
==== Front
Curr Opin Lipidol
Curr Opin Lipidol
COLIP
Current Opinion in Lipidology
0957-9672
1473-6535
Lippincott Williams & Wilkins Hagerstown, MD

39073599
MOL350503
10.1097/MOL.0000000000000944
00002
3
ATHEROSCLEROSIS: CELL BIOLOGY AND LIPOPROTEINS: Edited by Mohamad Navab and Menno de Winther
Evidence further linking the intestine to cardiovascular disease
Sulaiman Dawoud a
Reddy Srinivasa T. a b
Fogelman Alan M. a
a Division of Cardiology, Department of Medicine
b Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California, USA
Correspondence to Alan M. Fogelman, MD, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1736, USA. Tel: +1 310 825 6058; e-mail: afogelman@mednet.ucla.edu
10 2024
29 7 2024
35 5 223227
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0

Purpose of review

To review recent publications linking the intestine to cardiovascular disease.

Recent findings

Aromatic amino acid-derived metabolites produced by gut-bacteria were identified that increased or decreased the risk of cardiovascular events. Dietary phenylalanine was metabolized to phenylacetic acid by gut microbes, and converted into phenylacetylglutamine by the host, which increased thrombosis potential via adrenergic receptors and was associated with increased major adverse cardiovascular events. Another microbiota-associated metabolite of aromatic amino acids, indole-3-propionic acid, protected against heart failure with preserved ejection fraction. The mechanism by which dietary cholesterol is absorbed was found to involve the Nieman–Pick C1-like1 protein working together with a newly discovered protein called Aster. Levels of gut-derived bacterial lipopolysaccharide in serum that are an order of magnitude less than those seen in gram negative sepsis were shown to play a role in enhancing atherosclerosis and thrombosis.

Summary

Promising new therapeutic targets in the intestine for preventing or treating cardiovascular disease have been identified.

Keywords

atherosclerosis
cardiovascular disease
gut-microbiota
intestine
lipopolysaccharide
(A.M.F.) and the Castera, Laubisch, M.K. Grey and John and Shirley Luedde funds1R01 HL148286 OPEN-ACCESSTRUE
==== Body
pmcINTRODUCTION

Wright et al.[1] reported in 2000 that atherosclerosis in germ-free apoe−/− mice was not measurably different from that in conventionally raised apoe−/− mice. In 2011, Hazen et al.[2] published the seminal observation that gut flora metabolism of phosphatidylcholine promoted cardiovascular disease in humans and in apoe−/− mice by a process that involved trimethylamine N-oxide (TMAO) [2]. In 2018, Jonsson et al.[3] reported that in conventionally raised apoe−/− mice fed a Western diet supplemented with choline the plasma levels of TMAO were higher than in germ-free mice fed the same diet, but the size of the atherosclerotic lesions was not different. An accompanying editorial by Getz and Reardon [4] proposed a host of possible reasons for the discrepancies between the studies including differences in the gender of the mice, differences in the methods of lesion measurement and possible differences in the microbiota in different laboratories. This review will present recent data indicating that as Hazen et al. reported [2], the gut microbiota significantly affects cardiovascular disease in mice and humans. Additionally, we will review a novel mechanism for the absorption of dietary cholesterol and other recent evidence indicating the complexity of the gut microbiome and data linking the intestine to cardiovascular disease. 

