferation. Telmisartan, not losartan or fimasartan, increases p-AMPK-Thr172 in a PPARγ-independent manner. Increased AMPK activity downregulates p-mTOR-Ser2448 and p-p70S6K-Thr389 levels. Finally, the telmisartan-elevated AMPK activity inhibits VSMC proliferation by decreasing mTOR/p70S6K signaling axis.

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