The gut-heart axis: a correlation between Paneth cells’ dysfunction, microbiome dysbiosis, and cardiovascular diseases

Abstract

Gut microbiota dysbiosis is characterized by an imbalance in the core microbial equilibrium, leading to changes in the homeostasis of the gastrointestinal tract (GIT) environment. As guardians of the gut microbiota, Paneth cells (PCs) secrete antimicrobial peptides (AMPs) and play a crucial role in maintaining gut integrity and innate immunity in the small intestine. The gut-heart axis has emerged as a critical mediator in cardiovascular disease (CVD) pathogenesis and has drawn significant attention. In this regard, the reciprocal relationship between gut dysbiosis and PC dysfunction has been proposed, which may contribute to a compromised gut barrier and increased systemic inflammation, one of the main drivers of CVD development. It is also well-established that dysfunctional PCs disrupt gut homeostasis and subsequently permit the translocation of pro-inflammatory metabolites like trimethylamine N-oxide (TMAO) while reducing protective short-chain fatty acids (SCFAs), which correlates with atherosclerosis, hypertension, and heart failure.

A better understanding of the underlying mechanisms linking gut health, PCs function, and cardiovascular outcomes is warranted for developing novel gut-target therapies against major CVD risks. This review aimed to comprehensively discuss the predominant role of PCs in the gut-heart axis, some effective compounds on PC function and AMP modulation, and finally, a possible correlation between PC dysfunction and CVD pathogenesis, encouraging future research to further elucidate this crosstalk.

Introduction

As a leading cause of mortality, cardiovascular diseases (CVDs) pose a major public health challenge worldwide. In recent years, their impact has been particularly severe, resulting in nearly 20 million deaths in 2021 [1, 2]. This broad category of diseases encompasses conditions such as coronary artery disease (CAD), heart failure (HF), stroke, and peripheral arterial disease (PAD), each contributing to the high morbidity and mortality rates associated with CVD. The prevalence of these conditions is multifactorial and driven by a combination of genetic and environmental factors, including poor diet, limited physical activity, smoking, excessive alcohol consumption, and/or other lifestyle factors. Despite recent advances in medical research, CVD has remained a major leading cause of disability and mortality, particularly in low- and middle-income countries [3, 4]. Besides, the management of CVD becomes more complicated by the presence of comorbidities, the need for personalized therapeutic schedules, and the challenges of medication adherence due to accompanying complex pharmacotherapy regimens.

The gut microbiota, the community of microorganisms residing in the gastrointestinal tract (GIT), has recently garnered significant attention in medical research due to its substantial impact on human health. In this regard, the core microbiome signature, as a unique microbial composition pattern, characterized by dominant bacterial groups that co-exist in the ecosystem of healthy human guts. This signature represents the typical ecological configurations rather than generic diversity metrics or isolated biomarkers, which have been shown to correspond with functional roles in the gut microbiome, such as carbohydrate metabolism or fermentation, and can be used to diagnose deviations linked to health or disease [5]. The core microbial components remain consistent over time and across individuals, unlike transient microbiome elements, which fluctuate under environmental and host-related factors such as diet, genetics, and other exogenous factors [6]. Microbiome stability is defined by two key attributes: resistance (the ability to withstand disturbances) and resilience (the capacity to recover after a disruption). This stability operates on two scales—short-term maintenance within an individual and long-term evolutionary adaptation at the population level, reflecting shifts in the microbiome alongside host species evolution [6, 7]. Alternations in the gut microbiota composition, known as dysbiosis, have also been linked to cardiometabolic diseases. Modulating the gut microbiota through diet, pro-, and prebiotics, novel therapies targeting gut microbiota such as fecal microbiota transplantation (FMT), and engineered probiotics can help restore balance and improve metabolic health [8, 9]. In detail, engineered probiotics are genetically modified microorganisms created using synthetic biology to enhance gut health and various diseases treatment, such as cardiovascular conditions, by targeting some molecules, like enzymes, cytokines, and bactericidal peptides named antimicrobial peptides (AMPs) in response to gut signals, and allowing precise modulation of the intestinal environment [10]. Compared to natural probiotics, common strains, including Lactococcus lactis, Lactobacillus, Bacillus, and Escherichia coli, possess greater specificity, flexibility, and control. These engineered probiotics are being developed to address metabolic, digestive, cardiovascular, and neurodegenerative diseases. Advances also improve their gut colonization and acid resistance. However, future efforts focus on generating precise synthetic microbial communities for personalized medicine [10]. In addition, CRISPR-based microbial modulation refers to the use of CRISPR-Cas systems derived from bacterial adaptive immune mechanisms to regulate or modify the genomes of the gut microbiota to improve intestinal health with high specificity and efficiency [11].

Regarding the FMT modality, in a recent publication, 67 hypertensive patients who participated in a multicenter randomized clinical trial received FMT capsules three times during the study duration (30 days). Following a three-month follow-up, a significant bacterial transition was observed against harmful enteric bacteria. Furthermore, in comparison with placebo arm, the individuals who received FMT capsule exhibited a meaningful change in systolic blood pressure (SBP, 12.81mmHg in the FMT group vs. 5.14 mmHg in the placebo group; p = 0.029, and between-arm reduction [− 4.34 mmHg (95% CI, − 8.1 to − 0.58; p = 0.024)]), especially in patients over 48 years old [12].

Beyond the gut microbiota, Paneth cells (PCs), as a specialized secretory epithelial lineage, located at the base of the small intestinal crypts of Lieberkühn, play an imperative role in intestinal stability, gut microbiome balance, intestinal stem cell homeostasis, and response to the immune system and other stress conditions [13, 14].

To date, biologic-based therapeutics are regarded as one of the cutting-edge approaches for designing more efficient treatment strategies. In this context, modulating the gut microbiota represents a novel therapeutic strategy for a wide range of diseases, including cirrhosis, inflammatory bowel disease, and various cancers [15–17]. Within the field of CVD, creating new therapeutics that are both innovative and safe poses a significant challenge, necessitating a comprehensive approach to prevention, early diagnosis, and novel treatment options. This review explores the latest advancements in gut microbiome-based therapeutics to determine whether PC targeting will be effective in improving global health outcomes in the setting of CVD therapy.

