Table 3.

Animal experiments and cell culture studying the causal relationship between TMAO and AS.

	Experimental models	Intervention	Main observations	References
Positive results	C57BL6/J mice	Feeding with choline- or TMAO-enriched diet before transverse aortic constriction	Either TMAO- or choline-enriched diets enhanced heart failure severity	[45]
	ApoE−/− mice Germ-free mice	Feeding with additional choline	The AS plaque area was increased compared with mice fed with a control diet	[5]
	Peritoneal macrophages	The mice were fed a diet with TMAO, betaine, or choline	(1) The mRNA levels of CD36 and SR-A1 were increased (2) The macrophages had excess cholesterol accumulation and foam cell formation	[5]
	ApoE−/− mice	Redundant L-carnitine or antibiotics were introduced into the diet	TMAO could suppress RCT and levels of liver BA synthetase and BA transporters and modulate the activity of cholesterol transporters in macrophages	[25]
	LDLR−/− mice	Chronic choline supplementation	Plasma TMAO concentrations were increased and inflammatory gene expression in vascular cells was increased	[90]
	FeCl3-induced carotid artery injury mice Germ-free mice	i.p. TMAO or feeding with diet (0.12% TMAO or 1% choline supplementation)	(1) Intestinal flora promoted the conversion of choline to TMAO (2) There was an association of choline, TMAO, and thrombus risk	[26]
	Platelets	Platelets were exposed to TMAO	TMAO could enhance platelet activation from multiple agonists by increasing the release of Ca2+ from intracellular stores
	LDLR−/− mice ApoE−/− mice Il23−/− mice, Il22−/− mice	The mice were fed with Western diet	IL-23 and its downstream target IL-22 relieved AS by inhibiting TMAO	[46]
	ApoE−/− mice/peritoneal macrophages and RAW264.7	The mice were fed a high-fat diet with or without TMAO for 8 weeks/the cells were treated with TMAO or ox-LDL	(1) TMAO promoted the AS in vivo and in vitro (2) The CD36/MAPK/JNK pathway may play a crucial role in TMAO-induced formation of foam cells	[51]
	Mice at 20-24 months of age and mice at 8-10 weeks of age	The mice were treated for 3-4 weeks with broad-spectrum poorly absorbed antibiotics	(1) The gut microbiota was an important mediator of age-related arterial dysfunction (2) Plasma TMAO was higher in aged mice (3) Endothelial dysfunction and oxidative stress were elevated with aging	[47]
Negative results	ApoE−/− mice	The mice were supplemented with choline at 8 weeks of age	No association was observed between TMAO and the risk of AS	[48]
	ApoE−/− mice	The mice were transfected with an adeno-associated viral vector containing the human CETP gene	TMAO slowed the aortic lesion formation in ApoE−/− mice	[49]
	LDLR−/− mice ApoE−/− mice	Dietary intervention using extra choline, betaine, or TMAO	Dietary choline, betaine, or TMAO supplementation did not induce AS development	[50]

AS: atherosclerosis; TMAO: trimethylamine N-oxide; ApoE^−/−: apolipoprotein E-deficient; RCT: reverse cholesterol transport; BA: bile acids; LDLR^−/−: lipoprotein receptor-deficient; ADP: adenosine diphosphate; RAW264.7: a macrophage cell line; ox-LDL: low-density lipoprotein; CD36: cluster of differentiation 36; IL: interleukin; MAPK: mitogen-activated protein kinases; JNK: c-Jun N-terminal kinase; SR-A1: scavenger receptor A1; CETP: cholesteryl ester transfer protein.