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.


Reference

1. Locher R, Brandes RP, Vetter W, Barton M. Native LDL induces proliferation of human vascular smooth muscle cells via redoxmediated activation of ERK 1/2 mitogen-activated protein kinases. Hypertension. 2002; 39(2 Pt 2):645–650.
Article
2. Sachinidis A, Mengden T, Locher R, Brunner C, Vetter W. Novel cellular activities for low density lipoprotein in vascular smooth muscle cells. Hypertension. 1990; 15(6 Pt 2):704–711.
Article
3. Park IH, Hwang HM, Jeon BH, Kwon HJ, Hoe KL, Kim YM, et al. NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells. Exp Mol Med. 2015; 47:e168.
Article
4. Chidambara Murthy KN, Jayaprakasha GK, Kumar V, Rathore KS, Patil BS. Citrus limonin and its glucoside inhibit colon adenocarcinoma cell proliferation through apoptosis. J Agric Food Chem. 2011; 59:2314–2323.
Article
5. Vanamala J, Leonardi T, Patil BS, Taddeo SS, Murphy ME, Pike LM, et al. Suppression of colon carcinogenesis by bioactive compounds in grapefruit. Carcinogenesis. 2006; 27:1257–1265.
Article
6. Miller EG, Fanous R, Rivera-Hidalgo F, Binnie WH, Hasegawa S, Lam LK. The effect of citrus limonoids on hamster buccal pouch carcinogenesis. Carcinogenesis. 1989; 10:1535–1537.
Article
7. Tanaka T, Maeda M, Kohno H, Murakami M, Kagami S, Miyake M, et al. Inhibition of azoxymethane-induced colon carcinogenesis in male F344 rats by the citrus limonoids obacunone and limonin. Carcinogenesis. 2001; 22:193–198.
Article
8. Kim J, Jayaprakasha GK, Muthuchamy M, Patil BS. Structure-function relationships of citrus limonoids on p38 MAP kinase activity in human aortic smooth muscle cells. Eur J Pharmacol. 2011; 670:44–49.
Article
9. Wei LH, Wu G, Morris SM Jr, Ignarro LJ. Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation. Proc Natl Acad Sci U S A. 2001; 98:9260–9264.
Article
10. Peyton KJ, Ensenat D, Azam MA, Keswani AN, Kannan S, Liu XM, et al. Arginase promotes neointima formation in rat injured carotid arteries. Arterioscler Thromb Vasc Biol. 2009; 29:488–494.
Article
11. Ryoo S, Gupta G, Benjo A, Lim HK, Camara A, Sikka G, et al. Endothelial arginase II: a novel target for the treatment of atherosclerosis. Circ Res. 2008; 102:923–932.
12. Wang Y, Lindstedt KA, Kovanen PT. Mast cell granule remnants carry LDL into smooth muscle cells of the synthetic phenotype and induce their conversion into foam cells. Arterioscler Thromb Vasc Biol. 1995; 15:801–810.
Article
13. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91:406–413.
14. Ryoo S, Lemmon CA, Soucy KG, Gupta G, White AR, Nyhan D, et al. Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling. Circ Res. 2006; 99:951–960.
Article
15. Palmer AE, Tsien RY. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc. 2006; 1:1057–1065.
Article
16. Wu G, Pond WG, Flynn SP, Ott TL, Bazer FW. Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation. J Nutr. 1998; 128:2395–2402.
Article
17. Baydoun AR, Emery PW, Pearson JD, Mann GE. Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endothelial cells. Biochem Biophys Res Commun. 1990; 173:940–948.
Article
18. Block ER, Herrera H, Couch M. Hypoxia inhibits L-arginine uptake by pulmonary artery endothelial cells. Am J Physiol. 1995; 269(5 Pt 1):L574–L580.
Article
19. Gold ME, Bush PA, Ignarro LJ. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun. 1989; 164:714–721.
Article
20. Hecker M, Sessa WC, Harris HJ, Anggard EE, Vane JR. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: cultured endothelial cells recycle L-citrulline to L-arginine. Proc Natl Acad Sci U S A. 1990; 87:78612–78616.
