Gut Microbiota-Derived Metabolites in Atherosclerosis: Pathways, Biomarkers, and Targets

Lipopolysaccharides (LPS), major components of Gram-negative bacterial walls, are potent inducers of systemic inflammation. Their pro-atherogenic action is mainly mediated by binding to TLR4 on endothelial cells and macrophages, activating NF-κB and the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, which in turn stimulate cytokine release (IL-6, TNF-α, IL-1β). These effects promote endothelial dysfunction, monocyte recruitment, and plaque progression [30,31,32,33,34,35,36,37,38,39].

LPS, a component of Gram-negative bacterial membranes, induces systemic inflammation by binding to TLR4 on endothelial cells and macrophages, triggering NF-κB activation and cytokine release [40]. Clinically, endotoxemia has been associated with carotid atherosclerosis and cardiovascular events in prospective population studies, such as the Bruneck cohort [41]. These findings support a mechanistic and epidemiological link between low-grade endotoxemia and atherosclerosis.

While consistent in experimental models, the clinical impact of circulating LPS remains debated, as low-grade endotoxemia is often confounded by metabolic and renal disorders. Thus, LPS represent a mechanistically plausible but clinically variable contributor to atherosclerosis [42,43].

Trimethylamine-N-oxide (TMAO), generated from dietary choline and L-carnitine by gut microbiota and hepatic FMO3, is one of the most extensively studied microbial metabolites in cardiovascular disease. Mechanistically, TMAO enhances foam cell formation by upregulating scavenger receptors (CD36, SR-A1) and impairing cholesterol efflux (ABCA1, ABCG1), promotes platelet hyperreactivity through MAPK and Ca²⁺ signaling, and augments vascular inflammation [29].

TMAO contributes to atherosclerosis through multiple mechanisms. It promotes foam cell formation by upregulating the macrophage scavenger receptors CD36 and SR-A1, facilitating oxLDL uptake [29]. TMAO also enhances platelet hyperreactivity and thrombosis risk via calcium signaling and MAPK activation [44]. Additionally, it augments endothelial inflammation by activating NF-κB and altering bile acid metabolism [45]. These mechanistic insights provide a strong biological rationale for the observed association between elevated TMAO levels and adverse cardiovascular outcomes. These mechanistic findings have been validated and expanded in subsequent studies, linking TMAO to vascular inflammation and adverse cardiovascular outcomes [46,47,48,49].

Although multiple clinical studies link elevated TMAO to increased cardiovascular risk, recent large cohorts suggest heterogeneous associations, influenced by diet, kidney function, and microbiota composition. These discrepancies highlight the need for longitudinal human studies and interventional trials targeting TMAO metabolism [50,51,52,53,54,55,56,57,58].

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are fermentation products of dietary fibers and exert diverse effects on host metabolism and vascular health. Through activation of G-protein coupled receptors (GPR41/43) and inhibition of histone deacetylases, SCFAs modulate inflammation, lipid metabolism, and endothelial function. Butyrate, in particular, enhances intestinal barrier integrity and reduces systemic inflammation, whereas acetate and propionate display context-dependent actions on lipid and glucose metabolism [5,59,60,61,62,63,64].

SCFAs, mainly acetate, propionate, and butyrate, exert immunomodulatory and vasoprotective effects. Butyrate promotes colonic regulatory T cell (Treg) differentiation via histone deacetylase inhibition [65]. Propionate supplementation reduced hypertension-induced cardiovascular damage and vascular remodeling in animal models, with early clinical data supporting blood pressure reduction [66]. Collectively, SCFAs highlight the potential for microbiota-derived metabolites to act as protective mediators against cardiovascular disease.

While experimental evidence consistently supports protective effects against atherosclerosis, human studies remain limited and heterogeneous, with diet, microbiota composition, and interindividual variability influencing outcomes. Thus, SCFAs represent promising but incompletely validated mediators of cardiometabolic protection [67,68,69].

