Non-Lethal Sonodynamic Therapy Inhibits Atherosclerotic Plaque Progression in ApoE-/- Mice and Attenuates ox-LDL-mediated Macrophage Impairment by Inducing Heme Oxygenase-1

Cell Physiol Biochem. 2017;41(6):2432-2446. doi: 10.1159/000475913. Epub 2017 May 3.

Abstract

Background: Previous studies from our group showed that low-intensity sonodynamic therapy (SDT) has protective effects on atherosclerosis (AS). However, because the intensity of ultrasound passing through tissue is attenuated, the consequences of very low-intensity SDT, referred to as non-lethal SDT (NL-SDT), on atherosclerotic plaques are unclear. The aim of this study was to determine whether NL-SDT affects atherosclerotic plaques and to elucidate the possible underlying mechanisms.

Methods: An AS model was established using ApoE-/- mice fed a western diet. En face Oil Red O staining was used to measure atherosclerotic plaque size. Hematoxylin and eosin staining and immunohistochemical staining were used to observe plaque morphology and assess the location of macrophages and heme oxygenase 1 (HO-1). HO-1 mRNA and protein levels in AS plaques were evaluated by real-time PCR and western blotting. Human THP-1 cells and mouse peritoneal macrophages were used in this study. Western blotting was used to investigate the expression of cellular proteins after NL-SDT. Macrophage apoptosis was evaluated by TUNEL assays and flow cytometry with Annexin V/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with 2'-7'-dichlorofluorescein diacetate (DCFH-DA) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolyl carbocyanine iodide (JC-1) staining, respectively.

Results: NL-SDT significantly inhibited AS progression and reduced the necrotic core area. NL-SDT induced HO-1 expression in lesional macrophages and in cultured macrophages. NL-SDT activated the protein kinase B (AKT) and extracellular signal-related protein kinase (ERK) pathways and the transcription factor NF-E2-related factor 2 (Nrf2).NL-SDT significantly reduced oxidized LDL (ox-LDL)-induced macrophage MMP collapse, ROS production and cell apoptosis. Zinc protoporphyrin (ZnPP), a HO-1-specific inhibitor, reversed the protective effects of NL-SDT.

Conclusion: NL-SDT inhibits atherosclerotic plaque progression and increases plaque stability. In vitro, NL-SDT has a protective effect on ox-LDL-induced macrophage impairment via HO-1.

Keywords: AKT; Atherosclerotic plaque; ERK; HO-1; Macrophage; Nrf2; SDT.

MeSH terms

  • Animals
  • Apolipoproteins E / deficiency
  • Apolipoproteins E / genetics
  • Apoptosis / drug effects*
  • Atherosclerosis / metabolism
  • Atherosclerosis / pathology
  • Atherosclerosis / therapy*
  • Cells, Cultured
  • Disease Models, Animal
  • Disease Progression
  • Gene Expression Regulation, Enzymologic*
  • Heme Oxygenase-1 / antagonists & inhibitors
  • Heme Oxygenase-1 / genetics*
  • Heme Oxygenase-1 / metabolism*
  • Humans
  • Lipoproteins, LDL / toxicity*
  • Macrophages / cytology
  • Macrophages / drug effects
  • Macrophages / metabolism
  • Male
  • Membrane Potential, Mitochondrial / drug effects
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Protoporphyrins / toxicity
  • RNA Interference
  • Reactive Oxygen Species / metabolism
  • Ultrasonic Therapy

Substances

  • Apolipoproteins E
  • Lipoproteins, LDL
  • Protoporphyrins
  • Reactive Oxygen Species
  • oxidized low density lipoprotein
  • zinc protoporphyrin
  • Heme Oxygenase-1