Restricted accessResearch articleFirst published online 2023-01
Inhibition of Trimethylamine N-Oxide Attenuates Neointimal Formation Through Reduction of Inflammasome and Oxidative Stress in a Mouse Model of Carotid Artery Ligation
Trimethylamine-N-oxide (TMAO) is a metabolite generated from dietary choline, betaine, and l-carnitine, after their oxidization in the liver. TMAO has been identified as a novel independent risk factor for atherosclerosis through the induction of vascular inflammation. However, the effect of TMAO on neointimal formation in response to vascular injury remains unclear.
Results:
This study was conducted using a murine model of acutely disturbed flow-induced atherosclerosis induced by partial carotid artery ligation. 3,3-Dimethyl-1-butanol (DMB) was used to reduce TMAO concentrations. Wild-type mice were divided into four groups [regular diet, high-TMAO diet, high-choline diet, and high-choline diet+DMB] to investigate the effects of TMAO elevation and its inhibition by DMB. Mice fed high-TMAO and high-choline diets had significantly enhanced neointimal hyperplasia and advanced plaques, elevated arterial elastin fragmentation, increased macrophage infiltration and inflammatory cytokine secretion, and enhanced activation of nuclear factor (NF)-κB, the NLRP3 inflammasome, and endoplasmic reticulum (ER) stress relative to the control group. Mice fed high-choline diets with DMB treatment exhibited attenuated flow-induced atherosclerosis, inflammasome expression, ER stress, and reactive oxygen species expression. Human aortic smooth muscle cells (HASMCs) were used to investigate the mechanism of TMAO-induced injury. The HASMCs were treated with TMAO with or without an ER stress inhibitor to determine whether inhibition of ER stress modulates the TMAO-induced inflammatory response.
Innovation:
This study demonstrates that TMAO regulates vascular remodeling via ER stress.
Conclusion:
Our findings demonstrate that TMAO elevation promotes disturbed flow-induced atherosclerosis and that DMB administration mitigates vascular remodeling, suggesting a rationale for a TMAO-targeted strategy for the treatment of atherosclerosis. Antioxid. Redox Signal. 38, 215–233.
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References
1.
BakerM, PerazellaMA. NSAIDs in CKD: Are they safe?. Am J Kidney Dis, 2020; 76(4):546–557; doi: 10.1053/j.ajkd.2020.03.023.
2.
BennettBJ, de Aguiar VallimTQ, WangZ, et al.Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab, 2013; 17(1):49–60; doi: 10.1016/j.cmet.2012.12.011.
3.
BentzonJF, OtsukaF, VirmaniR, et al.Mechanisms of plaque formation and rupture. Circ Res, 2014; 114(12):1852–1866; doi: 10.1161/CIRCRESAHA.114.302721.
4.
BoiniKM, HussainT, LiPL, et al.Trimethylamine-N-oxide instigates NLRP3 inflammasome activation and endothelial dysfunction. Cell Physiol Biochem, 2017; 44(1):152–162; doi: 10.1159/000484623.
5.
BolisettyS, JaimesEA. Mitochondria and reactive oxygen species: Physiology and pathophysiology. Int J Mol Sci, 2013; 14(3):6306–6344; doi: 10.3390/ijms14036306.
6.
BrandMD. The sites and topology of mitochondrial superoxide production. Exp Gerontol, 2010; 45(7–8):466–472; doi: 10.1016/j.exger.2010.01.003.
7.
ChenCY, TsaiHY, TsaiSH, et al.Deletion of the FHL2 gene attenuates intima-media thickening in a partially ligated carotid artery ligated mouse model. J Cell Mol Med, 2020; 24(1):160–173; doi: 10.1111/jcmm.14687.
8.
ChenH, LiJ, LiN, et al.Increased circulating trimethylamine N-oxide plays a contributory role in the development of endothelial dysfunction and hypertension in the RUPP rat model of preeclampsia. Hypertens Pregnancy, 2019a;38(2):96–104; doi: 10.1080/10641955.2019.1584630.