Gut microbiota convert primary bile acids into secondary bile acids (e.g., deoxycholic acid, lithocholic acid), which interact with nuclear and membrane receptors such as the Farnesoid X receptor (FXR) and Takeda G protein-coupled receptor (TGR5). These pathways regulate cholesterol metabolism, glucose homeostasis, and inflammation. Experimental studies suggest dual roles: FXR activation may reduce triglyceride accumulation and vascular inflammation, while excessive secondary bile acids can promote oxidative stress and endothelial injury [46,70,71,72,73,74,75,76,77,78,79,80,81].

Secondary bile acids signal through FXR and TGR5 receptors, influencing lipid metabolism and vascular inflammation. FXR activation reduces triglyceride accumulation and suppresses inflammatory gene expression [82]. TGR5 activation enhances endothelial nitric oxide synthesis, improving vascular tone and energy balance [83]. Conversely, excessive deoxycholic acid has been linked to oxidative stress and vascular injury, suggesting a dual, context-dependent role of bile acids in cardiovascular disease.

Clinical data remain scarce and sometimes contradictory, with variability in bile acid profiles across individuals and disease states. Further studies are required to clarify whether targeting bile acid signaling can provide consistent therapeutic benefit in atherosclerosis [84,85].

To provide a comprehensive overview of the mechanistic links between gut microbiota-derived metabolites and atherosclerosis, we include a schematic flowchart (Figure 3). This illustration summarizes the main pathways through which TMAO, LPS, SCFAs, and secondary bile acids influence endothelial dysfunction, immune activation, and plaque development.

In addition to the well-characterized gut microbiota-derived metabolites such as TMAO, LPS, and SCFAs, several emerging metabolites have recently attracted attention for their potential roles in atherosclerosis. Among these, indole derivatives, produced by bacterial metabolism of tryptophan, show diverse and sometimes opposing vascular effects. Indoxyl sulfate, a protein-bound uremic toxin derived from indole metabolism, has been shown to promote oxidative stress, endothelial dysfunction, vascular smooth muscle proliferation, and inflammatory signaling, thereby contributing to vascular injury and atherosclerotic progression [86]. As summarized in Figure 2, indoxyl sulfate exerts pro-atherogenic actions by inducing oxidative stress and endothelial dysfunction. In contrast, indole-3-propionic acid (IPA) and indole-3-aldehyde (IAld) exhibit antioxidant and anti-inflammatory effects; IPA, in particular, has been associated with improved intestinal barrier function and reduced systemic inflammation, while IAld activates the aryl hydrocarbon receptor (AhR), enhancing mucosal immunity and potentially conferring protection against vascular inflammation [87].

Another important but understudied group comprises the polyamines, including spermidine, spermine, and putrescine, which are either synthesized directly by gut bacteria or modulated by microbial metabolism. Polyamines are involved in cellular growth, apoptosis, and immune modulation, and recent studies suggest they exert cardioprotective actions through the induction of autophagy, improved mitochondrial function, and suppression of inflammatory pathways [88]. Notably, dietary spermidine supplementation has been linked to extended lifespan, reduced arterial stiffness, and improved endothelial function in animal models, with preliminary evidence of cardiovascular benefits also observed in human cohorts [89]. Despite these promising findings, the mechanistic roles of indole derivatives and polyamines in human atherosclerosis remain incompletely defined, and their dual or context-dependent effects highlight the need for more targeted mechanistic and clinical studies. A schematic representation of these emerging metabolites and their pro- and anti-atherogenic effects is shown in Figure 4.

Table 1 summarizes the key gut microbiota-derived metabolites, their sources, mechanisms, and effects on atherosclerosis, highlighting their dual roles in disease progression.

Among gut microbiota-derived metabolites, TMAO is the most extensively studied candidate biomarker. Elevated plasma TMAO levels were first linked to adverse cardiovascular outcomes in coronary artery disease patients [52]. Subsequent prospective studies confirmed associations with myocardial infarction, stroke, and mortality, positioning TMAO as a potential independent risk factor. Mechanistically, TMAO promotes foam cell formation, platelet activation, and vascular inflammation, which supports its biomarker potential. Subsequent large prospective cohorts confirmed its predictive value for cardiovascular events and mortality across diverse populations [47,48,49]. A recent integrative study highlighted its role in microbiota–immune crosstalk and vascular inflammation. However, recent large cohorts revealed heterogeneous associations, influenced by renal function, diet, and gut microbiota composition, underscoring the need for standardized cut-off values before clinical translation [46].

