腸内細菌が動脈硬化性疾患に関与

近年、腸内細菌が炎症性腸疾患だけでなく糖尿病や脂質異常症といった代謝性疾患や悪性腫瘍、精神疾患などの発症に深く関係していることが分かってきているが、動脈硬化性疾患との関係も注目されている。神戸大学大学院循環器内科学分野教授の平田健一氏は第115回日本内科学会（4月13～15日）で、自身の研究成果も交えて腸内細菌と動脈硬化性疾患との関係を講演、「将来は患者の便のパターンによってプロバイオティクスなどによる腸内細菌で介入する個別化医療の時代が来る」という考えを示した。

冠動脈疾患でLactobacillales目細菌が増加

平田氏らは、糖尿病、脂質異常症、高血圧などの心血管危険因子を持つ患者（コントロール群）30例、健常者40例、冠動脈疾患患者30例から糞便提供を受け、細菌の16SrRNAを増幅して行う検査のT-RFLP法により腸内細菌パターンを調査した。その結果、コントロール群に比べ冠動脈疾患群ではLactobacillales目の細菌が増加し、一方でBacteroides門が減少していた（図）。

図. 3群でのLactobacillalesとBacteroidetesの割合

冠動脈疾患群の中でもLactobacillales目が極端に多い症例だけに限ると全て多枝病変症例であった（J Atheroscler Thromb 2016; 23: 908-921）。また、他の研究グループではあるが、糖尿病症例、脳梗塞症例でもLactobacillales目が増加しているという報告もある。冠動脈疾患群で少なかったBacteroides門を詳細に調べると、Bacteroides門のある2種類の菌が特徴的に少ないことが分かった。このように、冠動脈疾患患者での腸内細菌パターンが明らかになってきている。

腸内細菌の研究成果を生かし、既に臨床試験も始まる

平田氏によると、腸内細菌が動脈硬化を進展させる機序として①腸内細菌に腸管バリア機能が障害されリポポリサッカライド（LPS）などの菌体成分が血中に入り炎症を起こす②短鎖脂肪酸など腸内細菌の代謝産物が宿主に影響を与える③腸内細菌が制御性T細胞や樹状細胞などの免疫細胞に修飾を加え宿主側の炎症を調節する―などが考えられている。③に関しては、わが国の研究グループによりClostridumが制御性T細胞を誘導することが報告されている（Science 2011; 331: 337-341）。

　米・Cleveland Clinicの研究グループは、腸内細菌により心血管イベントの予測因子であるTMAO（トリメチルアミン-N-オキシド）が上昇する機序を解明している。肉、チーズ、卵などの食事由来のホスファチジルコリンから腸内細菌は、トリメチルアミンリアーゼという酵素を使ってトリメチルアミン（TMA）を産生。そのTMAは肝臓で酸化反応を受けTMAOとなり、マクロファージの泡沫化などにより動脈硬化を促進すると推測されている（N Engl J Med 2013; 368: 1575-1584、Nat Med 2013; 19: 576-585、Nature 2011; 472: 57-63、Cell 2015; 163: 1585-1595）。さらにトリメチルアミンリアーゼの阻害薬の候補物質も同研究グループは見つけており、既に前臨床試験が始まっているという。

　同氏らも腸内細菌を使って動脈硬化を抑制することに動物実験で成功している。動脈硬化モデルマウス（Apoe-/-マウス）に、Bacteroides門の2種類の菌を週5回、10週間にわたり経口投与をした結果、動脈硬化が抑制できた。また、血中のLPSやサイトカイン類の濃度が低下をしており、腸内細菌の投与が慢性炎症を抑制したと考えられた。

　同氏は最後に、腸内細菌と心不全との関係にも触れ、心不全でも血中のTMAO濃度が上昇しており、B型ナトリウムペプチド（BNP）とは独立した心不全マーカーとして今後期待されていることなどを紹介した。

Analysis of Gut Microbiota in Coronary Artery Disease Patients: a Possible Link between Gut Microbiota and Coronary Artery Disease.

Emoto T, Yamashita T, Sasaki N, Hirota Y, Hayashi T, So A, Kasahara K, Yodoi K, Matsumoto T, Mizoguchi T, Ogawa W, Hirata K.

J Atheroscler Thromb. 2016 Aug 1;23(8):908-21. doi: 10.5551/jat.32672. Epub 2016 Mar 5.

Abstract

CD4(+) T regulatory cells (T(regs)), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, T(regs) were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T(reg) cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β and affected Foxp3(+) T(reg) number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.