Box 1 no caption available

Helpful and harmful gut-derived metabolites

Hazen and colleagues [5▪▪] recently published an atlas of gut microbe-derived products of aromatic amino acids that are associated positively or negatively with cardiovascular mortality. For example, phenylacetylglutamine (PAGln) was shown to be derived from gut microbe metabolism of dietary phenylalanine into phenylacetic acid that was converted to PAGln by the host. PAGln was found to increase platelet responsiveness and increase thrombosis potential via adrenergic receptors [6]. In humans, increased PAGln levels were associated with an increase in major adverse cardiovascular events (MACE) that included myocardial infarction, stroke, or death [6]. Another microbiota-associated metabolite of aromatic amino acids is indole-3-propionic acid that was shown to be inversely related to diabetes risk [7]. Tryptophan is an essential amino acid that can be converted to indole-3-propionic acid by the action of gut bacteria such as Lactobacillus reuteri, Akkermansia muciniphila, and Clostridium sporogenes, and was shown by Wang et al.[8▪▪] to protect against heart failure with preserved ejection fraction. The atlas from Hazen and colleagues [5▪▪] presents outcome data (MACE and death) in humans that was associated with gut microbial metabolites derived from dietary phenylalanine (7 compounds), tyrosine (9 compounds) and tryptophan (9 compounds). An accompanying editorial by Stahli et al.[9▪] points out that a major strength of the data from Hazen et al.[5▪▪] is that the data were derived from two independent cohorts of stable subjects undergoing elective diagnostic cardiac evaluation, a cohort from the United States (GeneBank, n = 4000) and a European cohort (LipidCardio, n = 833). These studies unequivocally demonstrate that the gut microbiota acts on dietary constituents to generate metabolites that have clinically important consequences.

Chen et al.[10▪] examined stool samples from 77 ST-elevation myocardial infarction (STEMI) patients within 3 days of a percutaneous intervention and again approximately 28 days after the intervention. Comparing the two time points to samples from controls, they found increases in trimethylamine-associated and butyrate-producing bacteria in the STEMI groups. The results regarding the butyrate-producing bacteria were confirmed in a nonhuman primate model subjected to cardiac ischemia for 90 min followed by reperfusion. Subsequently, they transplanted human fecal samples from control and STEMI patients into germ-free (GF) mice. Following surgery to induce myocardial infarction there was a dramatic decrease in survival in the mice that received the STEMI samples. In a milder injury model in which angiotensin II was infused for 14 days, the mice receiving the STEMI samples had worsened left ventricular ejection fraction and fractional shortening with increased left ventricular stiffness. The mice receiving the STEMI samples had changes suggestive of increased gut inflammation. Metabolomics confirmed data from shotgun metagenomics and showed increased TAMO and β-hydroxybutyrate production post cardiac injury. Since butyrate-producing bacteria increased after cardiac injury in both humans and nonhuman primates, and others showed that butyrate and ketones improved cardiac function in rodents [11,12], Chen et al.[10▪] administered broad spectrum antibiotics to suppress the gut microbiome in some mice but not in others, they induced an MI in all mice, and then did or did not administer butyrate for 20 days post MI. The administration of butyrate resulted in improved cardiac function and smaller infarct size. The mice that did not receive antibiotics (i.e., they had an intact gut microbiome) responded even more robustly to the butyrate treatment. The authors concluded that the gut microbiome may play a direct role in post-MI outcomes that may be due to ketogenesis [10▪].

New insights into the absorption of dietary cholesterol

The cholesterol lowering drug ezetimibe was discovered in a program designed to identify acyl-coenzyme A cholesterol acyltransferase (ACAT) inhibitors [13]. Subsequently, it was determined that ezetimibe acts by binding to the sterol-sensing domain of Niemann-Pick C1-like1 (NPC1L1) in the brush border membrane of enterocytes. This binding prevents the formation of a tunnel-like structure that in the absence of ezetimibe forms in response to free cholesterol derived from the diet and delivers the cholesterol to the endoplasmic reticulum (ER) of the enterocyte where it is esterified by ACAT2 and incorporated into chylomicrons [14]. The mechanism by which the free cholesterol was transported from the NPC1L1 tunnel in the brush border membrane to the ER membrane in enterocytes was unknown. Peter Tontonoz et al. discovered a series of nonvesicular cholesterol transport proteins known as Aster-A, Aster-B, and Aster-C that are found in many cell types and facilitate cholesterol movement between membranes [15]. Focusing on the role of these proteins in enterocytes, the Tontonoz laboratory found that Aster-B was expressed along the entire small intestine and acted sequentially with NPC1L1 to deliver free cholesterol derived from the diet to the enterocyte ER where it was esterified and incorporated into chylomicrons [16▪▪]. Tontonoz et al. also demonstrated that a novel small molecule (AI-3d) that potently inhibits the Aster proteins was able to block the uptake of dietary cholesterol in mice suggesting that intestinal Aster may be a potentially important new therapeutic target [16▪▪].