Circulating endotoxin activity (a proxy for LPS translocation) has been associated with insulin resistance, metabolic syndrome, and higher cardiovascular risk. LPS induces systemic inflammation via TLR4/NF-κB and endothelial dysfunction, mechanistically linking it to atherosclerosis. Yet, its utility as a biomarker is limited by low sensitivity, variability in measurement assays, and confounding by obesity and diabetes. Thus, LPS may serve more as a risk enhancer rather than a stand-alone biomarker [95,96].

SCFAs such as acetate, propionate, and butyrate are detectable in plasma and stool, reflecting microbial fermentation of dietary fibers. Higher circulating SCFA levels correlate with reduced inflammation and improved metabolic profiles [67]. Propionate supplementation in humans lowered blood pressure and inflammatory markers, suggesting that SCFAs may function as protective biomarkers. However, interindividual variability and limited cardiovascular endpoint studies restrict their clinical applicability [97].

Alterations in bile acid profiles have been linked with dyslipidemia, insulin resistance, and vascular dysfunction. Secondary bile acids interact with FXR and TGR5, modulating cholesterol and glucose metabolism. Elevated plasma deoxycholic acid has been associated with vascular oxidative stress, while certain bile acid derivatives may exert anti-inflammatory effects. Despite mechanistic plausibility, human studies remain scarce and inconsistent, and bile acids are not yet validated as cardiovascular biomarkers [74,98].

A comparative summary of gut microbiota-derived metabolites and microbial signatures investigated as potential biomarkers for atherosclerosis is provided in Table 2.

The human gut microbiota, composed of ~100 trillion microorganisms (bacteria, viruses, fungi, parasites), begins colonization at birth and is shaped by delivery mode, maternal microbiota, age, genetics, geography, and especially diet. In healthy individuals, Firmicutes and Bacteroidetes predominate, with key genera such as Lactobacillus, Clostridium, and Bifidobacterium. A healthy microbiota shows high diversity, supporting barrier integrity, immune maturation, nutrient metabolism, and drug absorption [10].

Diet plays a central role: fiber-rich diets promote beneficial microbes (Bifidobacterium, Prevotella), while Western diets favor bile-tolerant, pro-inflammatory species (Bilophila, Bacteroides) at the expense of protective taxa (Roseburia). Dysbiosis, with reduced diversity and an altered Firmicutes/Bacteroidetes ratio, is common in obesity, often accompanied by decreased Akkermansia muciniphila, whose supplementation improves barrier integrity and inflammation [99,100].

Therapeutically, dietary interventions rich in fiber and anti-inflammatory components improve metabolic and inflammatory conditions. Moreover, fecal microbiota transplantation combined with an anti-inflammatory diet has shown superior efficacy in ulcerative colitis, underscoring the microbiome's therapeutic potential [101].

The gut microbiota plays a central role in nutrient metabolism and host health by transforming carbohydrates, proteins, and fats into metabolites that act locally and systemically. These include beneficial compounds such as short-chain fatty acids (SCFAs), as well as potentially harmful metabolites, depending on substrate type and microbial composition [102,103].

Bacteria dominate microbial metabolism, particularly in the colon, where fermentation produces SCFAs, B vitamins, and biotransformed polyphenols. These processes support colonocyte function, immune regulation, and barrier integrity. However, protein and fat fermentation may also yield toxic products that promote inflammation and epithelial injury [104].

Microbiota-derived metabolites influence distant organs via the gut–brain, gut–liver, gut–kidney, and gut–heart axes, with SCFAs being the best studied. While their anti-inflammatory effects are well established, the impact of protein and lipid metabolites on cardiovascular and metabolic disease remains less clear [105].

The gut microbiome's extensive genetic repertoire (≈3 million genes vs. ≈23,000 human genes) greatly expands host metabolic capacity, enabling functions that are absent in humans, such as polysaccharide degradation and vitamin synthesis [106]. Evidence from germ-free models and human studies confirms its role in immunity, energy balance, and disease susceptibility, including obesity, diabetes, IBD, and colon cancer [107].

