J Korean Med Sci.  2023 Oct;38(41):e335. 10.3346/jkms.2023.38.e335.

Far-Infrared Irradiation Decreases Proliferation in Basal and PDGFStimulated VSMCs Through AMPKMediated Inhibition of mTOR/p70S6K Signaling Axis

• Hwang YJ
• Park JH
• Cho DH

• KMID: 2547178
• DOI: http://doi.org/10.3346/jkms.2023.38.e335

Abstract

Background
Far-infrared (FIR) irradiation has been reported to improve diverse cardiovascular diseases, including heart failure, hypertension, and atherosclerosis. The dysregulated proliferation of vascular smooth muscle cells (VSMCs) is well established to contribute to developing occlusive vascular diseases such as atherosclerosis and in-stent restenosis. However, the effects of FIR irradiation on VSMC proliferation and the underlying mechanism are unclear. This study investigated the molecular mechanism through which FIR irradiation inhibited VSMC proliferation.
Methods
We performed cell proliferation and cell death assay, adenosine 5′-triphosphate (ATP) assay, inhibitor studies, transfection of dominant negative (dn)-AMP-activated protein kinase (AMPK) α1 gene, and western blot analyses. We also conducted confocal microscopic image analyses and ex vivo studies using isolated rat aortas.
Results
FIR irradiation for 30 minutes decreased VSMC proliferation without altering the cell death. Furthermore, FIR irradiation accompanied decreases in phosphorylation of the mammalian target of rapamycin (mTOR) at Ser2448 (p-mTOR-Ser²⁴⁴⁸ ) and p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr³⁸⁹ ). The phosphorylation of AMPK at Thr172 (p-AMPKThr¹⁷² ) was increased in FIR-irradiated VSMCs, which was accompanied by a decreased cellular ATP level. Similar to in vitro results, FIR irradiation increased p-AMPK-Thr¹⁷² and decreased p-mTOR-Ser 2448 and p-p70S6K-Thr³⁸⁹ in isolated rat aortas. Pre-treatment with compound C, a specific AMPK inhibitor, or ectopic expression of dn-AMPKα1 gene, significantly reversed FIR irradiation-decreased VSMC proliferation, p-mTOR-Ser²⁴⁴⁸ , and p-p70S6K-Thr³⁸⁹ . On the other hand, hyperthermal stimulus (39°C) did not alter VSMC proliferation, cellular ATP level, and AMPK/mTOR/p70S6K phosphorylation. Finally, FIR irradiation attenuated plateletderived growth factor (PDGF)-stimulated VSMC proliferation by increasing p-AMPK-Thr¹⁷² , and decreasing p-mTOR-Ser²⁴⁴⁸ and p-p70S6K-Thr³⁸⁹ in PDGF-induced in vitro atherosclerosis model.
Conclusion
These results show that FIR irradiation decreases the basal and PDGF-stimulated VSMC proliferation, at least in part, by the AMPK-mediated inhibition of mTOR/p70S6K signaling axis irrespective of its hyperthermal effect. These observations suggest that FIR therapy can be used to treat arterial narrowing diseases, including atherosclerosis and in-stent restenosis.

Keyword

Far-Infrared Irradiation; Vascular Smooth Muscle Cells; Proliferation; AMP-Activated Protein Kinase; Mammalian Target of Rapamycin; p70 S6 Kinase

Figure

• 

  Fig. 1 FIR irradiation decreases VSMC proliferation. (A) Rat VSMCs were exposed to FIR radiation for various times (0, 15, 30, or 45 minutes), and VSMC proliferation was measured by MTT assay as described in the Methods. (B) Rat VSMCs were exposed to FIR radiation for various times (0, 15, 30, or 45 minutes), and live or dead cells were measured using a LIVE/DEAD assay kit as described in the Methods. Scale bar indicates 50 µm. All experiments were independently performed at least four times, and the fluorescent images shown are representative of at least four experiments (n = 4). The bar graphs depict the mean percent alterations above/below the control levels (± standard deviation).FIR = Far-infrared, VSMC = vascular smooth muscle cell, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, RT = room temperature.Differences were considered statistically significant at *P < 0.01 and ***P < 0.001.

• 

  Fig. 2 FIR irradiation decreases mTOR/p70S6K phosphorylation and cellular ATP levels, increasing AMPK phosphorylation in VSMCs and isolated aortas. (A, B) Rat VSMCs were exposed to FIR radiation for various times (0, 15, or 30 minutes), and levels of p-mTOR-Ser2448, p-p70S6K-Thr389, and p-AMPK-Thr172 were evaluated by western blotting. (C) Rat VSMCs were irradiated with FIR ray or not for 30 minutes, cellular ATP levels were assessed using a luminescent ATP detection assay kit, as described in the Methods. (D, E) Endothelium-deprived rat aortas were irradiated with FIR ray or not for 30 minutes, and aortic proteins were extracted and subjected to western blot analyses. The levels of p-mTOR-Ser2448, p-p70S6K-Thr389, and p-AMPK-Thr172 were detected by western blotting. All experiments were independently performed at least four times, and the blots shown are representative of at least four experiments (n = 4). The bar graphs depict mean fold alterations above/below the controls (± standard deviation).FIR = Far-infrared, mTOR = mammalian target of rapamycin, p70S6K = p70 S6 kinase, ATP = adenosine 5′-triphosphate, AMPK = AMP-activated protein kinase, VSMC = vascular smooth muscle cell, RT = room temperature.Differences were considered statistically significant at *P < 0.05, #P < 0.05, ***P < 0.001, and ###P < 0.001.

