Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.
10.3349/ymj.2018.59.3.366
- Author:
Bon Hyeock KOO
1
;
Bong Gu YI
;
Wi Kwang WANG
;
In Young KO
;
Kwang Lae HOE
;
Young Guen KWON
;
Moo Ho WON
;
Young Myeong KIM
;
Hyun Kyo LIM
;
Sungwoo RYOO
Author Information
1. Department of Biological Sciences, Kangwon National University, Chuncheon, Korea. ryoosw08@kangwon.ac.kr
- Publication Type:Original Article
- Keywords:
Arginase inhibitor;
vascular smooth muscle cells;
cell proliferation;
NADPH oxidase;
native low-density lipoprotein;
limonin
- MeSH:
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
- From:Yonsei Medical Journal
2018;59(3):366-375
- CountryRepublic of Korea
- Language:English
-
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.