The microbiota also shapes mucosal and systemic immunity: SCFAs promote regulatory T cells (Tregs) via GPCR signaling and epigenetic regulation, while segmented filamentous bacteria induce Th17 differentiation. Beneficial taxa such as Bifidobacterium enhance tolerance and reduce inflammation [59,108,109]. Early colonization is critical for intestinal barrier maturation and immune homeostasis, involving epithelial cells, antimicrobial peptides, and gut-associated lymphoid tissue [110,111]. This lifelong crosstalk between microbes and host immunity underscores the therapeutic potential of microbiota-targeted strategies [112].

In health, the gut microbiota (~10¹⁴ cells, >1000 species, mainly Firmicutes and Bacteroidetes) maintains homeostasis and prevents pathogenic overgrowth. Dysbiosis may involve loss of beneficial microbes, expansion of harmful species, and reduced diversity, often occurring together. It has been associated with multiple diseases, including cardiovascular disease, IBD, obesity, diabetes, allergies, autism, and colorectal cancer [113].

Advances in microbiome research have enabled strategies to counteract dysbiosis, ranging from diet and antibiotics to probiotics, fecal microbiota transplantation (FMT), and environmental interventions. These approaches aim to restore microbial balance, strengthen barrier integrity, and modulate immunity. Future studies must integrate genetic, dietary, and environmental factors to develop targeted, personalized microbiota-based therapies [114].

Table 3 compares the gut microbiota composition in healthy versus dysbiotic states, illustrating microbial shifts linked to atherosclerosis.

Despite growing interest in gut microbiota-derived metabolites as cardiovascular biomarkers, several limitations hinder their clinical translation, as detailed below.

Interindividual variability. Microbiota composition is highly individual, influenced by genetics, age, diet, geography, medication use (antibiotics, statins), and comorbidities. Consequently, the same metabolite (e.g., TMAO) may show divergent associations with disease risk across populations [46,52].

Dietary and environmental confounders. Nutrient intake directly shapes microbial metabolites, making it difficult to disentangle diet-derived effects from microbiota-driven variability. For instance, red meat consumption strongly affects plasma TMAO levels, potentially confounding its predictive value [46,52,115,116].

Methodological heterogeneity. Biomarker studies often use different analytical techniques (16S rRNA sequencing vs. shotgun metagenomics for microbiota; ELISA vs. mass spectrometry for metabolites), limiting comparability. Lack of standardized assays and cut-off values hampers reproducibility across cohorts [117,118,119,120,121,122].

Functional redundancy. Multiple microbial taxa can produce the same metabolite, while a single species may contribute to multiple pathways. Thus, composition alone cannot predict function, and metabolite concentrations may not directly reflect biological effects [123].

Dynamic and context-dependent effects. Many microbial metabolites (e.g., SCFAs, secondary bile acids) have dual roles: they are protective in some contexts, and harmful in others. Without longitudinal and functional studies, cross-sectional measurements risk misinterpretation [124].

Limited clinical validation. While TMAO has robust epidemiological support, most other metabolites lack prospective or interventional studies. Translation into routine practice requires large, standardized, longitudinal trials assessing their incremental value beyond established risk factors [46].

Current evidence highlights promising microbiota-derived biomarkers, but their clinical use is constrained by variability, methodological inconsistencies, and lack of standardization. Future research should adopt integrated multi-omics approaches, harmonized protocols, and large-scale prospective studies to define reliable, clinically actionable microbiota-based biomarkers.

Diet is a major determinant of gut microbiota composition and function, capable of modulating microbial diversity, interactions, and resilience. Long-term consumption of plant-based, fiber-rich diets has been shown to enhance SCFA production, strengthen mucosal barriers, and increase beneficial bacteria such as Actinobacteria and Bacteroidetes. Interventions with Mediterranean diets can rapidly shift microbial profiles toward health-promoting genera like Butyricicoccus and Roseburia, while less healthy diets (Canadian-style) favor potentially dysbiotic taxa. Polyphenol- and bioactive-enriched diets, including green tea and orange juice, also enhance microbiota diversity and support SCFA-producing microbes [99,128].