The role of PCs in gut microbiome homeostasis

The gut microbiota refers to a complex component of the GIT, comprised of over 100 trillion living microorganisms under a balanced status, which is crucial for maintaining overall gut homeostasis at the host-microbial interface [18]. The gut microbiota is associated with various health issues, such as dysbiosis, and supports multiple functions, including digestion, regulation of the immune response, detoxification, and protection against pathogens [19]. In this regard, the PCs, located ubiquitously at the base of crypts alongside the leucine-rich repeat-containing receptor 5 intestinal stem cells (Lgr5⁺ ISC), play a pivotal role in maintaining the balance of the gut microbiota by engaging the cholinergic system and through constitutive production of various proteins in their specialized secretory dense core vesicles (DCVs). The main products of the DCVs are various AMPs such as lysozyme, α-defensins, secretory phospholipase A2, and regenerating islet-derived protein 3 beta and gamma (Reg3β and − 3γ), which maintain a balanced interface between the host and the gut microbiota by targeting various endotoxins (Fig. 1) [20, 21].

Fig. 1.

The impacts of PCs on Gut Microbiota. The PCs, strategically located at the base of the intestinal crypts of Lieberkühn, play a substantial role in maintaining a healthy gut ecosystem. (A) These specialized epithelial cells are defined as the first line of defense against pathogens and further contribute to intestinal hemostasis and promote microbial diversity— by establishing a balance in the gut microbiota composition. PCs prevent gut dysbiosis and contribute to the host’s well-being through (1) AMPs secretion and immune regulatory activities, (2) Lgr5 + stem cell support (niche maintenance), (3) CVA morphogenesis, and (4) Maintenance of gut barrier integrity. (B) There is a close cross-link between gut dysbiosis and PCs dysfunction in which the reduced AMP secretion allows pathogenic bacteria to overgrow, leading to the microbial imbalance. Also, disruption in gut barrier integrity due to PCs dysfunction can result in bacterial translocation, recruitment of immune cells, and excessive cytokine/chemokine production and further exacerbating dysbiosis and systemic inflammation. Abbreviations: AMPs, anti-microbial peptides; CVA, crypt-villus axis; CD74+, cluster of differentiation 74+; Lgr5+, Leucine-rich repeat-containing G-protein coupled receptor 5+

The α-defensins regulation mediated by PCs can mainly occur through Toll-like receptor 9 (TLR-9) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) [22], which is a key pattern recognition receptor (PRR) in PCs. The NOD-2 senses a component of bacterial peptidoglycan named muramyl dipeptide (MDP), thereby triggering innate immune responses within PCs. NOD2 activation can influence the expression of various AMPs through the nuclear factor-κB (NF-κB)-dependent pathway. To facilitate lysozyme sorting, NOD2 also interacts with some factors such as leucine-rich repeat kinase 2 (LRRK2) and Rab2a (Fig. 2). In this regard, Wang et al. also showed that Rip2, an adaptor protein in NF-κB and MAPKs activation, is also required in NOD2/ LRRK2/Rab2a-mediated lysozyme sorting into the DCVs [23].

Fig. 2.

Comprehensive signaling pathways in PCs. (A) Autophagy plays a critical role in the function of PCs. The coordinated action of ATG5, ATG7, and ATG16, as key genes in the autophagy process, leads to the formation of double-membrane structures known as autophagosomes. Autophagy machinery especially contributes to the regulation of AMPs secretion and the degradation of intracellular pathogens beyond the recycling of cellular components. (B) This part illustrates the canonical Wnt signaling pathway in PCs. In the absence of Wnt ligands to stimulate Frizzled receptors, the inhibitory complex, composed of APC, Axin, GSK-3β, and CK1, targets β-catenin for phosphorylation. Following Wnt signaling activation, stabilized β-catenin (phosphorylated form) translocates into the nucleus and forms a complex of TCF/LEF transcription factors, which in turn, activate the transcription of Wnt target genes. (C) NOD2, a key pattern recognition receptor, plays a crucial role in directing both gene expression and proper sorting and secretion of AMPs i.e., lysozyme, as a key enzyme for degrading bacterial cell walls (D) The presence of some TLRs (TLR-9, TLR-3, TLR-4) in regulating AMPs expression, highlighting the key role of TLRs, and related downstream transcription factors named TRIF, MyD88 in this regard. Abbreviations: Akt-1, Akt serine-threonine protein kinase-1; AMPs, antimicrobial peptides; APC, antigen-presenting cell; ATG, autophagy-related gene; CK1, Casein Kinase 1; c-Kit, c-receptor tyrosine kinase; CRP, C-reactive protein; DCVs, dense core vesicles; ER, Endoplasmic reticulum; GSK-3β, glycogen synthase kinase 3β; IRF3/7, IFN regulatory factors 3 and 7; LEF, lymphoid enhancer factor; LRRK2, leucine-rich repeat kinase 2; NF-κB, nuclear factor ‘kappa-light-chain-enhancer of activated B-cells; PI3K, phosphatidylinositol-3 kinase; Rab2a, Ras-related protein Rab-2A; NOD2, nucleotide-binding oligomerization domain-containing protein 2; Reg3 β/γ, regenerating islet-derived protein 3 beta/gamma; SCF, Stem cell factor; TCF, T-cell factor; TLR, Toll-like receptor; Trif, TIR domain-containing adaptor inducing interferon

Notably, TLRs also play a crucial role in AMPs expression. For instance, following bacterial sensing, TLR-9 can stimulate the gene expression of defensin [24]. Additionally, TLR-3 and − 4 have the potential to recognize the viral double-stranded RNA and bacterial lipopolysaccharide (LPS), respectively, through TIR-domain-containing adaptor-inducing interferon-β- Myeloid differentiation factor 88 (TRIF-MyD88) adaptor proteins and transcription factors NF-κB and IRF3/7, which ultimately upregulate the other AMPs such as Reg3-β, Reg3-γ, and CRP-ductin [25].