9.
ChenML, YiL, ZhangY, et al.Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota. mBio, 2016; 7(2):e02210-15; doi: 10.1128/mBio.02210-15.
10.
ChenML, ZhuXH, RanL, et al.Trimethylamine-N-oxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-MtROS signaling pathway. J Am Heart Assoc, 2017; 6(9); doi: 10.1161/JAHA.117.006347.
11.
ChenS, HendersonA, PetrielloMC, et al.Trimethylamine N-oxide binds and activates PERK to promote metabolic dysfunction. Cell Metab, 2019b;30(6):1141–1151.e5; doi: 10.1016/j.cmet.2019.08.021.
12.
ChouRH, ChenCY, ChenIC, et al.Trimethylamine N-oxide, circulating endothelial progenitor cells, and endothelial function in patients with stable angina. Sci Rep, 2019; 9(1):4249; doi: 10.1038/s41598-019-40638-y.
13.
CollinsHL, Drazul-SchraderD, SulpizioAC, et al.L-carnitine intake and high trimethylamine N-oxide plasma levels correlate with low aortic lesions in ApoE(−/−) transgenic mice expressing CETP. Atherosclerosis, 2016; 244:29–37; doi: 10.1016/j.atherosclerosis.2015.10.108.
14.
DarlingNJ, CookSJ. The role of MAPK signalling pathways in the response to endoplasmic reticulum stress. Biochim Biophys Acta, 2014; 1843(10):2150–2163; doi: 10.1016/j.bbamcr.2014.01.009.
15.
DelmotteP, SieckGC. Endoplasmic reticulum stress and mitochondrial function in airway smooth muscle. Front Cell Dev Biol, 2019; 7:374; doi: 10.3389/fcell.2019.00374.
16.
Dos Anjos CassadoA. F4/80 as a Major Macrophage Marker: The case of the peritoneum and spleen. Results Probl Cell Differ, 2017; 62:161–179; doi: 10.1007/978-3-319-54090-0_7.
17.
GennaroG, MenardC, MichaudSE, et al.Inhibition of vascular smooth muscle cell proliferation and neointimal formation in injured arteries by a novel, oral mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor. Circulation, 2004; 110(21):3367–3371; doi: 10.1161/01.CIR.0000147773.86866.CD.
18.
GomezC, MartinezL, MesaA, et al.Oxidative stress induces early-onset apoptosis of vascular smooth muscle cells and neointima formation in response to injury. Biosci Rep, 2015; 35(4); doi: 10.1042/BSR20140122.
GuptaN, BuffaJA, RobertsAB, et al.Targeted inhibition of gut microbial trimethylamine N-oxide production reduces renal tubulointerstitial fibrosis and functional impairment in a murine model of chronic kidney disease. Arter Thromb Vasc Biol, 2020; 40(5):1239–1255; doi: 10.1161/ATVBAHA.120.314139.
21.
HalperJ, KjaerM. Basic components of connective tissues and extracellular matrix: Elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins. Adv Exp Med Biol, 2014; 802:31–47; doi: 10.1007/978-94-007-7893-1_3.
22.
HasegawaY, ChenSY, ShengL, et al.Long-term effects of western diet consumption in male and female mice. Sci Rep, 2020; 10(1):14686; doi: 10.1038/s41598-020-71592-9.
23.
HuangCY, LaiCH, KuoCH, et al.Inhibition of ERK-Drp1 signaling and mitochondria fragmentation alleviates IGF-IIR-induced mitochondria dysfunction during heart failure. J Mol Cell Cardiol, 2018a;122:58–68; doi: 10.1016/j.yjmcc.2018.08.006.
24.
HuangF, ZhangF, XuD, et al.Enterococcus faecium WEFA23 from infants lessens high-fat-diet-induced hyperlipidemia via cholesterol 7-alpha-hydroxylase gene by altering the composition of gut microbiota in rats. J Dairy Sci, 2018b;101(9):7757–7767; doi: 10.3168/jds.2017-13713.