Animal studies highlight that dietary timing is critical: pre-, during, and post-antibiotic consumption of fiber-rich foods (oats) better preserves microbial diversity and mitigates dysbiosis. Similarly, high-fiber diets in humans enhance post-antibiotic microbiome recovery and reduce the abundance of antibiotic resistance genes. Vegan and omnivorous diets that are rich in fermentable fibers increase Firmicutes abundance and butyrate production, contributing to microbial resilience [129]. However, despite these promising findings, the precise mechanisms and key dietary drivers that promote long-term microbiome stability and recovery remain poorly understood, limiting targeted nutritional strategies for microbiome restoration [130].

It was demonstrated that an high-fat diet (HFD), particularly its low fiber content, induces gut microbiota dysbiosis, reducing SCFA production and impairing intestinal immune defenses. This promotes systemic inflammation and atherosclerosis by enhancing gut-derived immune cell trafficking to vascular sites. FMT experiments establish a causal link between HFD-shaped microbiota and atherosclerosis, independent of metabolic alterations. Fiber supplementation (with fructooligosaccharide) mitigates these effects by restoring microbial balance and SCFA levels. These findings underscore dietary fiber's therapeutic potential in preventing microbiota-driven CVDs and highlight the gut–immune–vascular axis as a critical pathway in atherosclerosis [131,132].

A recent study in a Southeast Asian population linked healthy plant-based diets rich in dietary fiber to reduced cardiometabolic risk, unlike unhealthy plant-based foods (refined grains, fried snacks, sugar-sweetened beverages). Hutchison et al. demonstrated that dietary fiber attenuates atherosclerosis through gut microbiome modulation, while Sakurai et al. showed that alpha-cyclodextrin, a glucose-based cyclic polymer, reduces atherosclerosis by altering cecal bacteria composition. Additional studies confirm that high intakes of fibers like inulin and pectin similarly mitigate atherosclerosis, suggesting that dietary fiber plays a critical role in halting the onset and progression of atherosclerosis via microbiome-mediated mechanisms [26,133].

Strengthening the stability and resilience of the gut microbiome may be achieved through synbiotic interventions, which combine live microorganisms with substrates selectively utilized by host microbes. While prebiotics and probiotics can increase the abundance of beneficial bacteria such as Bifidobacterium and Lactobacillus, their effects on overall microbiota composition are often modest and usually persist only during the intervention [134].

The type, dosage, and duration of prebiotic intake influence the enrichment of specific bacterial groups. Soluble fibers like pectin and inulin are fermented by beneficial microbes to produce SCFAs, which acidify the colon, inhibit pathogens, and contribute to immune modulation, epithelial barrier function, and metabolic health. Insoluble fibers like cellulose and lignin support Prevotella and Ruminococcus, leading to other anti-inflammatory metabolites [135].

Novel prebiotics, such as bifidobacterial-galacto-oligosaccharides (B-GOS), show promise in reducing travelers' diarrhea, while polysaccharides from the mushroom Dictyophora indusiata (DIP) have demonstrated the ability to restore microbiota after antibiotic-induced dysbiosis and reduce inflammation. DIP promotes beneficial bacterial families and SCFA production while suppressing harmful taxa, as confirmed by both in vivo mouse studies and in vitro fermentation assays. These findings underscore the potential of targeted synbiotic and fiber-based strategies to foster a resilient, health-promoting gut microbiome [136].

Recent studies underscore the therapeutic potential of polysaccharides/oligosaccharides, particularly when combined with zinc, in addressing obesity-related metabolic disorders and atherosclerosis through gut microbiome modulation. Research indicates that obese individuals have lower zinc levels, linked to metabolic disorders, and zinc supplementation may mitigate obesity. A novel Ulva oligosaccharide-based zinc supplement was shown to improve intestinal flora, reduced dyslipidemia, and lowered body weight in obese mice. Additionally, mannose oligosaccharides were found to prevent atherosclerosis progression by lowering serum cholesterol, increasing cecal butyrate, and enhancing bile acid excretion via microbiome changes. Moreover, Laminaria japonica polysaccharides suppressed atherosclerosis by boosting autophagy pathways, while red algal polysaccharides are emerging as a potential treatment. These findings highlight innovative, microbiome-mediated strategies using prebiotic polysaccharides and zinc supplementation for preventing and treating obesity and atherosclerosis [137,138].