Intriguingly, the PCs provide a niche to support the Lgr5⁺stem cells located in the intestinal crypts, consisting of epidermal growth factor (EGF), Wnt3a, and Notch signaling ligands [e.g., delta-like 4 (DLL4)]. The DLL4 participates in the proliferation, differentiation, and retention of the stemness feature of ISCs, which is also necessary for the integrity and function of the intestinal epithelium [26]. The gut microbiome immune responses derived from PCs can also be mediated by lysozyme 1 (Lyz1) regulation [27]. An experimental study showed that Lyz1 deficiency in Lyz1 ^−/− mice can influence the intestinal immunity and reduce the mucosal response to bacterial molecular patterns, which is accompanied by the expansion of mucolytic bacteria, one of the leading causes of bowel inflammatory conditions such as Crohn’s disease [27].

Maintaining the GI barrier integrity

PCs are also involved in the physical GIT barrier by maintaining the integrity of the epithelial layer against invasive and exogenous pathogens [28]. Previously, it has been well-established that PCs can directly sense the gut microbiota via PRRs, TLRs 3/4, by increasing the anti-microbial factors expression through the MyD88-TRIF dependent pathway, which subsequently confines the enteric bacteria-induced mucosal penetration into the host tissues, as well as decreases pathogen translocation into the mesenteric lymph nodes [18]. Hence, PCs augment the GIT’s ability to combat pathogens in the face of bacterial infections, consequently maintaining a balanced symbiotic relationship [18]. Also, the proliferation rate, adhesion potential, and stiffness of PC-rich domains accompanied by the cluster of Lgr5⁺ stem cells are the main factors in promoting the crypt cells’ fission, which is a pivotal process for intestinal expansion [29].

Immunomodulatory impacts of PCs

Regarding the immunomodulatory impact of PCs, it has been shown that IL-17 receptor activation, by T-helper 17 (Th17, which produces IL-17), plays a substantial role [30]. Furthermore, Brabec et al. found that genetic mutation of IL-17 can alter the gut microbiota toward gut dysbiosis due to a reduced number of lysozyme-expressed PCs in the ileum [30]. However, it has been revealed that the specific type of PC subset (CD74⁺ PCs) increased after the GIT inflammatory diseases [14]. Besides the undeniable role of the CD74⁺ PCs subset in the progression of GIT infection and inflammation, the pathogenic microbes can also potentially increase the expression of PC-specific mucosal pentraxin (Mptx2) in activated PCs following increased cytokine release and reactive oxygen species (ROS) generation. By targeting MyD88 and Mptx2, the CD74⁺ PC population is dramatically mitigated, leading to improved pathogen-induced inflammatory disease [14].

Additionally, in response to inflammatory diseases such as inflammatory bowel syndrome (IBS) and Lgr5⁺ stem cell loss, it has been discovered that the PCs have the potential to de-differentiate through SCF/c-Kit/Wnt signaling accompanied by PI3K/Akt activation and GSK3β inhibition, as well as further attain the stem cell characteristics to regenerate the plasticity of the intestinal epithelium (Fig. 2) [31].

Role of autophagy in pcs’ immune modulation

It has been well documented that PCs can exert a desirable antimicrobial effect by modulating innate immunity in the face of various infections. Noteworthy, the interplay between autophagy and PCs is crucial for enhancing the production of antibacterial substances in the gut, protecting the intestinal epithelial barrier, and maintaining intestinal homeostasis [32].

In this line, INF-γ-dependent immune responses derived from PCs-specific autophagy, a preserved catabolic process to maintain energy balance by turnover of worn-out organelles, play a rigorous protective role, particularly against intestinal TNF-α-mediated inflammation and acute infections [33]. Also, Araujo et al. revealed that INF-γ, one of the main inflammatory mediators, plays a crucial role in compromising PC function and inducing cell death through non-canonical and mTOR-dependent signaling pathways. In particular, mTOR, as a serine/threonine kinase and master regulator of cellular metabolism [34], has a key role in the regulation of the autophagy process through mTORC1 [35] (Fig. 2).

However, PCs dysfunction induced by autophagy gene knockout, ATGE5^−/−, contributes to acute mortality following cytokine-mediated GIT infection [33]. During starvation, the levels of PC-secreted AMPs remarkably reduce, secondary to the failed secretory DCVs and autolysosome degradation [36, 37]. The development of chronic inflammatory diseases, including inflammatory bowel disease (IBD), is also associated with the PCs’ dysfunction and dysregulated autophagy in AMP production [38]. In addition, a direct association has been established between intestinal inflammation and the impaired PC-derived autophagy process, in which the mutations in Atg16L1 can lead to PC dysfunction, and consequently cause ileitis development [39]. Furthermore, deletion of Atg7 in intestinal epithelial cells led to alterations in the behavior of secretory vesicles and decreased crypt microenvironment AMP levels [40].

In the clinical setting, it has also been proven that the level of AMPs secreted by PCs in obese individuals is reduced following unfolded protein accumulation. On the other hand, similar to α-defensins deficiency, the increased population of Firmicutes rather than Bacteroidetes can be observed during obesity. Together, the enhanced rate of unfolded protein response (UPR) and subsequent PC dysfunction are assumed to correlate with impaired autophagy activity [41].

The pcs’ regulation and differentiation

As part of the innate immune system, the intricate process of regulation by PC depends on multiple factors. In this regard, the Wnt/β-catenin pathway, as a multi-potential signal in intestinal epithelium pathophysiology [42], has a pivotal role in the maturation program of PCs and ISCs [43].

In line with this scenario [44], it has also been revealed that Sox9, as a transcription factor in the intestinal epithelium, is considered another modulator of PCs’ differentiation in a Wnt/β–catenin–Tcf dependent manner [45]. In turn, silencing the Sox9 gene affected the PCs and Gablet cells, resulting in substantial changes in the colon epithelium morphology [45].

For the first time, Vidrich et al. revealed the regulatory effect of fibroblast growth factor receptor-3 (FGFR-3) on the PCs’ characteristics, intestinal crypt cells morphogenesis, and stem cell proliferation via both β-catenin/Tcf-4-dependent and independent manners [46]. Another factor that plays a crucial role in the maturation of both goblet cells and PCs is the terminal differentiation known as the SAM pointed domain ETS transcription factor (Spdef). The Spdef is a Tcf4-responsive gene downstream of Math1, which targets a specific subset of goblet and PCs genes, including Cryptdins, Mmp7, Ang4, Kallikreins, and Muc2 [47].

