Table 1.

Characteristics of animal studies.

Authors	n	Marker of VA	Intervention	Duration	Effect on Vascular Ageing	Mechanisms Linked to Microbiota
Guirro, M., 2020 [77]	48	Neuraminidase circulating levels	Hesperidin treatment; two diets for 9 wk (n = 24): standard diet and cafeteria (CAF) diet	9 weeks of diet + 8 weeks of hesperidin	CAF feeding resulted in increased endothelial dysfunction, arterial stiffness, and inflammation. Hesperidin supplementation reduced SBP and markers of arterial stiffness in CAF-fed rats	Urinary metabolites of hesperidin were positively correlated with Bacteroidaceae family.
Liu, H., 2020 [78]	35	PWV at the left common carotid artery	Gavage with feces from either healthy donors (controls) or myocardial infarction patients (CAD) + high fat diet	12 weeks	Mice treated with CAD feces had higher vascular stiffness than controls (Controls: 2.75 ± 0.29 m/s vs. CAD: 3.59 ± 0.27 m/s; p = 0.043). No BP data.	In mice treated with CAD feces: increased LPS and pro-inflammatory cytokines; increased activated TH17 cells; reduced Treg cells.
Battson, M.L., 2019 [79]	40	AorticPWV (doppler)	Fecal transplantation, 10 controls and 10 obese mice received healthy microbiota, and 10 and 10 received obese microbiota	8 weeks	Control mice receiving microbiota of obese subjects had higher PWV. Akkermansia abundance inversely related to PWV. No BP data.	Obese mice had reduced Clostridia and Oscillospira. Control mice and obese mice receiving microbiota of obese subjects had higher level of Bacteroides sp. Control mice receiving microbiota of obese subjects had reduced level of Akkermansia.
Natarajan, N., 2016 [29]	10	Aortic stiffness (PWV by doppler and ex vivo)	Gpr41 KO group vs Grp41 WT group	3 and 6 months	At 6 months PWV was significantly higher in KO mice vs WT mice, with similar compliance in ex vivo analysis, suggesting functional vascular alteration. KO mice presented isolated systolic hypertension at baseline	Gpr41 (SCFA receptor) localizes in the vascular endothelium. Vascular endothelium is essential for SCFA-mediated vasodilation to occur, as vasodilation is absent in endothelium-denuded vessels ex vivo.
Edwards, J.M., 2020 [83]	12	Resistance arteries stiffness (ex vivo)	Ex vivo evaluation of vascular stiffness		Resistance arteries from male GF mice present increased vascular stiffness. No changes in vascular stiffness in arteries from female mice. No BP data.	Microbiota influenced the vasoconstriction response.
Cross, T.W.L., 2017 [74]	40	Aortic PWV (doppler)	Ovariectomy vs sham surgery; soy-rich vs soy-free diet	28 weeks	PWV was lowered with soy feeding but was not affected by ovariectomy. No BP data.	Soy-rich diet modified intestinal microbiota composition (lower F:B ratio).
Battson, M.L., 2018 [81]	36	Aortic PWV (doppler)	Standard diet (SD) (n 12) or Western diet (WD) (n 24) for 5 months, then WD mice were randomized to receive broad-spectrum antibiotic cocktail (WD + Abx) or placebo (n 12/group) for 2 months	7 months	PWV progressively increased in WD mice during the 7-month intervention. In WD + Abx, PWV was completely normalized to SD levels. No BP data.	WD had increased Firmicutes and decreased Bacteroidetes and Actinobacteria. Abundance of numerous bacterial taxa were altered by diet; in particular, Bifidobacterium spp. were significantly more abundant in SD animals compared with WD.
Brunt, V.E., 2019 [82]	73	Aortic PWV (doppler); ex vivo intrinsic mechanical stiffness	Cocktail of broad-spectrum, poorly absorbed antibiotics in drinking water vs placebo. 4 groups: young controls (YC); young antibiotics (YA); old controls (OC); old antibiotics (OA).	3–4 weeks	At baseline, PWV was higher in OC and OA vs YC (p < 0.01). PWV increased in YC but not in YA during intervention. In OA, PWV was reduced at the end of the intervention. Antibiotic treatment in old mice was associated with a partial improvement back towards young levels (p = 0.047 vs. OC). Aortic elastin protein expression was lower in OC vs. YC (p = 0.02), but was restored in OA. No BP modifications were registered.	Ageing was associated with greater alpha diversity. Old mice demonstrated several bacterial markers of gut dysbiosis and/or inflammation. Three-fold age-related increase in circulating plasma TMAO levels. In both young and old mice, antibiotic treatment suppressed TMAO levels.
Lee, D.M., 2018 [75]	47	Aortic PWV (doppler); ex vivo	(1) standard diet; (2) standard diet + dapagliflozin (60 mg dapagliflozin/kg diet). Controls (n = 11); Controls + dapa (n = 12); Diabetics (Db) (n = 12); Db + dapa (n = 12).	8 weeks	Dapagliflozin treatment improved both endothelium-dependent dilatation (EDD) and Endothelium-independent dilation (EID) in Db mice. PWV was negatively and EID-EDD positively correlated with Akkermansia abundance. PWV was positively correlated with Firmicutes and F:B ratio. No BP data.	Significantly reduced richness and diversity in the Db + dapa group compared to controls. Bacteroidetes and Proteobacteria were influenced by dapagliflozin treatment in Db + dapa. Db + dapa had a significantly lower F:B ratio than the other treatment groups. Oscillospira was significantly reduced in the Db + dapa compared to all other groups
Lee, D.M., 2020 [76]	48	Aortic PWV (doppler)	Standard (SD) vs Western diet (WD). Indole-3-propionic acid (IPA) vs placebo. (1) SD + placebo, (2) WD +placebo, (3) SD + IPA, 4) WD + IPA. (n = 12 mice/group).	5 months	IPA supplementation did not affect PWV in WD, but impaired PWV in SD. Bifidobacterium reduction by WD was related to PWV. No BP data.	WD feeding decreased Bifidobacterium. Reduced abundance of Bifidobacterium was observed in SD + IPA.
Trikha, S.R.J., 2021 [80]	10	Aortic PWV (doppler)	2 age-matched male and 2 female (1 of each lean [LM], and 1 obese [OBM]) microbiota donors to form cohorts 1 and 2 of inoculated mice.		PWV was increased in OBM mice vs. GF mice. In cohort 2, OBM mice displayed a marked increase in PWV vs. LM mice. No BP data.	Mouse microbiota profiles clustered according to their transplant donor groups. Taxa appear to be driving this separation, Bacteroides ovatus and Parabacteroides diastonis were consistently associated with LM mice.

VA stands for Vascular ageing; SBP, systolic blood pressure; PWV, pulse wave velocity; LPS, lipopolysaccharides; Gpr, G-protein coupled receptor; SCFA, short-chain fatty acid; GF, germ-free; F:B, Firmicutes/Bacteroidetes ratio; TMAO, trimethylamine-N-oxide.