Probiotics support gut health by competing with pathogens, enhancing mucosal integrity, modulating immunity, and producing beneficial metabolites and antimicrobial peptides. However, clinical evidence on their role in restoring the human gut microbiome (HGM) post-antibiotics or enhancing its resilience remains inconsistent. Some studies suggest that probiotics may delay microbiome recovery due to competition with native taxa and immune activation, while others report benefits such as reduced antibiotic-associated diarrhea and increased SCFA production [139].

These conflicting results highlight the complexity of host–microbe interactions and the limitations of current probiotic strains. Meta-analyses show limited evidence supporting the role of probiotics in preventing travelers' diarrhea, with only S. boulardii showing consistent efficacy. Most clinical interventions have focused on co- or post-treatment administration, with few exploring pre-antibiotic probiotic use. Some studies show modest improvement in microbial diversity, while others, including Suez et al. (2018) [139], report delayed recovery and increased inflammation with multi-strain probiotic blends [140].

New approaches propose assessing functional rather than compositional shifts and exploring next-generation probiotics such as B. uniformis, A. muciniphila, and F. prausnitzii, which have shown promise in preclinical models for restoring microbiota, reducing inflammation, and promoting mucosal healing. Synergistic combinations of Lactobacillus, Bifidobacterium, Bacteroides, and Akkermansia strains demonstrate superior recovery outcomes in antibiotic-treated mice compared to single-strain therapies. These findings underscore the potential of advanced probiotic formulations and functional assessment metrics in developing effective strategies for gut microbiome resilience and recovery [141].

Probiotics, live microorganisms found in fermented foods like yogurt, kefir, and sauerkraut, confer health benefits by modulating gut microbiota, lipid profiles, endothelial function, oxidative stress, and inflammation, offering potential for atherosclerosis (AS) prevention and treatment. Strains like Lactobacillus and Bifidobacterium reduce AS risk by lowering cholesterol, trimethylamine N-oxide (TMAO), and inflammatory markers while enhancing gut barrier integrity and short-chain fatty acid (SCFA) production. In ApoE−/− mice, Lactiplantibacillus plantarum ATCC 14,917 (10⁹ CFU, 12 weeks) prevented plaque formation by improving intestinal integrity, while multi-strain probiotics reduced vascular inflammation. L. plantarum ZDY01 lowered TMAO in choline-fed mice, and L. reuteri and Bifidobacterium species increased fecal SCFAs, correlating with reduced hepatic cholesterol [142,143].

Human trials show that Lactobacillus and Bifidobacterium strains improve HDL, lower total cholesterol, and enhance endothelial function in coronary artery disease patients. Probiotics also reduce oxidative stress (lower ox-LDL, MDA) and inflammation (reduced IL-1, TNFα) by inhibiting NF-κB signaling. Emerging Lactobacillus fermentum strains combined with phenolic compounds like quercetin and resveratrol show enhanced antioxidant and anti-inflammatory effects, suggesting novel nutraceutical potential. However, optimal strain combinations, treatment duration, and long-term safety require further clinical validation [144,145,146].

Targeting gut microbiota through dietary fiber supplementation, probiotics, or prebiotics could reduce systemic inflammation and atherosclerosis risk. Further research into personalized microbiota interventions and immune cell trafficking mechanisms may enhance CVD prevention strategies [14].

Recent research highlights innovative methods for regulating the gut microbiome to mitigate atherosclerosis, focusing on probiotics, prebiotics, and small molecules. While probiotics and prebiotics are widely used, their safety, colonization stability, and precise control over microbial composition remain challenging. Scientists at Scripps Research Institute developed a screening method to identify d- and L-α-peptide cyclic molecules that selectively inhibit bacterial growth by disrupting cell membrane function.