In addition to the importance of Notch signal in the regulation of stem cells and their differentiation into the secretory lineage (i.e., PCs, Gablet, and enteroendocrine cells), Shp2-mediated MAPK signaling plays a crucial role in Gablet cells and PCs expression through regulation of the Wnt/β-catenin pathway (Fig. 3) [42].

Fig. 3.

Immunomodulatory impact of Paneth cells. Abbreviations: AMPs, Antimicrobial peptides; Dact1, dishevelled binding antagonist of beta-catenin 1; EGF, Epidermal growth factor; FGF-R3, fibroblast growth factor receptor 3; FXR, farnesoid X receptor; Gfi-1, growth factor independence-1; ICS, intestine stem cell; INF, interferon; HNF4α, hepatocyte nuclear factor 4α; IL-17 A, interleukin-17 A, IL-17 A R, interleukin-17 A receptor; Lgr5+, Leucine-rich repeat-containing G-protein coupled receptor 5; MAPK, mitogen-activated protein kinase; Nur77, Nuclear receptor 77; Shp2, Src homology 2-conatining protein tyrosine phosphatase 2; Wnt3a, wingless-type MMTV integration site family 3a, DII4, Delta-like 4; Math 1, mouse atonal homolog 1; Spdef, SAM pointed domain-containing Ets transcription factor; SOX-9, SRY-box transcription factor 9; Th17, T-helper 17

Recently, a novel PC’ regulator, named nuclear receptor 77 (Nur77), has also been found to play a crucial role in PC differentiation and function by modulating disheveled binding antagonist of beta-catenin 1 (Dact1), an antagonist to Wnt/β-catenin signaling [48]. Following Nur77 deficiency, a remarkable impairment in the epithelial stem cell niche and AMP expression was also identified. In this regard, Jones et al. uncovered the upstream transcriptional regulator of PCs, specifically Wnt3-related signaling HNF4-α, in jejunal enteroids. Besides, it has been shown that HNF4a has a potent potential to regulate homeostasis by promoting PC differentiation and epithelium renewal even in the absence of compensatory mesenchymal signals, and canonical Wnt signaling (Fig. 3) [49].

The role of the gut microbiota on PC biology and population was also evaluated by Alexi et al. They interestingly found that the gut microbiota increases PC numbers and AMPs expression (e.g., Reg3γ) without significantly altering ISC proliferation in vivo [50]. In turn, the remodeling of the gut microbiota is also associated with AMPs regulation. In this sense, it has been found that increased levels of FABP4 in PCs can influence the α-defensins expression in rats receiving a high-fat diet. Indeed, FABP4-mediated α-defensins downregulation is directly related to the increased risk of pathogenic bacteria and gut reprogramming, and further predisposition to inflammatory diseases [51].

Gut dysbiosis following PC dysfunction

Gut dysbiosis, an imbalance in the gut microbiota, can be mainly caused by prolonged use of antibiotics, proton pump inhibitors (PPIs), non-steroidal anti-inflammatory, and certain anti-diabetic medications, which have the potential to distort the gut microbiota signature [52–54]. Also, high intake of carbohydrates, processed foods, and poor dietary fiber consumption are considered other causes of dysbiosis [55, 56]. In addition, various inflammatory diseases, exposure to environmental toxins, and psychological stress, such as anxiety and depression, contribute to gut microbiota imbalance due to the overgrowth of harmful bacteria [57–59].

In light of PC’s crucial role in constituting the gut microbiota composition, and the GIT mucosal interface between host and environment, PC dysfunction (driven by cellular stress, genetic mutations, e.g., ATG16L1, NOD-2, LRRK2, and UPR), or environmental factors can trigger gut dysbiosis [60], such as diet-induced obesity and long-term consumption of the Western diet, developing PC dysfunction likely through over-activation of farnesoid X receptor (FXR) and type I interferon (IFN) signaling, mediated by increased levels of bile acid deoxycholic acid in the ileum section [61].

Therapeutic options to augment PC function

Considering the important role in retaining the gut microbiota hemostasis, PCs dysfunction and subsequent AMPs secretion impairment are associated with a broad array of intestinal and non-intestinal pathologies. Although lifestyle modification and pre-/probiotic intake are highly recommended and can be helpful to restore a balance in the gut microbiota, some pharmacotherapies also exert benefits in this regard.

Azathioprine

A recent study has investigated the therapeutic potential of azathioprine, a medication used for the treatment of Crohn’s disease, to modify PC dysfunction [62]. The results of the study showed that azathioprine administration augmented the PCs’ function and differentiation by enhancing the level of AMPs expression and mitochondrial oxidative phosphorylation in the experimental settings [62]. These findings unraveled the therapeutic potential of azathioprine, which could be considered for further research in the field of PC dysfunction.

Lysozyme supplements

To note, PCs are the main source of C-type lysozyme enzyme, a β-1, 4-N-acetylmuramoylhydrolase, mainly involved in breaking down the bacterial cell walls [27].

As a novel therapeutic approach, lysozyme supplementation improves PC function by enhancing lysozyme expression, reducing intestinal permeability, inflammation, apoptosis, and pathological markers [63]. The gut microbiota remodeling also plays a crucial role in restoring gut barrier injury and bacterial translocation following lysozyme administration [63, 64].

Noteworthy, the potential of lysozyme antimicrobial defense can change when exposed to bacterial infection. In this context, the autophagy-based secretory pathway plays a substantial role in likelihood through Atg16L1 up-regulation and innate lymphoid cell recruitment in response to the invasive pathogens-induced endoplasmic reticulum stress [65].

The association between CVD and gut microbiota dysbiosis

Recent studies have underscored the significant impact of the gut microbiota on various systemic diseases, including CVD. It is also well-documented that dysbiosis has been linked to the development of atherosclerosis, hypertension, and other cardiovascular conditions [66]. Through modification of bile acid and cholesterol metabolism, it has been implied that gut dysbiosis potentially increases the risk of coronary artery disease [67, 68]. Also, gut microbiome-derived metabolites following dietary supplements play a critical role in CVD risk prediction [69]. In this regard, Wang et al. studied the pro-atherogenic phosphatidylcholine metabolism with gut flora and subsequent increasing the risk of atherosclerotic heart diseases most likely through up-regulation of macrophage scavenger receptors. Consequently, they found a positive correlation between increased levels of phosphatidylcholine metabolite and the pathogenesis of atherosclerosis [70].