Low levels of gut-derived bacterial lipopolysaccharide in atherosclerosis and thrombosis

Mukherjee et al.[17] reported that feeding a Western diet (WD) to Ldlr−/− mice resulted in dramatic changes in the microbiome of the small intestine including the virtual disappearance of Akkermansia muciniphila while overall bacteria numbers and LPS levels in the mucus of the small intestine and in plasma increased. The increase in LPS levels was associated with a decrease in gene expression and protein levels in the small intestine for antimicrobial proteins and peptides. These changes were associated with decreased gut barrier function and an increase in the levels of oxidized phospholipids in the small intestine. These WD-mediated changes were ameliorated by addition to the WD of a concentrate of transgenic tomatoes expressing the apolipoprotein mimetic peptide 6F [17] or by intestine specific knockout of the Enpp2 gene that encodes for phospholipase D, which plays a major role in the formation of lysophosphatidic acid [18].

Yang et al.[19▪] recruited 215 patients with coronary artery disease (CAD) that included 56 patients with stable CAD, 106 patients with unstable angina, 53 patients with MI, and 156 control subjects without CAD. The feces of each subject were subjected to metagenomic sequencing. The feces of all the CAD groups showed a dramatic decrease in the presence of Faecalibacterium prausnitzii compared to the control group. To determine if these bacteria played a role in atherogenesis, apoe−/− mice on an atherogenic diet received vehicle alone (phosphate buffered saline) or the vehicle containing Faecalibacterium prausnitzii 5 days per week by gastric gavage. The mice receiving Faecalibacterium prausnitzii had significantly less lesion area throughout the aorta and significantly less lesion area in the aortic root without any significant difference in plasma lipids [19▪]. There was a decrease in LPS biosynthesis pathways in the gut microbiota of mice receiving Faecalibacterium prausnitzii that was accompanied by a significant decrease in fecal LPS levels, decreased serum LPS levels and increased levels of gene and protein expression in the ileum for the tight junction protein ZO-1 [19▪]. The authors concluded that the decreased aortic atherosclerosis in the mice receiving Faecalibacterium prausnitzii was due to improved barrier function in the intestine associated with reduced gut microbiota synthesis of LPS, reduced uptake of LPS, and reduced serum levels of LPS [19▪].

The conclusions of Yang et al.[19▪] are in line with the findings of Li et al.[20] who reported that feeding a WD to apoe−/− mice reduced the fecal abundance of Akkermansia muciniphila that could be offset by administering Akkermansia muciniphila by stomach gavage to these mice, and which significantly reduced the enhanced systemic inflammation and aortic atherosclerosis caused by the WD. Li et al.[20] also reported that administration of Akkermansia muciniphila was accompanied by decreased levels of circulating LPS in the mice that the authors attributed to an induction of tight junction proteins including ZO-1. Furthermore, they found that infusion of LPS into the mice reversed the protective effect of administering the Akkermansia muciniphila[20]. These studies suggest that specific bacteria in the gut-microbiota can influence gut-barrier function and the absorption of LPS into the circulation that contributes to systemic inflammation and enhances atherosclerosis.

The two examples described above [19▪,20] raise the question addressed by Wang et al.[21▪▪]. How can a small number of specific bacteria among a microbiome that includes hundreds of bacterial strains, which exist at abundance levels that vary by many orders of magnitude possibly alter function? To answer this question, Wang et al.[21▪▪] eliminated two strains that occupy the bile acid 7α-dehydroxylation niche, Clostridium scindens and Clostridium hylemonae. Eliminating these two strains eliminated secondary bile acid production and caused eight strains to change in relative abundance by >100-fold. Removing one but not the other of these two strains revealed that either strain alone was capable of dehydroxylating the entire pool of primary bile acids, and the resulting bile acid profiles were indistinguishable [21▪▪]. When the niche was mono-colonized with Clostridium scindens versus Clostridium hylemonae only six strains differed in relative abundance. However, when Clostridium scindens was eliminated from the community there was an unexpected proliferation of Clostridium sporogenes, which increased >1000-fold in abundance and led to a large increase in reductive phenylalanine metabolites; this did not happen if Clostridium hylemonae was eliminated from the community [21▪▪]. The authors concluded that altering a particular species of bacteria that are in a functional niche together with other species, even when the other species in the niche are functionally redundant, may result in dramatic differences outside of the niche. Therefore, in trying to achieve a desired phenotype, it would be imprudent to just focus on the strains that are responsible for the desired phenotype without also determining the interactions of the individual strains with their partners in the community that are outside of the functional niche [21▪▪].

