Atherosclerosis, a cardiovascular, inflammatory disease characterized by arterial lumen narrowing owing to plaque formation, is a major cause of death globally [1
]. Endothelial dysfunction occurs early in atherosclerosis development and is involved in plaque formation and disease progression [3
]. Therefore, improving endothelial function might help prevent cardiovascular disease in at-risk individuals.
Polyamines (putrescine, spermidine, and spermine), which are involved in the synthesis and stabilization of nucleic acids and stimulation of cell proliferation and differentiation [4
], have become a recent focus in the field of atherosclerosis. Autophagy induction by oral spermidine administration reduces blood pressure and cardiovascular disease risk in wild-type mice and salt-sensitive hypertensive rats [5
]. The intracellular production and concentration of polyamines in tissues and organs decrease with age [6
]; therefore, older individuals are at higher risk of developing atherosclerosis. Polyamines, especially spermine, inhibit inflammatory activity by suppressing pro-inflammatory cytokine synthesis [7
] and leukocyte function-associated antigen-1 [8
]. Dietary polyamines have also been found to be negatively associated with cardiovascular disease [9
]. Therefore, the intake of exogenous polyamines from foods and intestinal bacterial metabolites might benefit cardiovascular health [10
]. Most dietary polyamines are absorbed in the small intestine [12
], and putrescine and spermidine in the colon are provided by intestinal bacteria [13
]. However, high levels of polyamines are observed in tumors [14
], and the selective inhibition of ornithine decarboxylase, a key enzyme in polyamine synthesis, is effective against malignancies, including colon cancer [14
]. Hence, whether polyamines are harmful or beneficial for health remains controversial.
To verify the effect of microbial polyamines on human health, we developed a reliable method to provide polyamines to the body by regulating intestinal bacterial metabolism [15
]. This treatment, which involves arginine (Arg) and Bifidobacterium animalis
administration, increased colonic luminal putrescine levels, and blood spermidine concentrations [15
]. This effect is primarily modulated by a pathway involving the independent metabolic systems of three types of bacteria (hybrid putrescine biosynthesis system): Escherichia coli
(arginine-dependent acid resistance system), Enterococcus faecalis
(agmatine deiminase-associated energy production system), and Bifidobacterium
spp. (acid production system) [16
]. Thus, putrescine cannot be stably produced by intestinal bacteria unless both B. animalis
and free Arg (not the constituent Arg in dietary proteins) exist simultaneously in the intestine. Furthermore, B. animalis
showed the most potent ability to induce putrescine production among Bifidobacterium
]. While our previous studies focused on how polyamine production can be regulated, the application of this method to modulate autophagy and prevent/treat atherosclerosis was not examined.
Here, we evaluated the effects of putrescine produced by the intestinal microbiome following B. animalis subsp. lactis and Arg administration on endothelial function during the early stages of atherosclerosis development in a randomized, double-blinded, placebo-controlled, parallel-group comparative study.
We investigated the effect of consuming yogurt containing B. animalis
and Arg on endothelial function in healthy adults. Endothelial dysfunction increases the risk of developing cardiovascular disease [37
]. Key RHI values <1.67 indicate endothelial dysfunction [22
]. Here, the RHI increased from 1.50 to 1.81 after Bifal + Arg YG consumption, suggesting that Bifal + Arg YG intake restored endothelial function to normal levels. Moreover, blood pressure and cardiovascular disease are closely related, with systolic and diastolic blood pressure higher than 115 mmHg and 75 mmHg, respectively, being associated with an increased risk of disease development [38
]. In the Bifal + Arg YG group, blood pressure decreased to near the baseline level, suggesting that the consumption of this yogurt improved blood pressure. Endothelial function and blood pressure strongly influence each other, and both deteriorate during atherosclerosis progression [39
]. Therefore, simultaneous improvement in both parameters is not surprising and indicates that Bifal +
Arg YG intake might help prevent the early process of atherosclerosis development. However, the observed changes in blood pressure could also be related to changes in smooth muscle cell reactivity and arterial compliance; further studies are needed to clarify this effect.
Colonic cells can absorb putrescine [40
], which is utilized for spermidine and spermine biosynthesis [41
]. Previously, we demonstrated that oral Arg administration in rats increased both fecal putrescine and blood spermidine concentration [15
]. Moreover, various physiological activities of polyamines depend on the magnitude of their polarity [42
]. Thus, although considerably substantial changes in spermidine and spermine levels induce autophagy, minor changes in putrescine levels do not [43
]. Therefore, the improvement in endothelial function might be mediated by a putrescine-induced increase in serum spermidine biosynthesis. Blood spermidine-induced autophagy might be involved in both blood pressure reduction [5
] and endothelial function improvement in mice [45
], supporting our results. Thus, Bifal + Arg YG intake may induce microbial putrescine production, which is absorbed from the intestinal lumen and transported into the blood. Consequently, spermidine is biosynthesized, which promotes autophagy in endothelial cells, thereby improving endothelial function.
Other biomarkers further support the improvement of vascular endothelial function after Bifal + Arg YG consumption. Serum platelet [46
], and triglyceride concentrations [47
], two risk factors for atherosclerosis, decreased after Bifal + Arg YG consumption. Serum HDL-cholesterol, which is inversely correlated with cardiovascular disease incidence [48
], increased significantly after Bifal + Arg YG consumption. These support the improvement of endothelial function by this yogurt.
Trimethylamine might be involved in the progression of atherosclerosis [49
]. B. animalis
consumption has been shown to decrease the relative abundance of Lachnospiraceae and other Clostridiales, which might be involved in trimethylamine production [34
]. However, in this study, the relative abundance of these bacterial groups and fecal trimethylamine concentration were unchanged. This might be attributed to the fecal collection method, which differed between the previous and current study. In addition, because dietary carnitine and choline influence microbial trimethylamine production [49
], all subjects in the present study were provided identical meals on the day before feces collection. However, the subjects in the previous study ingested different meals. Therefore, further studies are required to clarify this discrepancy.