Noticeably, the microbiota community also changes during the CVD, highlighting how gut dysbiosis impacts CVD pathogenesis. For instance, during HF, the population of butyrate-producing bacteria decreases, while the numbers of pathogen bacteria such as Salmonella, Campylobacter, Shigella, Yersinia enterocolitica, and Candida species increase [71–73]. The clinical importance of trimethyllysine, a precursor of carnitine, in the production of atherogenic-related metabolites was also investigated in the prognosis of patients with stable chronic HF [74]. The findings of this study pointed out that the serum levels of trimethyllysine were positively correlated with the serum NT-proBNP biomarker and could be considered a reliable indicator for major adverse cardiac events, including all-cause mortality, re-admission, and cardiovascular death [74].

As a biological intervention, in a clinical study by Malik et al., it has been revealed that Lactobacillus plantarum 299v supplementation has the potential to improve vascular endothelial function and reduce inflammatory responses in men with stable CAD [75]. In detail, following oral administration of the liquid form of this supplement once daily for six weeks, the remarkable vasodilation effect in the esistance arteries, along with serum reduction of IL-8 and IL-12, was observed without influencing TMAO or other traditional biomarkers [75].

Additionally, a correlation between IBD and the higher risk of acute myocardial infarction (AMI) has also been found in the clinical setting [76]. Following histopathological examination, some microscopic changes such as carotid intimal thickness, endothelial dysfunction, and increased serum levels of homocysteine (~ 4–5 fold) were also identified [77]. Emoto et al. also found a rational correlation between altered gut microbiota profile and CAD, assessed on bacterial DNA of fecal samples using data mining analysis, which could be considered a diagnostic indicator [78].

Notably, in patients with advanced heart failure (cardiac cachexia), intestinal blood flow, especially in the superior mesenteric artery, decreases, contributing to the growth of GI bacteria in the justamucosal zone following increased levels of immunoglobulin A-antilipopolysaccharide [79]. All clinical and preclinical studies investigating dietary and microbial interventions on gut microbiota modulation and subsequent cardiometabolic outcomes have been presented in Table 1.

Table 1.

Clinical and preclinical studies investigating dietary and microbial interventions on gut microbiota and cardiometabolic outcomes

Authors	Year	Type of study	Sample	Disease	Interventions	Outcome(s)
Organ et al., [111]	2016	In vivo	Mice (n = 10–12)	HF	Diet containing choline (1.2%) or TMAO (0.12%)	HF severity is significantly increased in mice fed diets supplemented with either choline or the TMAO.
Sandek et al., [79]	2014	Clinical trial	Patients (n = 65)	CHF	-	Due to reduced Intestinal blood flow in patients with CHF, the higher rate of juxtamucosal bacterial growth and GI symptoms has been observed, which is complicated by cachexia.
Marques et al., [174]	2017	In vivo	Mice (n = 13)	HF and hypertension	High-fiber diet and acetate supplementation	High-fiber diet significantly reduced systolic and diastolic blood pressures, cardiac fibrosis, and hypertrophy, most likely through the short-chain fatty acid acetate.
Wan et al., [105]	2018	Clinical trial	Healthy adults (aged 18–35 years, n = 217)	-	Low and high-fat diet	Higher-fat is appear to be associated with increased plasma proinflammatory factors, unfavorable changes in gut microbiota, and fecal metabolomic profiles.
Fan et al., [12]	2025	Multi-central clinical study	Patient (n = 67)	Hypertension	FMT	Unstable BP reduction was observed in the FMT-received hypertension. SBP reduction: [-4.34 (95% CI, − 8.1 to − 0.58; p = 0.024) mmHg]
Malik et al., [75]	2018	Clinical trial	Male patients (n = 20)	Stable CAD	Lp299v supplementation	Improved endothelium-dependent vasodilation and reduced systemic inflammation in men with stable CAD were found following Lp299v supplementation.
Wang et al., [108]	2023	Clinical trial	Young adults (n = 17)	Healthy	Adding Lean Red Meat to LOV-HDP	The favorable changes in gut microbial composition in association with improvements in cardiovascular risk factors were observed.
Hayashi et al., [72]	2018	Clinical trial	Patient (n = 22)	Comp or Decomp HF	-	Bifiodobacterium was abundant in HF patients. TMAO was also increased in HF patients (Decomp HF vs. control, P = 0.003; Comp HF vs. control, P = 0.004). Scherichia/Shigella was more abundant in Decomp than in Comp HF (P = 0.030).
Djekic et al., [106]	2020	Clinical trial	Patient (n = 27)	Ischemic heart disease	VD/MD	The VD reduced levels of ox-LDL-C, improved cardiometabolic risk factors, and related to the Ruminococcaceae, Lachnospiraceae, and Akkermansiaceae abundance in patients with IHD.
Hill et al., [175]	2024	Clinical trial	Patients (n = 41)	Overweight and obesity	HDP +/- unprocessed meat	In both groups, the improvements in multiple risk factors for the development of cardiometabolic conditions were observed.
D Shah et al., [176]	2020	Clinical trial	Patient (n = 104)	Overweight and obesity	Soy consumption (3 mg/kg)	Depending on gut microbiota composition, especially the abundance of Prevotella species, soy consumption is associated with reduced systolic blood pressure. (p < 0.05)

Abbreviations: Compensated Heart Failure, Comp HF; Decompensated Heart Failure, Decomp HF; IHD, Ischemic Heart Disease; Lp299v supplementation, Lactobacillus plantarum 299v Supplementation; LOV, lacto-ovo vegetarian diet, MD, Meat Diet; HDP, Healthy U.S.-Style Dietary Pattern, Ox-LDL-C, Oxidized Low-density lipoprotein cholesterol; TMAO, Trimethylamine N-oxide; Vegetarian Diet, VD

Proposed therapeutic options

There are several therapeutic options to improve the gut microbiota dysbiosis associated with CVD, including fiber-rich diets to enhance SCFAs, and probiotics (e.g., Bacteroides vulgatus, B.dorei, Lactobacillus helveticus, and L. casei) with cardiovascular benefits [80, 81]. Small molecule antimicrobial enzyme targeting TMA and TMAO can also reduce the risk of atrial fibrillation, atherosclerosis, and subsequent stroke event [82, 83], most likely through modulation of microbiota compositions and microbiome-derived circulating metabolites. The FMT from a healthy donor is considered a novel therapeutic option to improve systolic blood pressure, endothelial dysfunction, vasculitis, oxidative stress, and the immune system (a balance between Th17 and Tregs) [84]. However, there are some limitations due to the donor variability in FMT, such as the variation in fecal microbial composition, which can influence its therapeutic potential among different donors, particularly in complex conditions like IBD [85]. Additionally, donor screening protocols are also necessary to minimize the risk of pathogen transmission. To our knowledge, all research in the field of FMT assessed the short-term efficacy and safety; therefore, the long-term efficacy and durability of FMT remain uncertain, with limited data on sustained microbiota engraftment and clinical benefits over time. Addressing these challenges is critical for optimizing FMT standardization and wider clinical application.