25.
IshimuraS, FuruhashiM, MitaT, et al.Reduction of endoplasmic reticulum stress inhibits neointima formation after vascular injury. Sci Rep, 2014; 4:6943; doi: 10.1038/srep06943.
26.
JohriAM, HeylandDK, HetuMF, et al.Carnitine therapy for the treatment of metabolic syndrome and cardiovascular disease: Evidence and controversies. Nutr Metab Cardiovasc Dis, 2014; 24(8):808–814; doi: 10.1016/j.numecd.2014.03.007.
27.
KimSY, KwonYW, JungIL, et al.Tauroursodeoxycholate (TUDCA) inhibits neointimal hyperplasia by suppression of ERK via PKCalpha-mediated MKP-1 induction. Cardiovasc Res, 2011; 92(2):307–316; doi: 10.1093/cvr/cvr219.
28.
KoethRA, LevisonBS, CulleyMK, et al.Gamma-butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO. Cell Metab, 2014; 20(5):799–812; doi: 10.1016/j.cmet.2014.10.006.
29.
KoethRA, WangZ, LevisonBS, et al.Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med, 2013; 19(5):576–585; doi: 10.1038/nm.3145.
30.
KokaS, XiaM, ChenY, et al.Endothelial NLRP3 inflammasome activation and arterial neointima formation associated with acid sphingomyelinase during hypercholesterolemia. Redox Biol, 2017; 13:336–344; doi: 10.1016/j.redox.2017.06.004.
31.
KuDN, GiddensDP, ZarinsCK, et al.Pulsatile flow and atherosclerosis in the human carotid bifurcation. Positive correlation between plaque location and low oscillating shear stress. Arteriosclerosis, 1985; 5(3):293–302; doi: 10.1161/01.atv.5.3.293.
32.
KurilshikovA, van den MunckhofICL, ChenL, et al.Gut microbial associations to plasma metabolites linked to cardiovascular phenotypes and risk. Circ Res, 2019; 124(12):1808–1820; doi: 10.1161/CIRCRESAHA.118.314642.
33.
KuzanA, WisniewskiJ, MaksymowiczK, et al.Relationship between calcification, atherosclerosis and matrix proteins in the human aorta. Folia Histochem Cytobiol, 2021; 59(1):8–21; doi: 10.5603/FHC.a2021.0002.
34.
LansingAI, AlexM, RosenthalTB. Calcium and elastin in human arteriosclerosis. J Gerontol, 1950; 5(2):112–119; doi: 10.1093/geronj/5.2.112.
35.
LibbyP, RidkerPM, HanssonGK. Progress and challenges in translating the biology of atherosclerosis. Nature, 2011; 473(7347):317–325; doi: 10.1038/nature10146.
36.
LimS, ParkS. Role of vascular smooth muscle cell in the inflammation of atherosclerosis. BMB Rep, 2014; 47(1):1–7; doi: 10.5483/bmbrep.2014.47.1.285.
37.
MaG, PanB, ChenY, et al.Trimethylamine N-oxide in atherogenesis: Impairing endothelial self-repair capacity and enhancing monocyte adhesion. Biosci Rep, 2017; 37(2); doi: 10.1042/BSR20160244.
MenteA, ChalcraftK, AkH, et al.The relationship between trimethylamine-N-oxide and prevalent cardiovascular disease in a multiethnic population living in Canada. Can J Cardiol, 2015; 31(9):1189–1194; doi: 10.1016/j.cjca.2015.06.016.
40.
MessengerJ, ClarkS, MassickS, et al.A review of trimethylaminuria: (Fish Odor Syndrome). J Clin Aesthet Dermatol, 2013; 6(11):45–48.
41.
MohammadiA, GholamhoseyniannajarA, YaghoobiMM, et al.Expression levels of heat shock protein 60 and glucose-regulated protein 78 in response to trimethylamine-N-oxide treatment in murine macrophage J774A.1 cell line. Cell Mol Biol Noisy—Gd, 2015; 61(4):94–100.
