J Lipid Atheroscler.  2019 Sep;8(2):267-276. 10.12997/jla.2019.8.2.267.

Anti-malarial Drugs Reduce Vascular Smooth Muscle Cell Proliferation via Activation of AMPK and Inhibition of Smad3 Signaling

• Lee H
• Han JH
• Kim S
• Kim S
• Cho DH
• Woo CH

• KMID: 2458392
• DOI: http://doi.org/10.12997/jla.2019.8.2.267

Abstract

OBJECTIVE
The aim of this study was to investigate the effects of 2 anti-malarial drugs, chloroquine (CQ) and hydroxychloroquine (HCQ), on inhibition of vascular smooth muscle cell (VSMC) proliferation both in vivo and in vitro via Adenosine monophosphate-activated protein kinase (AMPK) activation.
METHODS
Protein and mRNA levels were determined by western blot analysis and real-time reverse transcription-polymerase chain reaction in primary rat VSMCs treated with CQ and HCQ, respectively. Cell proliferation was measured by flow cytometry and cell counting. Mice carotid arteries were ligated and treated with CQ or HCQ every other day for 3 weeks. Pathological changes of carotid arteries were visualized by both microscopy and fluorescence microscopy.
RESULTS
CQ and HCQ increase AMPK phosphorylation in VSMCs. Both CQ and HCQ decrease platelet-derived growth factor-induced VSMC proliferation and cell cycle progression in an AMPK-dependent manner. In addition, CQ and HCQ inhibit Smad3 phosphorylation and VSMC proliferation induced by transforming growth factor-β1. Moreover, CQ and HCQ diminished neointimal proliferation in a mouse model of carotid artery ligation-induced neointima formation.
CONCLUSION
The results demonstrated that CQ and HCQ inhibit cell proliferation and cell cycle progression in VSMCs via the AMPK-dependent signaling pathway. Carotid artery ligation-induced intima thickness was reduced in mouse arteries treated with CQ or HCQ, suggesting a role for antimalarial drugs in treating atherosclerosis and restenosis.

Keyword

Chloroquine; Hydroxychloroquine; Vascular smooth muscle; AMP-activated protein kinase; Intima formation

MeSH Terms

Adenosine
AMP-Activated Protein Kinases*
Animals
Antimalarials
Arteries
Atherosclerosis
Blotting, Western
Carotid Arteries
Cell Count
Cell Cycle
Cell Proliferation*
Chloroquine
Flow Cytometry
Hydroxychloroquine
In Vitro Techniques
Mice
Microscopy
Microscopy, Fluorescence
Muscle, Smooth, Vascular*
Neointima
Phosphorylation
Protein Kinases
Rats
RNA, Messenger
AMP-Activated Protein Kinases
Adenosine
Antimalarials
Chloroquine
Hydroxychloroquine
Protein Kinases
RNA, Messenger

Figure

• 

  Fig. 1 Anti-malarial drugs increase AMPK phosphorylation in primary rat VSMCs. Primary rat VSMCs were treated with CQ or HCQ at the indicated concentrations for 1 hour. Phosphorylation of AMPK was determined by immunoblotting with anti-phospho-AMPK antibody. Amount of protein was normalized to total amount of AMPK and tubulin expression. Bar graphs present the densitometric quantification of western blot bands. Results are expressed as mean±standard deviation and are representative of 3 independent experiments. AMPK, adenosine monophosphate-activated protein kinase; VSMC, vascular smooth muscle cell; CQ, chloroquine; HCQ, hydroxychloroquine; p-AMPK, phospho-adenosine monophosphate-activated protein kinase. *p<0.05; †p<0.01 compared with controls.