As mentioned earlier, gut dysbiosis can lead to immune impairment and intestinal inflammation, consequently affecting heart health. Additionally, PC-derived IL-17a appears to participate in ischemic reperfusion injury (IRI), which can be considered another therapeutic target.

α-defensin supplement

The AMPs produced by PCs, particularly α-defensins, are critical factors in maintaining intestinal homeostasis and innate immunity [1]. These peptides are secreted in response to bacterial and other stimuli, providing the first line of defense against pathogens while preserving the beneficial commensal microbiota [1, 86]. The α-defensin (also known as cryptdin), derived from neutrophils, macrophages, and PCs, appears as a main constituent of PCs’ secreted AMPs. The prominent bactericidal activity of α-defensins against pathogens helps modification of the gut microbiota composition, which is essential for overall intestinal health [1]. Intriguingly, the role of PC-derived α-defensins in modulating the gut microbiota composition suggests a potential therapeutic option to promote cardiovascular health [87]. It is worth noting that where PCs topographically reside can affect the potency of bactericidal activity and innate immune responses. Hence, the PCs have maximum reach in the distal part of the ileum. In addition, the antibacterial potential and the expression of α-defensins isoform 1 and 4 in ileal PCs are higher than in other sections of the small intestine, consisting of the jejunum and duodenum, in preclinical settings [88].

One proposed mechanism is likely through maintaining the gut microbiota hemostasis, and intestinal permeability, as well as the prevention of systemic inflammation responses [89, 90], derived from the translocation of bacterial endotoxins into the blood circulation and mesenteric lymph nodes.

Short-chain fatty acids (SCFAs)

Specific gut microbiota-derived metabolites produced by healthy gut microbiota, named short-chain fatty acids (SCFAs), including butyrate, acetate, and propionate, have been shown to exert a protective effect against CVD [91]. These metabolites can influence blood pressure regulation, lipid metabolism, and anti-inflammatory pathways [86].

SCFAs exert a desirable cardiovascular protective effect beyond intestinal integrity support and anti-inflammatory properties, mainly through histone deacetylase inhibition and metabolic benefits. In turn, the α-defensins, by providing a beneficial microbial environment, may enhance the production of SCFAs, as protective metabolites, thereby contributing to cardiovascular health. Given these advantages, SCFAs, especially butyrate salts, could be considered the essential stimuli for α-defensins secretion [92, 93].

Beyond the desirable impacts, including anti-inflammatory (by modulating IL-6, TNF-α, IL-10, and TGF-β), immunomodulatory (by targeting T and B cells), and metabolic benefits (such as anti-obesity and anti-diabetic effects), some cardioprotective benefits of SCFAs, like vasodilation through G-protein receptor (GPR) 41/43 activation, and reduction of atherosclerosis by inhibiting cholesterol synthesis also discovered [94]. There are several in vitro (1 µM, 0.2–10 mmol) [95–98] and in vivo (100–200 mmol/L in drinking water, 200 mg/kg, gavage) [97–104] studies with various concentrations regarding the beneficial cardioprotective effects of SCFAs.

For example, in both in vivo study and double-blind RCT, the cardioprotective effect of calcium-propionate supplement in apolipoprotein E−/− (ApoE −/−) mice (n = 23) under HFD (crude fat 34.6%, cholesterol 290 mg/kg; Ssniff, Soest, Germany, E1574) and 62 patients with dyslipidemia (elevated baseline LDL cholesterol levels) (500 mg, twice daily) for 6 and 8 weeks, respectively, has been observed by modifying cholesterol homeostasis [100].

Together, SCFAs have wide-ranging health benefits but need to be maintained at appropriate levels to prevent negative side effects. Increasing dietary fiber is the safest way to enhance SCFA production, whereas direct supplementation should be approached carefully due to potential risks.

In a randomized controlled-feeding trial for six months, young healthy individuals (n = 245), aged 18–35 years, were fed by iso-caloric lower- or higher-fat diets. Intriguingly, the results showed that SCFA serum levels in the higher-fat diet (fat 40% energy) group significantly reduced when compared with the lower-fat diet group (fat 20% energy; p < 0.001). Also, in fecal examination, increased number of beneficial bacteria such as Faecalibacterium (p = 0.04) and Blautia (p = 0.007) were observed, while the higher-fat diet was associated with increased harmful bacteria, named Bacteroides (p < 0.001), Alistipes (p = 0.04), and decreased levels of Faecalibacterium (p = 0.04) beyond the increased serum levels of pro-inflammatory factors [105].

In a study by Djekic et al., the compositions of gut microbiota were compared in two groups who received either lactovo-vegetarian or beef (145 g of meat) diet (n = 31). Beyond the significant difference in lipid profile, including mean total cholesterol (-5.03 mg/dL), ox-LDL-C (-2.73 U/L), and LDL-C (-3.87 mg/dL), the results indicated that the community of Ruminococcaceae, Lachnospiraceae, and Akkermansiaceae, responsible for SCFA, is higher in the VD group [106].

In case of the carotid intima-media thickness (IMT), it has also been reported that the Firmicutes/Bacteroidetes ratio was greater in the group with high IMT values (> 0.9) than in those with normal IMT (IMT < 0.9) (2.299 vs. 1.436, respectively; p = 0.031) [107].