42.
MohammadiA, VahabzadehZ, JamalzadehS, et al.Trimethylamine-N-oxide, as a risk factor for atherosclerosis, induces stress in J774A.1 murine macrophages. Adv Med Sci, 2018; 63(1):57–63; doi: 10.1016/j.advms.2017.06.006.
43.
NodaT, MaedaK, HayanoS, et al.New endoplasmic reticulum stress regulator, gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury. Arter Thromb Vasc Biol, 2015; 35(5):1246–1253; doi: 10.1161/ATVBAHA.114.304923.
44.
NowinskiA, UfnalM. Trimethylamine N-oxide: A harmful, protective or diagnostic marker in lifestyle diseases?. Nutrition, 2018; 46:7–12; doi: 10.1016/j.nut.2017.08.001.
45.
OchoaCD, WuRF, TeradaLS. ROS signaling and ER stress in cardiovascular disease. Mol Asp Med, 2018; 63:18–29; doi: 10.1016/j.mam.2018.03.002.
46.
PattenRD. Models of gender differences in cardiovascular disease. Drug Discov Today Models, 2007; 4(4):227–232; doi: 10.1016/j.ddmod.2007.11.002.
47.
PengW, CaiG, XiaY, et al.Mitochondrial dysfunction in atherosclerosis. DNA Cell Biol, 2019; 38(7):597–606; doi: 10.1089/dna.2018.4552.
48.
QiuL, TaoX, XiongH, et al.Lactobacillus plantarum ZDY04 exhibits a strain-specific property of lowering TMAO via the modulation of gut microbiota in mice. Food Funct, 2018; 9(8):4299–4309; doi: 10.1039/c8fo00349a.
49.
RandrianarisoaE, Lehn-StefanA, WangX, et al.Relationship of serum trimethylamine N-oxide (TMAO) levels with early atherosclerosis in humans. Sci Rep, 2016; 6:26745; doi: 10.1038/srep26745.
50.
RavaniA, WerbaJP, FrigerioB, et al.Assessment and relevance of carotid intima-media thickness (C-IMT) in primary and secondary cardiovascular prevention. Curr Pharm Des, 2015; 21(9):1164–1171; doi: 10.2174/1381612820666141013121545.
51.
RobertsAB, GuX, BuffaJA, et al.Development of a gut microbe-targeted nonlethal therapeutic to inhibit thrombosis potential. Nat Med, 2018; 24(9):1407–1417; doi: 10.1038/s41591-018-0128-1.
52.
RossR, GlomsetJA. Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science, 1973; 180(4093):1332–1339; doi: 10.1126/science.180.4093.1332.
53.
RyanMJ, DidionSP, DavisDR, et al.Endothelial dysfunction and blood pressure variability in selected inbred mouse strains. Arter Thromb Vasc Biol, 2002; 22(1):42–48; doi: 10.1161/hq0102.101098.
54.
SamulakJJ, SawickaAK, HartmaneD, et al.L-carnitine supplementation increases trimethylamine-N-oxide but not markers of atherosclerosis in healthy aged women. Ann Nutr Metab, 2019; 74(1):11–17; doi: 10.1159/000495037.
55.
SeldinMM, MengY, QiH, et al.Trimethylamine N-oxide promotes vascular inflammation through signaling of mitogen-activated protein kinase and nuclear factor-kappaB. J Am Heart Assoc, 2016; 5(2); doi: 10.1161/JAHA.115.002767.
56.
SharmaRB, DarkoC, AlonsoLC. Intersection of the ATF6 and XBP1 ER stress pathways in mouse islet cells. J Biol Chem, 2020; 295(41):14164–14177; doi: 10.1074/jbc.RA120.014173.
57.
ShiY, HuJ, GengJ, et al.Berberine treatment reduces atherosclerosis by mediating gut microbiota in ApoE−/− mice. Biomed Pharmacother, 2018; 107:1556–1563; doi: 10.1016/j.biopha.2018.08.148.