PMID: 21205640 PMCID: PMC3969237 DOI: 10.1126/science.1198469

Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk.

Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, Wu Y, Hazen SL.

N Engl J Med. 2013 Apr 25;368(17):1575-84. doi: 10.1056/NEJMoa1109400.

Abstract

BACKGROUND:

Recent studies in animals have shown a mechanistic link between intestinal microbial metabolism of the choline moiety in dietary phosphatidylcholine (lecithin) and coronary artery disease through the production of a proatherosclerotic metabolite, trimethylamine-N-oxide (TMAO). We investigated the relationship among intestinal microbiota-dependent metabolism of dietary phosphatidylcholine, TMAO levels, and adverse cardiovascular events in humans.

METHODS:

We quantified plasma and urinary levels of TMAO and plasma choline and betaine levels by means of liquid chromatography and online tandem mass spectrometry after a phosphatidylcholine challenge (ingestion of two hard-boiled eggs and deuterium [d9]-labeled phosphatidylcholine) in healthy participants before and after the suppression of intestinal microbiota with oral broad-spectrum antibiotics. We further examined the relationship between fasting plasma levels of TMAO and incident major adverse cardiovascular events (death, myocardial infarction, or stroke) during 3 years of follow-up in 4007 patients undergoing elective coronary angiography.

RESULTS:

Time-dependent increases in levels of both TMAO and its d9 isotopologue, as well as other choline metabolites, were detected after the phosphatidylcholine challenge. Plasma levels of TMAO were markedly suppressed after the administration of antibiotics and then reappeared after withdrawal of antibiotics. Increased plasma levels of TMAO were associated with an increased risk of a major adverse cardiovascular event (hazard ratio for highest vs. lowest TMAO quartile, 2.54; 95% confidence interval, 1.96 to 3.28; P<0.001). An elevated TMAO level predicted an increased risk of major adverse cardiovascular events after adjustment for traditional risk factors (P<0.001), as well as in lower-risk subgroups.

CONCLUSIONS:

The production of TMAO from dietary phosphatidylcholine is dependent on metabolism by the intestinal microbiota. Increased TMAO levels are associated with an increased risk of incident major adverse cardiovascular events. (Funded by the National Institutes of Health and others.).

Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis.

Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, DiDonato JA, Chen J, Li H, Wu GD, Lewis JD, Warrier M, Brown JM, Krauss RM, Tang WH, Bushman FD, Lusis AJ, Hazen SL.

Nat Med. 2013 May;19(5):576-85. doi: 10.1038/nm.3145. Epub 2013 Apr 7.

Abstract

Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary L-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of L-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma L-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.

Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.

Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, Wu Y, Schauer P, Smith JD, Allayee H, Tang WH, DiDonato JA, Lusis AJ, Hazen SL.

Nature. 2011 Apr 7;472(7341):57-63. doi: 10.1038/nature09922.

Abstract

Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine--choline, trimethylamine N-oxide (TMAO) and betaine--were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidaemic mice. Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.

Non-lethal Inhibition of Gut Microbial Trimethylamine Production for the Treatment of Atherosclerosis.

Wang Z, Roberts AB, Buffa JA, Levison BS, Zhu W, Org E, Gu X, Huang Y, Zamanian-Daryoush M, Culley MK, DiDonato AJ, Fu X, Hazen JE, Krajcik D, DiDonato JA, Lusis AJ, Hazen SL.

Cell. 2015 Dec 17;163(7):1585-95. doi: 10.1016/j.cell.2015.11.055.

Abstract

Trimethylamine (TMA) N-oxide (TMAO), a gut-microbiota-dependent metabolite, both enhances atherosclerosis in animal models and is associated with cardiovascular risks in clinical studies. Here, we investigate the impact of targeted inhibition of the first step in TMAO generation, commensal microbial TMA production, on diet-induced atherosclerosis. A structural analog of choline, 3,3-dimethyl-1-butanol (DMB), is shown to non-lethally inhibit TMA formation from cultured microbes, to inhibit distinct microbial TMA lyases, and to both inhibit TMA production from physiologic polymicrobial cultures (e.g., intestinal contents, human feces) and reduce TMAO levels in mice fed a high-choline or L-carnitine diet. DMB inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in apolipoprotein e(-/-) mice without alterations in circulating cholesterol levels. The present studies suggest that targeting gut microbial production of TMA specifically and non-lethal microbial inhibitors in general may serve as a potential therapeutic approach for the treatment of cardiometabolic diseases.