Noteworthy, a clinical study was designed to unveil the possible effect of adding unprocessed/processed lean red meats to a healthy U.S.-style dietary pattern (HDP) on SCFA levels. Despite no change in gut microbiota and fecal SCFA level, some changes in lipid profile were observed, associated with lowering cardiovascular risk factors [108].

Trimethylamine-N-oxide (TMAO) inhibitors

Evidence suggests that the high serum concentration of TMAO, as a toxic gut microbial metabolite, is associated with an increased risk of CVD, especially atherosclerosis [109]. In better words, TMAO is known to actively participate in atherosclerotic plaque formation by promoting platelet accumulation, increased oxidative stress, and inflammatory responses [110]. In ApoE-deficient mice, it has also been shown that choline or TMAO-rich supplements exacerbated the pressure overload HF characterized by cardiac hypertrophy, pulmonary edema, reduced EF, and myocardial fibrosis [111]. In turn, the reduction of plasma TMAO using choline TMA lyase and TMA oxidation inhibitors could provide CV benefits by stabilizing atherosclerotic plaques [110].

Another study in bovine aortic endothelial cells also indicated that TMAO can induce endothelial dysfunction by targeting vasodilatory mechanisms through modulating nitric oxide (NO) release, purinergic-activated intracellular Ca²⁺/eNOS pathway, highlighting a new concept of the gut–heart axis [112]. To ascertain the TMAO potential in the first stroke prediction, Nie et al. designed a study to determine the serum concentration of TMAO in hypertensive patients who were admitted due to the first stroke. According to the obtained data, a linear correlation was found between the first stroke occurrence and increased levels of TMAO [113]. Compelling evidence also noted that the serum levels of TMAO and its precursor, TMA, significantly increased in patients who underwent aortic valve replacement when compared with healthy people [114]. Noticeably, a correlation between higher TMA serum levels and cardiomyocyte death, accompanied by LDH and albumin-increased serum levels, was also found [114].

Also, a correlation between the gut microbiota-derived metabolites and the prognosis of cardiovascular risks is well-documented in the clinical setting. For example, the plasma levels of phenylalanine and phenylacetylglutamine significantly increased in patients with coronary in-stent restenosis [AUC = 0.732; 95% confidence interval (CI), 0.606–0.858; P = 0.002, and AUC = 0.861; 95% CI, 0.766–0.957; P < 0.001, respectively) [115]. The results further emphasized the prognostic value of microbiota-derived metabolites as valuable indicators for predicting coronary artery problems.

Therefore, remodeling of gut microbiota using therapeutic agents can regulate TMAO synthesis and minimize cardiovascular complications. For instance, Xie et al. showed that the natural product, berberine, attenuated the serum levels of choline diet-induced TMAO and subsequent platelet accumulation, as well as thrombotic events by targeting ERK1/2 and JNK signaling pathways and enhancing the Lactobacillus community [116]. Noteworthy, the gut microbiota is also considered a contributing factor for the progression of arterial thrombosis in a TLR-dependent manner, particularly TLR-2 [117], which is associated with the microbial-associated molecular patterns, and influences the synthesis of adhesion molecules in the vasculature [118]. It has also been well-established that bacterial LPS and higher serum levels of TMAO, following consumption of a choline-rich diet, play a key role in arterial thrombosis modulation, which is transmissible during FMT [119]. Mechanistically, following the augmented serotonin biosynthesis in the colon, agonist-induced secretion reaction of platelets and related hemostatic function increase. In this pattern, platelets present reduced type I collagen-induced platelet activation and increased ADP-induced platelet aggregation. In addition, reduced P-selectin surface expression, collagen-triggered granulophysin release, and exposure of activated integrin αIIbβ3 on the platelet surface have been detected [120]. It has also been pointed out that the Cut-c gene in gut commensals has a crucial role in the progression of arterial thrombosis [119].

TMA lyase inhibitors

TMA lyase inhibitors, as small molecules, represent a promising therapeutic strategy for addressing gut dysbiosis and potentially restoring PC function. By selectively modulating microbial metabolism and composition, these agents can improve host metabolic health and re-establish the gut ecosystem, suggesting a novel approach to treating diseases linked to microbial metabolites and intestinal barrier dysfunction.

Compounds such as iodomethylcholine (IMC, 0.06%, wt/wt), in mice with low and high fat diets, selectively blocking the microbial conversion of choline to TMA, reduce TMA and consequently TMAO levels by about 10,000 times more effectively than 3, 3-Dimethyl-1-butanol (DMB). Following some metabolic benefits, an effective transition toward enhancing the beneficial bacteria commensals was also observed 48 h after the IMC administration (31 mg/kg) [121].

Since TMAO is considered a risk factor for platelet activation and thrombus formation, notably, adding either FMC (0.006% wt/wt, n = 10) or IMC (0.006% wt/wt, n = 11) on a choline supplemented diet (1% wt/wt), showed a marked reduction in thromboembolic events without increasing bleeding risk, in vivo, beyond a desirable shift within microbiota composition [FMC (100 mg/kg; n = 11) or IMC (100 mg/kg; n = 11)] [122]. Although IMC and FMC appear well-tolerated in animal models with no significant side effects or toxicity [122], clinical safety data are not yet available, and further studies are highly recommended to characterize potential side effects in this subset of patients.

FMO3 enzyme inhibitors

Flavin-containing monooxygenase 3 (FMO3) is a key enzyme for converting TMA to TMAO in the liver [123]. Based on pre-clinical data, FMO3 inhibitors, including quercetin (100–500 mg per day), methimazole (in vitro 200 µM [124], in vitro 50–100 mg/kg intraperitoneal [125]) represent a promising therapeutic approach to mitigate the harmful effects of elevated TMAO levels derived from gut microbial metabolism, more likely followed by reducing systemic and intestinal inflammation, improving metabolic parameters, and helping restore PC function.

3,3-Dimethyl-1-butanol (DMB)

In an overload-induced HF animal model (n = 9 in each group), orally 1% DMB by inhibiting p65 NF-κB and TGF-β1/Smad3 signaling pathways could reduce the TMAO serum levels and improve cardiac remodelling [126]. Albeit the TMAO inhibitors exert a promising effect in reducing cardiovascular and metabolic risks associated with high TMAO serum levels, the possible side effects are not yet well-characterized.

