Korean J Physiol Pharmacol.  2020 May;24(3):241-248. 10.4196/kjpp.2020.24.3.241.

Heat shock protein 90 inhibitor AUY922 attenuates platelet-derived growth factor-BB-induced migration and proliferation of vascular smooth muscle cells

• Kim J
• Lee KP
• Kim BS
• Lee SJ
• Moon BS
• Baek S

• KMID: 2502506
• DOI: http://doi.org/10.4196/kjpp.2020.24.3.241

Abstract

Luminespib (AUY922), a heat shock proteins 90 inhibitor, has anti-neoplastic and antitumor effects. However, it is not clear whether AUY922 affects events in vascular diseases. We investigated the effects of AUY922 on the platelet-derived growth factor (PDGF)-BB-stimulated proliferation and migration of vascular smooth muscle cells (VSMC). VSMC viability was detected using the XTT (2,3-bis-(2-methoxy- 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reagent. To detect the attenuating effects of AUY922 on PDGF-BB-induced VSMCs migration in vitro, we performed the Boyden chamber and scratch wound healing assays. To identify AUY922- mediated changes in the signaling pathway, the phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) 1/2 was analyzed by immunoblotting. The inhibitory effects of AUY922 on migration and proliferation ex vivo were tested using an aortic ring assay. AUY922 was not cytotoxic at concentrations up to 5 nM. PDGF-BB-induced VSMC proliferation, migration, and sprout outgrowth were significantly decreased by AUY922 in a dose-dependent manner. AUY922 significantly reduced the PDGF-BB-stimulated phosphorylation of Akt and ERK1/2. Furthermore, PD98059 (a selective ERK1/2 inhibitor) and LY294002 (a selective Akt inhibitor) decreased VSMC migration and proliferation by inhibiting phosphorylation of Akt and ERK1/2. Greater attenuation of PDGF-BB-induced cell viability and migration was observed upon treatment with PD98059 or LY294002 in combination with AUY922. AUY922 showed anti-proliferation and anti-migration effects towards PDGF-BBinduced VSMCs by regulating the phosphorylation of ERK1/2 and Akt. Thus, AUY922 is a candidate for the treatment of atherosclerosis and restenosis.

Keyword

Atherosclerosis; AUY922; Heat shock protein 90; Platelet-derived growth factor; Vascular disease

Figure

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  Fig. 1 Effect of AUY922 on platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cells (VSMCs) proliferation. (A) Structure of AUY922. Molecular weight: 465.5 g/ml. (B) VSMCs were treated with 0.005% sodium dodecyl sulfate (vehicle, veh), 1% Triton X-100, and 5 nM AUY922 for 24 h. The cell morphologies were captured using the microscopy. Magnification, ×200. (C) VSMCs (2 × 104 cells/ml) were seeded into a 96-well plate for 24 h. Cells were exposed to difference concentrations of AUY922 (1, 2, 3, 5, 10, 20, 30, and 50 nM) for 24 h. Cell viability was determined using 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. (D) VSMCs were incubated with serum-free media for 24 h and treated with or without PDGF-BB (10 ng/ml) and AUY922 (1, 2, 3, and 5 nM) for 24 h. The viability at the quiescent state is expressed as 100%. Data are expressed as means ± standard deviation. Different superscript letters indicate significant differences based on Tukey's multiple range test (p < 0.05).

• 

  Fig. 2 Effect of AUY922 on platelet-derived growth factor (PDGF)-BB-induced in vitro and ex vivo models. (A) Vascular smooth muscle cells (VSMCs) treated with AUY922 (1, 2, 3, and 5 nM) for 90 min at 37°C. Cells were stained using a Diff-Quik Kit. (B) Cells were scratched with 200-µl tips and co-treated with PDGF-BB (10 ng/ml) and AUY922 (1, 2, 3, and 5 nM) for 24 h. The black solid line shows the scratch at 0 h and the white dotted line shows the scratch at 24 h. The wound healing area is expressed as 100%. Data are expressed as means ± standard deviation (SD). *Significant difference compared to the PDGF-BB group (p < 0.05). (C) Rat aortas were sliced to obtain 1-mm sections and embedded in Matrigel. The aortic rings were treated with or without PDGF-BB (10 ng/ml) and AUY922 (1, 2, 3, and 5 nM) for 4 days. The sprout area in the vehicle (veh) group is expressed as 100%. The photographs were captured using the microscopy. Magnification, ×200. Data are expressed as means ± SD. Different superscript letters indicate significant differences based on Tukey's multiple range test (p < 0.05).

• 

  Fig. 3 Western blot analysis of extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylation. (A) Vascular smooth muscle cells (VSMCs) were incubated in serum-free media and stimulated with platelet-derived growth factor (PDGF)-BB (10 ng/ml) and AUY922 (2, 3, and 5 nM) for 15 min. The cell lysates were separated by SDS-PAGE. Protein expression was detected using specific antibodies, such as anti-phosphorylated (p) ERK1/2, p-ERK1/2, Akt, and p-Akt. Graphs show the intensity of p-ERK1/2 and p-Akt, respectively. (B) VSMCs were stimulated in the presence or absence of PDGF-BB (10 ng/ml), PD98059 (30 μM), LY294002 (20 μM), and AUY922 (5 nM) for 30 min. Cells were lysed and protein expression was analyzed by western blotting. Data in graphs correspond to the images on the left. The viability at the quiescent state is expressed as 100%. Data are expressed as means ± standard deviation. Different superscript letters indicate significant differences based on Tukey's multiple range test (p < 0.05). Veh, vehicle.

• 

  Fig. 4 Extracellular signal-regulated kinase (ERK) 1/2 and Akt inhibitors decreased platelet-derived growth factor (PDGF)-BB-induced increases in vascular smooth muscle cells (VSMC) proliferation and migration. (A) VSMCs were treated with PDGF-BB, PD98059 (30 μM), LY294002 (20 μM), and AUY922 (5 nM) for 24 h. The proliferation of VSMCs was determined by the XTT reagent. The viability at the quiescent state is expressed as 100%. (C) Graphs were obtained by Panel B. Data are expressed as means ± standard deviation (SD). *Significant difference compared to the PDGF-BB group (p < 0.05). (B) VSMCs (1 × 106 cells/ml) incubated with PD98059 (30 μM), LY294002 (20 μM), and AUY922 (5 nM) in the upper chamber and co-stimulated with PDGF-BB (10 ng/ml) in the lower chamber for 90 min. Magnification, ×200. Data are expressed as means ± SD. Different superscript letters indicate significant differences based on Tukey's multiple range test (p < 0.05). Veh, vehicle.

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