• 

  Fig. 3 FIR irradiation-activated AMPK inhibits VSMC proliferation by decreasing mTOR/p70S6K signaling pathway. (A) Rat VSMCs were exposed to FIR radiation for 30 minutes in the absence or presence of 10 µM compound C, and levels of p-mTOR-Ser2448 and p-p70S6K-Thr389 were detected by western blotting. (B, C) Cells were transfected with rat dn-AMPKα1 (D157A) gene or empty vector and exposed to FIR radiation for 30 minutes. Levels of p-AMPK-Thr172, p-mTOR-Ser2448, and p-p70S6K-Thr389 were assessed by western blotting. (D) Rat dn-AMPKα1 (D157A) gene- or empty vector-transfected VSMCs were exposed to FIR radiation for 30 minutes, and cell proliferation was measured using an MTT assay, as described in the Methods. 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 above/below the controls (± standard deviation).FIR = Far-infrared, AMPK = AMP-activated protein kinase, VSMC = vascular smooth muscle cell, mTOR = mammalian target of rapamycin, p70S6K = p70 S6 kinase, dn = dominant negative, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, RT = room temperature.Differences were considered statistically significant at *P < 0.05, #P < 0.05, and **P < 0.01.

• 

  Fig. 4 Inhibitory action of FIR irradiation on VSMC proliferation, cellular ATP levels, and AMPK/mTOR/p70S6K signaling axis is FIR ray’s peculiar effect, not the hyperthermal effect. (A, B) Rat VSMCs were incubated for 30 minutes at 39°C using the heat block, at 37°C in the culture incubator, or at 25°C (RT), or exposed to FIR radiation for 30 minutes at 25°C. Levels of p-mTOR-Ser2448, p-p70S6K-Thr389, and p-AMPK-Thr172 were detected by western blotting. (C) The cells were treated as described above, and cellular ATP levels were assessed using a luminescent ATP detection assay kit as described in the Methods. (D) Cells were treated as described above, and cell proliferation was measured using an MTT assay as described in the Methods. All experiments were independently performed at least four times, and blots shown are representative of at least four experiments (n = 4). Bar graphs depict the mean fold alterations above/below the controls (± standard deviation).FIR = Far-infrared, VSMC = vascular smooth muscle cell, ATP = adenosine 5′-triphosphate, AMPK = AMP-activated protein kinase, mTOR = mammalian target of rapamycin, p70S6K = p70 S6 kinase, RT = room temperature, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.Differences were considered statistically significant at **P < 0.01, ***P < 0.001, and ###P < 0.001.

• 

  Fig. 5 FIR irradiation attenuates PDGF-stimulated VSMC proliferation by inhibiting AMPK/mTOR/p70S6K signaling axis. (A) Rat VSMCs were irradiated with FIR ray for 30 minutes, followed by incubation for 24 hours in the absence or presence of 10 ng/mL PDGF, and then cell proliferation was measured by MTT assay, as described in the Methods. (B, C) After rat VSMCs were transfected with rat dn-AMPKα1 (D157A) gene or empty vector, cells were irradiated with FIR ray for 30 minutes, followed by incubation for 24 hours in the absence or presence of 10 ng/mL PDGF. The levels of p-AMPK-Thr172, p-mTOR-Ser2448, and p-p70S6K-Thr389 were detected by western blotting. (D) Cells were treated as described above, and 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). The bar graphs depict mean fold alterations above/below the controls (± standard deviation).FIR = Far-infrared, PDGF = platelet-derived growth factor, VSMC = vascular smooth muscle cell, AMPK = AMP-activated protein kinase, mTOR = mammalian target of rapamycin, p70S6K = p70 S6 kinase, MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, dn = dominant negative, RT = room temperature, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.Differences were considered statistically significant at *P < 0.05, #P < 0.05, **P < 0.01, and ##P < 0.01.

• 

  Fig. 6 A schematic illustration of FIR irradiation-inhibited VSMC proliferation. FIR irradiation reduces the cellular ATP level, which increases p-AMPK-Thr172. Increased AMPK activity decreases p-mTOR-Ser2448 and p-p70S6K-Thr389. Finally, FIR irradiation-activated AMPK inhibits basal and PDGF-stimulated VSMC proliferation by decreasing mTOR/p70S6K signaling pathway.FIR = Far-infrared, ATP = adenosine 5′-triphosphate, AMPK = AMP-activated protein kinase, mTOR = mammalian target of rapamycin, p70S6K = p70 S6 kinase, PDGF = platelet-derived growth factor, VSMC = vascular smooth muscle cell.

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