Nie et al.[22▪] reported that feeding apoe−/− mice a high-fat high-cholesterol diet decreased colon expression of the transcriptional factor KrÜppel-like factor (KLF) 4 and decreased colon expression of tight junction proteins that were associated with an increase in circulating levels of LPS. Using intestinal epithelial specific transgenic apoe−/− mice constructed with adeno-associated virus encoding villin promoter-mediated Klf4 they demonstrated amelioration of the diet-induced increase in gut-permeability, a reduction in circulating LPS levels, and a reduction in aortic atherosclerosis [22▪]. The benefits of the overexpression of Klf4 were lost if exogenous LPS was administered suggesting that the benefits of Klf4 expression were due to its role in regulating serum LPS levels. Their studies also implicated miRNA-34a in the Klf4-mediated changes [22▪].

In 2020, Hazen et al.[23] reviewed molecular pathways and host receptors that link gut microbiota-derived products and metabolites with cardiovascular and cardiometabolic disease phenotypes. More recently, Bucci et al.[24▪] reported on the role of low-grade endotoxemia on the risk for thrombosis in primary antiphospholipid syndrome. The gut is the major source of circulating LPS in animals and humans [25▪]. Bucci et al.[24▪] reported that the median concentration of serum LPS levels in 21 healthy control subjects was 19 pg/ml compared to 19.7 pg/ml in 16 primary antiphospholipid carriers and compared to 26.3 pg/ml in 97 patients with primary antiphospholipid syndrome. There was no significant difference between the carriers and the healthy control subjects, but the differences in serum LPS levels between the controls and those with primary antiphospholipid syndrome and between the carriers and those with the syndrome was significant (P < 0.001 for syndrome versus controls and P = 0.02 for syndrome versus carriers) [24▪]. After 4.7 years of follow up, 11 composite outcome events occurred in those with the syndrome compared to none in the carriers. In the group with the syndrome there was one cardiovascular death, three patients had ischemic strokes, two patients had myocardial infarctions, three patients had a peripheral arterial thrombosis and two patients had venous thrombotic emboli [24▪]. On multivariate Cox-regression analysis adjusted for sex, diabetes, and the adjusted global antiphospholipid syndrome score, serum LPS levels were significantly associated with outcome when considered as a continuous variable or as a dichotomic variable [24▪]. The authors concluded that gut derived LPS provided a plausible ‘second hit’ that appears to be required for thrombosis in primary antiphospholipid syndrome. They also concluded that study of larger cohorts was required to confirm their findings [24▪].

CONCLUSION

Recent research provides strong support to the evidence indicating that there are important links between the intestine and cardiovascular disease. The gut microbiota can modify dietary constituents to products that can increase or decrease the risk for cardiovascular disease. A new mechanism for the uptake of dietary cholesterol by Aster presents new therapeutic targets. Serum levels of LPS are significantly determined by diet interacting with the microbiome and regulating gut barrier function. Serum levels of LPS at an order of magnitude less than those seen in gram negative sepsis have been shown to influence systemic inflammation, atherosclerosis, and thrombosis. The complexity of the gut microbiome provides both opportunities and challenges for targeting the intestine to improve cardiovascular risk.

Acknowledgements

None.

Financial support and sponsorship

1R01 HL148286 (A.M.F.) and the Castera, Laubisch, M.K. Grey and John and Shirley Luedde funds at UCLA.

Conflicts of interest

S.T.R. and A.M.F. are inventors of patents held by the Regents of the University of California.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest
==== Refs
REFERENCES

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