Arg is the substrate of NO synthase, which synthesizes the NO essential for blood pressure regulation and maintenance of endothelial function via vascular smooth muscle regulation [50
]. A meta-analysis of clinical trials showed that oral administration of Arg lowers blood pressure effectively [51
]. However, an Arg intake of 6 g/day does not affect blood pressure [52
], and to our knowledge, no study has reported Arg intake below this level to be effective in improving blood pressure. The Bifal + Arg YG used in our study contained only 0.6 g Arg. This suggests that the improved endothelial function and decreased blood pressure after Bifal + Arg YG consumption was not caused by Arg-derived NO but might be modulated by the putrescine-induced increase in serum spermidine. Moreover, the serum NO2
concentration was unchanged after Bifal + Arg YG consumption, further supporting our conclusions.
According to the response to injury theory [53
], the first stage of atherosclerosis is endothelial dysfunction, followed by the induction of an inflammatory response and atherosclerotic plaque formation. In this study, no differences were observed in serum TNF-α or IL-1β concentrations between the treated and placebo groups. The risk of cardiovascular disease was shown to increase when the blood TNF-α concentration was >6 pg/mL [54
]. However, the subjects in the present study only had a maximum of 0.94 pg/mL, indicating the absence of a significant inflammatory environment. Therefore, the anti-inflammatory activity of polyamines [7
] was probably not related to the improvement in endothelial function in this study.
Regarding fecal microbiota, the relative abundance of Citrobacter
, representing bacteria possessing an arginine-dependent acid resistance system (acid-tolerance system), and Enterococcus
, representing bacteria possessing an agmatine deiminase system (energy production system), was higher in the Bifal + Arg YG group than in the placebo group. This indicated that Bifal + Arg YG intake might enhance the intestinal microbiota composition to be more suitable for putrescine production by acid-producing bacteria, including B. animalis
]. However, the reason for the observed reductions in Bacteroidetes and the Bacteroidetes/Firmicutes ratio in Bifal + Arg YG-treated subjects is unclear. Although a controversial association between obesity and decreased Bacteroidetes/Firmicutes ratios in the intestinal microbiota has been suggested [55
], the BMI of these individuals did not change in the present study. Thus, this relationship needs to be clarified in future studies. There are several recent human studies on heart failure and alteration in intestinal microbiome [56
]. Similar to our study, Luedde at al. [57
] studied fecal microbiome, and analyzed data using 16S rRNA gene amplicon sequencing, and reported that patients with heart failure experienced significant decreases in levels of Coriobacteriaceae, Erysipelotrichaceae, and Ruminococcaceae at the family level, and decreases in levels of Blautia
, unclassified Erysipelotrichaceae, and unclassified Ruminococcaceae at the genus level, as compared to healthy controls. Kamo et al. [58
] found that patients with heart failure had significantly decreased levels of Clostridium
at the genus level as compared to healthy controls. Kummen et al. [59
] reported significant increases in the levels of Prevotella
, and Succiniclasticum
and decreases in the levels of Anaerostipes
, Eubacterium hallii
group, and an unknown genus in Lachnospiraceae at the genus level, in patients with heart failure as compared to healthy controls. However, in this study, the relative abundance of these bacterial groups was unchanged. Furthermore, although one of these studies found that bacterial diversity in patients with heart failure was decreased as compared to that in healthy controls, bacterial diversity was not changed during the experimental period. These analyses suggest that improvement of endothelial function triggered by this yogurt is independent of the intestinal bacterial composition.
The current study has some limitations. First, the subjects were all healthy Japanese individuals (BMI <30); we cannot be sure that the same results would be obtained if the subjects had severe atherosclerosis or were from other countries or ethnicities, with different dietary cultures. It is possible that the difference in the change in RHI was caused by the difference in the baseline RHI level between both groups. Therefore, the accurate effect needs to be confirmed by increasing the number of participants in future studies. Because there is a controversy regarding whether EndoPAT values are correlated with the results of FMD—a gold standard test of endothelial function—an additional study using FMD is indispensable in subsequent studies. Moreover, further studies are required to compare the administration of B. animalis subsp. lactis or Arg alone and together to confirm whether this strategy depends on the hybrid putrescine biosynthesis system. We do not know the reason why abdominal circumference in both groups was increased. It is estimated that this is caused by measurer’s bias because this value of all subjects unnaturally and uniformly increased more than 2 cm, and the measurer at week 12 differed from the one at week 0. Finally, as endo-peripheral arterial autophagy could not be analyzed, the possibility that other functions of spermidine led to this effect cannot be excluded.
Multiple clinical studies have reported the efficacy of probiotics in improving the blood lipid profile [60
]. However, to our knowledge, direct improvement of endothelial function by probiotics has not yet been reported. Although one clinical test using Lactobacillus casei
Shirota was conducted to assess the improvement of endothelial function, its efficacy was not observed [61
]. Therefore, Bifal + Arg YG is the first functional food to demonstrate efficacy in improving endothelial function during the early process of atherosclerosis development in humans. Controlling intestinal bacterial metabolites might be a novel strategy to maintain host health. While it is challenging to control bacterial metabolites because of vast individual differences in human microbiomes and diets, our yogurt supplementation was effective in controlling target metabolites derived from intestinal microbiota and in enhancing endothelial function via altered physiological activity (autophagy). This study provides insights into atherosclerosis development and highlights a novel treatment option for at-risk patients.