Article
21. Bleeke T, Zhang H, Madamanchi N, Patterson C, Faber JE. Catecholamine-induced vascular wall growth is dependent on generation of reactive oxygen species. Circ Res. 2004; 94:37–45.
Article
22. Lavigne MC, Malech HL, Holland SM, Leto TL. Genetic demonstration of p47phox-dependent superoxide anion production in murine vascular smooth muscle cells. Circulation. 2001; 104:79–84.
Article
23. Touyz RM, Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res. 2002; 35:1001–1015.
Article
24. Yamakawa T, Tanaka S, Yamakawa Y, Kamei J, Numaguchi K, Motley ED, et al. Lysophosphatidylcholine activates extracellular signal-regulated kinases 1/2 through reactive oxygen species in rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2002; 22:752–758.
Article
25. Adachi T, Pimentel DR, Heibeck T, Hou X, Lee YJ, Jiang B, et al. S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells. J Biol Chem. 2004; 279:29857–29862.
Article
26. Dang PM, Stensballe A, Boussetta T, Raad H, Dewas C, Kroviarski Y, et al. A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites. J Clin Invest. 2006; 116:2033–2043.
Article
27. Ni W, Zhan Y, He H, Maynard E, Balschi JA, Oettgen P. Ets-1 is a critical transcriptional regulator of reactive oxygen species and p47(phox) gene expression in response to angiotensin II. Circ Res. 2007; 101:985–994.
Article
28. Dikalova AE, Bikineyeva AT, Budzyn K, Nazarewicz RR, McCann L, Lewis W, et al. Therapeutic targeting of mitochondrial superoxide in hypertension. Circ Res. 2010; 107:106–116.
Article
29. Joo HK, Lee YR, Choi S, Park MS, Kang G, Kim CS, et al. Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells. Korean J Physiol Pharmacol. 2017; 21:377–384.
Article
30. Nguyen MC, Park JT, Jeon YG, Jeon BH, Hoe KL, Kim YM, et al. Arginase inhibition restores peroxynitrite-induced endothelial dysfunction via L-arginine-dependent endothelial nitric oxide synthase phosphorylation. Yonsei Med J. 2016; 57:1329–1338.
Article
31. Wei LH, Jacobs AT, Morris SM Jr, Ignarro LJ. IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells. Am J Physiol Cell Physiol. 2000; 279:C248–C256.
Article
32. Durante W, Liao L, Reyna SV, Peyton KJ, Schafer AI. Transforming growth factor-beta(1) stimulates L-arginine transport and metabolism in vascular smooth muscle cells: role in polyamine and collagen synthesis. Circulation. 2001; 103:1121–1127.
Article
33. Durante W, Liao L, Peyton KJ, Schafer AI. Lysophosphatidylcholine regulates cationic amino acid transport and metabolism in vascular smooth muscle cells. Role in polyamine biosynthesis. J Biol Chem. 1997; 272:30154–30159.
Article
34. Durante W, Liao L, Reyna SV, Peyton KJ, Schafer AI. Physiological cyclic stretch directs L-arginine transport and metabolism to collagen synthesis in vascular smooth muscle. FASEB J. 2000; 14:1775–1783.
Article
35. Yoon J, Ryoo S. Arginase inhibition reduces interleukin-1β-stimulated vascular smooth muscle cell proliferation by increasing nitric oxide synthase-dependent nitric oxide production. Biochem Biophys Res Commun. 2013; 435:428–433.
Article
36. Dudek D, Legutko J, Heba G, Bartus S, Partyka L, Huk I, et al. L-arginine supplementation does not inhibit neointimal formation after coronary stenting in human beings: an intravascular ultrasound study. Am Heart J. 2004; 147:E12.
Article
37. Walker HA, McGing E, Fisher I, Böger RH, Bode-Böger SM, Jackson G, et al. Endothelium-dependent vasodilation is independent of the plasma L-arginine/ADMA ratio in men with stable angina: lack of effect of oral L-arginine on endothelial function, oxidative stress and exercise performance. J Am Coll Cardiol. 2001; 38:499–505.
Article
38. Wilson AM, Harada R, Nair N, Balasubramanian N, Cooke JP. Larginine supplementation in peripheral arterial disease: no benefit and possible harm. Circulation. 2007; 116:188–195.
Article
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