58.
ShihDM, WangZ, LeeR, et al.Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis. J Lipid Res, 2015; 56(1):22–37; doi: 10.1194/jlr.M051680.
59.
SunX, JiaoX, MaY, et al.Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome. Biochem Biophys Res Commun, 2016; 481(1–2):63–70; doi: 10.1016/j.bbrc.2016.11.017.
60.
TangWH, HazenSL. The contributory role of gut microbiota in cardiovascular disease. J Clin Invest, 2014; 124(10):4204–4211; doi: 10.1172/JCI72331.
61.
TangWH, WangZ, LevisonBS, et al.Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med, 2013; 368(17):1575–1584; doi: 10.1056/NEJMoa1109400.
62.
ThomsonJ, SinghM, EckersleyA, et al.Fibrillin microfibrils and elastic fibre proteins: functional interactions and extracellular regulation of growth factors. Semin Cell Dev Biol, 2019; 89:109–117; doi: 10.1016/j.semcdb.2018.07.016.
63.
TripoltNJ, LeberB, TrieblA, et al.Effect of Lactobacillus casei Shirota supplementation on trimethylamine-N-oxide levels in patients with metabolic syndrome: An open-label, randomized study. Atherosclerosis, 2015; 242(1):141–144; doi: 10.1016/j.atherosclerosis.2015.05.005.
64.
VanderLaanPA, ReardonCA, GetzGS. Site specificity of atherosclerosis: Site-selective responses to atherosclerotic modulators. Arter Thromb Vasc Biol, 2004; 24(1):12–22; doi: 10.1161/01.ATV.0000105054.43931.f0.
65.
VeeravalliS, KaruK, ScottF, et al.Effect of flavin-containing monooxygenase genotype, mouse strain, and gender on trimethylamine N-oxide production, plasma cholesterol concentration, and an index of atherosclerosis. Drug Metab Dispos, 2018; 46(1):20–25; doi: 10.1124/dmd.117.077636.
66.
WangZ, KlipfellE, BennettBJ, et al.Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature, 2011; 472(7341):57–63; doi: 10.1038/nature09922.
67.
WangZ, RobertsAB, BuffaJA, et al.Non-lethal inhibition of gut microbial trimethylamine production for the treatment of atherosclerosis. Cell, 2015; 163(7):1585–1595; doi: 10.1016/j.cell.2015.11.055.
68.
WangZ, TangWH, BuffaJA, et al.Prognostic value of choline and betaine depends on intestinal microbiota-generated metabolite trimethylamine-N-oxide. Eur Heart J, 2014; 35(14):904–910; doi: 10.1093/eurheartj/ehu002.
69.
WarrierM, ShihDM, BurrowsAC, et al.The TMAO-generating enzyme flavin monooxygenase 3 is a central regulator of cholesterol balance. Cell Rep, 2015; 10(3):326–338; doi: 10.1016/j.celrep.2014.12.036.
70.
YangS, LiX, YangF, et al.Gut microbiota-dependent marker TMAO in promoting cardiovascular disease: inflammation mechanism, clinical prognostic, and potential as a therapeutic target. Front Pharmacol, 2019; 10:1360; doi: 10.3389/fphar.2019.01360.
71.
YoshidaH, MatsuiT, YamamotoA, et al.XBP1 MRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell, 2001; 107(7):881–891; doi: 10.1016/s0092-8674(01)00611-0.
72.
ZhuW, GregoryJC, OrgE, et al.Gut microbial metabolite TMAO enhances platelet hyperreactivity and thrombosis risk. Cell, 2016; 165(1):111–124; doi: 10.1016/j.cell.2016.02.011.
73.
ZhuW, WangZ, TangWHW, et al.Gut microbe-generated trimethylamine N-oxide from dietary choline is prothrombotic in subjects. Circulation, 2017; 135(17):1671–1673; doi: 10.1161/CIRCULATIONAHA.116.025338.
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