• 

  Fig. 2 Anti-malarial drugs inhibit PDGF-induced cell proliferation and cell cycle progression in rat VSMCs. (A) Rat VSMCs were pretreated with CQ or HCQ (10, 20 μM) for 2 hours and then incubated with PDGF (10 ng/mL) for indicated times (1, 2, or 3 days). Cell proliferation was determined by cell counting. (B) Serum-starved rat VSMCs were pretreated with CQ or HCQ (20, 40 μM) for 2 hours and then incubated with PDGF (10 ng/mL) for 24 hours. Protein expression of cyclin D was determined by immunoblotting with anti-cyclin D antibody. Amount of protein was normalized to tubulin expression. Bar graphs present the densitometric quantification of western blot bands. Results are expressed as mean±standard deviation and are representative of 3 independent experiments. (C) Serum-starved rat VSMCs were pretreated with CQ or HCQ (20, 40 μM) for 2 hours and then incubated with PDGF (10 ng/mL) for 24 hours. For cell cycle analysis, cells were detached, stained with PI, and subjected to flow cytometry. Results are representative of 3 independent experiments that yielded similar results. PDGF, platelet-derived growth factor; VSMC, vascular smooth muscle cell; CQ, chloroquine; HCQ, hydroxychloroquine. *p<0.05; †p<0.01 compared with cells treated with PDGF.

• 

  Fig. 3 Anti-malarial drugs inhibit TGF-β1-induced Smad3 phosphorylation in rat VSMCs. (A) Rat VSMCs were pretreated with CQ or HCQ (40 μM) for 2 hours and then incubated with TGF-β1 (2 ng/mL) for 10 or 30 minutes. Phosphorylation of Smad3 and AMPK was determined by immunoblotting with anti-phospho-Smad3 and anti-phospho-AMPK antibodies, respectively. Amount of protein was normalized to total amount of Smad3, AMPK, and tubulin. Bar graphs present densitometric quantification of western blot bands. Results are expressed as mean±standard deviation and are representative of 3 independent experiments. (B) Rat VSMCs were pretreated with CQ or HCQ (10 μM) for 2 hours and then incubated with TGF-β1 (2 ng/mL) for 3 days. Cell viabilities were determined with the MTT assay. The data represent the mean values of 3 wells. (C) Rat VSMCs were pretreated with CQ or HCQ (10 μM) for 2 hours and then incubated with TGF-β1 (2 ng/mL) for 3 days. Cell proliferation was determined by cell counting. Results are representative of 3 independent experiments that yielded similar results. TGF-β1, transforming growth factor-β1; VSMC, vascular smooth muscle cell; CQ, chloroquine; HCQ, hydroxychloroquine; AMPK, adenosine monophosphate-activated protein kinase; p-AMPK, phospho-adenosine monophosphate-activated protein kinase; p-Smad3, phospho-Smad3. *p<0.05; †p<0.01 compared with controls. ‡p<0.01 vs. TGF-β1 2 ng/mL-treated. §p<0.01 vs. control, ∥p<0.01 vs. TGF-β1 2 ng/mL-treated.

• 

  Fig. 4 Carotid artery ligation-induced neointimal hyperplasia is significantly attenuated in animals treated with anti-malarial drugs. (A) Partial carotid ligation was performed on 8-week-old male C57BL/6 mice. After surgery, mice were treated with CQ (50 mg/kg, i.p.) or HCQ (50 mg/kg, i.p.) every other day for 3 weeks. Top panel: H&E staining of C57BL/6 arteries under 200× magnification. Elastin was visualized by autofluorescence. Images are representative of n=4. Bottom panel: graphical representation of the media, intima, and intima/media ratio (n=4; mean±standard error of mean). (B) C57BL/6 arteries were isolated and homogenized. Protein levels were measured by western blot analysis with specific antibodies against collagen I, collagen III, and tubulin. Results are representative of 3 independent experiments. CQ, chloroquine; HCQ, hydroxychloroquine; i.p., intraperitoneal; R, non-ligated right common carotid artery; L, ligated left common carotid artery; I, intima; M, media; H&E, hematoxylin and eosin; LCA, left common carotid artery; RCA, right common carotid artery. *p<0.05.

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