In high-fat diet-induced atherosclerotic mice, these peptides reduced cholesterol by 36% after 2 weeks and atherosclerotic plaque area by ~40% after 10 weeks, shifting gut microbiota toward a low-fat diet profile. Additionally, Wang et al. showed that 3,3-dimethyl-1-butanol, a choline analog, attenuated atherosclerosis by inhibiting microbial TMA-lyase activity and TMA/TMAO production. A Ganoderma meroterpene derivative was identified that enriched Parabacteroides merdae, enhancing branched-chain amino acid catabolism and improving obesity-related atherosclerosis. These findings reveal novel microbiota-mediated mechanisms of atherosclerosis progression and propose targeted small-molecule interventions for cardiovascular health improvement [26,57,147,148].

Phenolic compounds, particularly polyphenols like quercetin, resveratrol, curcumin, gallic acid, naringin, procyanidin, geraniin, and protocatechuic acid, show significant potential in preventing and treating atherosclerosis (AS) by modulating gut microbiota and related metabolic pathways [149].

Quercetin, found in foods like apples and red wine, reduces atherosclerotic lesions, cholesterol, and inflammatory markers (TNF-α, IL-6) in ApoE−/− and LDLr−/− mice by increasing gut microbial diversity and beneficial genera like Akkermansia and Bacteroides, while altering bile acid and coprostanol metabolism. Resveratrol, abundant in grapes and berries, lowers TMAO, cholesterol, and LDL, enhances HDL, and improves endothelial function via eNOS and PKA signaling, with gut microbiota modulation increasing Bacteroides and Lactobacillus [150].

Curcumin restores the Firmicutes/Bacteroidetes ratio and reduces TMAO and LPS levels, while gallic acid shows sex-specific reductions in plaque formation. Naringin enhances bile acid excretion and shifts microbial composition, favoring Lactobacillus and reducing TMA-producing bacteria. Procyanidin A2 and geraniin increase microbial diversity and beneficial genera like Akkermansia, reducing plaque area, while protocatechuic acid decreases inflammation without affecting TMAO [151].

These compounds collectively improve gut barrier integrity, reduce pro-atherogenic metabolites (TMAO, LPS), and enhance anti-inflammatory and antioxidant effects, suggesting that dietary intake of polyphenol-rich foods could be a promising strategy for AS prevention and treatment, although further studies are needed to explore synergistic effects with probiotics and precise mechanisms [152].

Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, is a recognized risk factor for atherosclerosis (AS), prompting research into strategies to reduce its levels through dietary interventions, gut flora regulation, inhibition of TMAO precursor production, fecal microbiota transplantation (FMT), and pharmacological approaches, including traditional Chinese medicine [153].

Dietary interventions like the Mediterranean diet, vegan diets, and intermittent fasting lower TMAO by modulating gut microbiota, with studies showing reduced TMAO in vegetarians and those on fasting-mimicking diets due to shifts in microbial composition (lower Clostridia, higher Trichospira). However, nutrients like choline and L-carnitine, while essential, can increase TMAO, necessitating a balance to retain their cardiovascular benefits. Regulating intestinal flora with antibiotics suppresses TMAO but risks dysbiosis and resistance, while probiotics like Lactobacillus plantarum ZDY04 and Bifidobacterium species reduce TMAO and improve lipid metabolism, although results vary by strain [154].

Inhibiting TMAO precursor production targets microbial choline TMA lyase (CutC) with inhibitors like 3,3-dimethyl-1-butanol (DMB), iodomethylcholine (IMC), and fluoromethylcholine (FMC), which reduce TMAO and plaque formation without harming gut flora. Inhibiting TMA conversion to TMAO by targeting flavin-containing monooxygenase 3 (FMO3) with compounds like trigonelline or antisense oligonucleotides decreases TMAO and thrombosis risk, although safe inhibitors are needed [52]. The structural analogue 3,3-dimethyl-1-butanol (DMB) was first shown to inhibit TMA lyases and reduce atherosclerosis in mice [57]. More recently, Roberts et al. developed a mechanism-based inhibitor, iodomethylcholine, that irreversibly targets microbial CutC/D enzymes, suppresses TMA formation, and attenuates atherosclerosis without altering gut microbial viability [155].

