Yonsei Med J.  2018 May;59(3):366-375. 10.3349/ymj.2018.59.3.366.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation

Affiliations
  • 1Department of Biological Sciences, Kangwon National University, Chuncheon, Korea. ryoosw08@kangwon.ac.kr
  • 2Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea. hyunkyolim@yonsei.ac.kr
  • 3Department of Medical Biotechnology, Kangwon National University, Chuncheon, Korea.
  • 4Department of New Drug Discovery and Development, Chungnam National University, Daejeon, Korea.
  • 5Department of Biochemistry, Yonsei University, Seoul, Korea.
  • 6Department of Neurobiology, Kangwon National University, Chuncheon, Korea.
  • 7Department of Molecular and Cellular Biochemistry, Kangwon National University, Chuncheon, Korea.

Abstract

PURPOSE
Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms.
MATERIALS AND METHODS
Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin.
RESULTS
Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation.
CONCLUSION
Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.

Keyword

Arginase inhibitor; vascular smooth muscle cells; cell proliferation; NADPH oxidase; native low-density lipoprotein; limonin

MeSH Terms

Animals
Aorta
Arginase*
Arginine
Blotting, Western
Bromodeoxyuridine
Cell Proliferation*
Chromatography, High Pressure Liquid
Chromatography, Liquid
Lipoproteins
Luminescence
Membranes
Muscle, Smooth, Vascular*
NADP*
NADPH Oxidase*
Phosphorylation
Phosphotransferases
Protein Kinase C
Rats
Reactive Oxygen Species
Superoxides
Arginase
Arginine
Bromodeoxyuridine
Lipoproteins
NADP
NADPH Oxidase
Phosphotransferases
Protein Kinase C
Reactive Oxygen Species
Superoxides

Figure

  • Fig. 1 Limonin prevents nLDL-induced VSMC proliferation. (A) Structure of limonin. Effects of limonin on inhibition of (B) arginase I (C) and II (D) activity at difference concentrations. Enzyme solutions for arginase I and II were prepared from liver and kidney lysates of rats, respectively. DMSO was used as a control. Effects of limonin treatment on nLDL-induced VSMC proliferation in WST-1 (E) and BrdU (F) assays. Effects of limonin on upregulation of p21Waf1/Cip1 (G) and arginase isoforms (H). *p<0.01 vs. untreated cells; †p<0.01 vs. nLDL. VSMC, vascular smooth muscle cell; nLDL, native low-density lipoprotein.

  • Fig. 2 Limonin attenuates NADPH oxidase-dependent reactive oxygen species generation induced by nLDL stimulation. Effects of pre-incubation with limonin on PKCβII (A) and ERK1/2 (B) phosphorylation in nLDL-stimulated vascular smooth muscle cells. Effects of limonin on the translocation of p47phox from the cytosol to the membrane in response to nLDL (C). Effects of limonin on nLDL-induced superoxide production (D). *p<0.01 vs. untreated cells; †p<0.01 vs. nLDL. nLDL, native low-density lipoprotein; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; MAPK, mitogenactivated protein kinase.

  • Fig. 3 Limonin inhibits mitochondrial ROS-dependent PKCβII phosphorylation. (A) Effects of nLDL and limonin on mitochondrial ROS generation in vascular smooth muscle cells stained with MitoSOX-Red (100 nM) (Scale bar: 50 µm). (B) Effects of the mitochondrial ROS scavenger Mito-TEMPO on nLDL-induced PKCβII phosphorylation. *p<0.01 vs. untreated cells, †p<0.01 vs. nLDL. ROS, reactive oxygen species; PKC, protein kinase C; nLDL, native low-density lipoprotein.

  • Fig. 4 L-arginine inhibits nLDL-induced PKCβII phosphorylation, but not ERK1/2 phosphorylation. (A) The cells were incubated with various concentrations of limonin for 24 h, and arginase activity was measured. *p<0.01 vs. untreated cells. (B) Vascular smooth muscle cells were incubated with limonin for 24 h, and intracellular polyamine and L-arginine contents were measured using high-performance liquid chromatography. *p<0.01 vs. untreated cells; †p<0.01 vs. nLDL. (C) Effects of L-arginine pretreatment (1 mM, 30 min) on PKCβII and ERK1/2 phosphorylation following nLDL treatment (D). *p<0.01 vs. untreated cells, †p<0.01 vs. nLDL. nLDL, native low-density lipoprotein; PKC, protein kinase C; ERK, extracellular signal-regulated kinase.

  • Fig. 5 Schematic demonstrating the proposed mechanisms by which limonin prevents nLDL-stimulated VSMCs proliferation. In nLDL-stimulated VSMCs, ERK1/2 MAPK and PKCβII phosphorylation involved in the p47phox phosphorylation and NADPH oxidase activation. The superoxide anion from activated NADPH oxidase induced VSMCs proliferation. Limonin as an arginase inhibitor prevented phosphorylation of PKCβII by nLDL stimulation via decrease in mitochondrial reactive oxygen species formation. Therefore, arginase inhibitor, limonin, showed anti-proliferative effect in nLDL-stimulated VSMC. VSMC, vascular smooth muscle cell; nLDL, native low-density lipoprotein; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase.


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