FMT shows potential to alter microbiota and reduce TMAO, but limited studies and ethical concerns restrict its use. Pharmacological approaches, including metformin and resveratrol, modulate gut microbiota and lower TMAO, while traditional Chinese medicines like berberine and Ganoderma reduce TMAO by adjusting microbial composition and inhibiting TMA synthesis, showing promise but requiring further clinical validation. These strategies highlight the potential of targeting TMAO to mitigate AS, with ongoing research needed to optimize efficacy and safety [156].

Fecal microbiota transplantation (FMT), a method involving the transfer of filtered fecal samples from healthy donors or autologous sources to restore gut microbiota balance, has gained traction as a therapeutic approach for dysbiosis-related conditions, including atherosclerosis. Initially developed in the 1960s, FMT is highly effective for treating Clostridium difficile infections and shows promise in extra-intestinal diseases like cardiovascular diseases (CVDs) by altering bile acid composition and reducing plasma triglycerides, as seen in obese recipients receiving FMT from lean donors [116,157].

These changes suggest anti-atherosclerotic effects by mitigating dyslipidemia and pro-atherogenic metabolites like trimethylamine N-oxide (TMAO). Preclinical studies demonstrate that FMT from healthy donors reduces plaque development and systemic inflammation in atherosclerosis models. However, human evidence is limited, with small-scale studies showing microbiota shifts but no significant improvements in TMAO, lipids, or endothelial function. One ongoing clinical trial (NCT04410003) is investigating FMT for severe atherosclerosis, but larger trials are needed to confirm efficacy. Risks, including endotoxin transfer and long-term safety concerns, currently limit FMT's widespread use, necessitating further research to optimize its therapeutic potential in atherosclerosis management [158,159,160].

Table 4 summarizes therapeutic interventions targeting the gut microbiota, including their mechanisms, evidence from studies, and current challenges.

While preclinical models consistently show that microbiota-targeted interventions such as probiotics, prebiotics, and fecal microbiota transplantation (FMT) attenuate atherosclerosis, the clinical applicability remains uncertain, as detailed below.

Probiotics and prebiotics: Some randomized controlled trials suggest modest benefits. For instance, supplementation with Lactobacillus plantarum reduced LDL-C and inflammatory markers in hypercholesterolemic patients [161], whereas in other cohorts effects were minimal [162]. Prebiotic fibers such as inulin improved lipid and glucose metabolism and lowered TMAO levels in overweight adults [164], although interindividual variability remained high.

FMT: Although FMT reshapes gut microbiota composition, cardiovascular endpoints have not yet been systematically evaluated in humans. A pilot randomized trial in obese men [163] demonstrated safety and improvements in insulin sensitivity, but cardiovascular outcomes were not assessed.

Dietary interventions: The Mediterranean diet, rich in fibers and polyphenols, lowers circulating TMAO and increases SCFA production. In the landmark PREDIMED trial (>7000 participants), adherence to a Mediterranean diet reduced major cardiovascular events and was associated with favorable shifts in microbiota-derived metabolites [95].

Collectively, these findings underscore that microbiota-based therapies are promising but remain in an early translational phase, with the need for well-powered, long-term randomized trials in cardiovascular populations. These findings are summarized in Table 5, which highlights both the modest benefits observed in small-scale RCTs and the robust evidence from large dietary intervention trials.

Ongoing clinical trials investigating microbiota-targeted strategies are summarized in Table 6. Despite promising results in preclinical and early clinical studies, translation into cardiovascular practice remains challenging. Several ongoing studies have been identified in ClinicalTrials.gov, highlighting the growing interest in microbiota-targeted strategies for cardiometabolic disease.

To summarize the therapeutic section, Figure 5 illustrates how dietary, microbial, and pharmacological interventions converge on common pathways—including increased SCFA production, reduced TMAO, improved gut barrier function, and immune modulation—that ultimately lead to reduced vascular inflammation and atherosclerosis.