A link between CVD and PCs dysregulation

PC dysfunction and CVD may exacerbate each other through a feedback loop involving gut barrier disruption and systemic inflammation. As mentioned earlier, PCs dysfunction can disrupt the gut microbiota, which is associated with increased intestinal permeability and failure to control gut inflammation, allowing pathogen endotoxins to enter the blood circulation. The zonulin, a tight junction protein that regulates the intestinal permeability associated with bacterial translocation, may collaborate with PCs in gut barrier integrity. In this regard, Li et al. revealed that in patients with CAD, bacterial 16 S ribosomal RNA genes belonging to Enterobacteriaceae have been detected in atherosclerotic plaques due to bacterial DNAemia and increased intestinal permeability associated with the higher plasma levels of zonulin [127], which is more common in the elderly population [128]. Similarly, a positive correlation was also detected between the progression of chronic HF complications (e.g., physical disability) and plasma levels of zonulin [129]. It is also worth noting that the microbial signature in atherosclerotic plaques could be different in acute and chronic coronary syndromes [130]. Calling attention, among intestinal permeability plasma indicators, it has been found that zonulin exclusively correlates with gut permeability and cardiometabolic dysfunction [131].

According to the recent evidence, patients with IBD, a condition associated with PCs dysfunction, have an increased cardiovascular risk likely due to endothelial cell dysfunction [132, 133]. In turn, this phenomenon results in systemic inflammation, a key driver of endothelial dysfunction, plaque formation, and other cardiovascular conditions. In addition, PCs impairment indirectly increases the risk of metabolic syndrome (e.g., dyslipidemia and insulin resistance), one of the major risk factors of CVD progression. On the other hand, chronic HF and atherosclerosis can lead to neuro-hormonal changes concerning the sympathetic and renin-angiotensin-aldosterone systems, chronic systemic inflammation, oxidative stress, and simultaneous ischemic conditions, which in turn, influence the PC function following the alteration in gut barrier and microbiota composition [134].

Although little is known about the link between PCs dysfunction and CVD, some studies unveiled the prognostic value and predictive potential of PC-derived AMPs in the case of CVD progression. Meanwhile, it can be assumed that the dysregulation of PCs and subsequent excessive AMPs secretion into the blood circulation have the potential to participate in the progression of coronary artery syndromes.

In a retrospective clinical study, the baseline α-defensin, as a neutrophil peptide 1–3 (HNP1-3), was measured in patients with stable CAD. The results surprisingly showed that higher serum levels of α-defensin are strongly associated with mortality rate and recurrent percutaneous intervention [135]. Similar results were also observed in patients with dyslipidemia who are at higher risk of atherosclerosis [136]. Hence, it can be implied that α-defensin contributes to accelerates atherosclerosis progression likely by linking inflammation and lipid metabolism [137]. In detail, α-defensin can enhance the attachment and buildup of LDL and lipoprotein A on vascular matrix, preventing its breakdown and metabolism [138]. Additionally, α-defensin inhibit fibrinolysis mediated by tissue plasminogen activator (tPA), decreasing fibrin degradation and development of a pro-thrombotic state inside the plaques [139]. On the other hand, neutrophils releasing α-defensin induce more stress and programmed cell death in endothelial cells, further encouraging the recruitment of monocytes and the formation of foam cells, both essential for plaque expansion [140].

Besides, the amount of skin α-defensin can also be considered an independent predictor for CAD severity, highlighting the correlation between neutrophil over-activity and atherosclerosis progression (p = 0.016, OR 5.97, 95% CI 1.4–24.2) [137]. Noteworthy, it has been documented that serum levels of α-defensin could be another reliable biomarker for the prognosis of CHF and all-cause mortality in addition to NT-proBNP [141].

Critical appraisal of current evidence

As shown in Table 2, clinical studies examining AMPs and their potential role in CVD have been illustrated. In a study conducted by Maneerat et al., plasma levels of α-defensin (HNP 1–3) were significantly higher in CHD patients than in both healthy controls and those with hyperlipidemia [136]. This study assessed α-defensin gene expression as a predictor of CHD risk using DNA microarray, qRT-PCR, and ELISA. Their findings suggested a link between α-defensin levels and atherosclerosis progression. However, the study faced significant limitations: the small sample size (7–17 per group) diminished statistical power. CHD patients were considerably older (median 66 vs. 42 years, p = 0.000), with no age-matched comparison. Only male subjects were included. Age was not adjusted for in correlation analyses, raising concerns that elevated α-defensin levels in CHD patients might reflect age-related inflammatory processes rather than CHD-specific mechanisms (Table 2).

Table 2.

Effects of Paneth cell-derived anti-microbial peptides in CVD

Study	Year	Study design	Patients (sample size)	Sample	Type of AMP	Level	Disease	Outcome(s)
Shapira et al., [135]	2024	Retrospective observational study	174	Serum	α-defensin	10,859 pg/ml IQR [6,920 to 23,320] in dead, vs. 9,020 pg/ml IQR [5,540 to 16,180] in surviving patients (P = 0.15, 20% higher),	Stable CAD	Baseline plasma α-defensin is a novel biomarker in CV risk assessment among Patients with stable CAD.
Maneerat et al., [136]	2016	Cross-sectional study	7	PBMCs	α-defensin	Two-fold increase in RNA expression of α-defensin in CAD patients (r = 0.429, p = 0.023)	CAD	The α-defensin may predict the risk of CHD development in hyperlipidemia patients.
Heidi et al., [141]	2012	Prospective observational study	194	Plasma	α-defensin	599 mg/L in NYHA III-IV vs. 486 mg/L NYHA I-II All-cause mortality (HR 1.65, 95% CI 1.19–2.28, P = 0.002)	CHF	Both high α-defensin and NT-proBNP levels provide reliable prognostic information in heart failure.
Joseph et al., [142]	2008	Prospective observational study	389	Plasma	α-defensin	Patients with nephropathy 305 µg/L vs. 223 µg/L CVD-related morbidity and mortality to an HR of 2.8 (1.3–5.9) (median and 95% CI, P = 0.006). In patients without nephropathy	Type 1 diabetic patients with CVD risk	The α-defensin may serve as a risk marker for CVD-related morbidity and mortality in type 1 diabetes. HR of 2.8 (1.3–5.9) with 95% CI
Janket et al., [144, 177]	2004, 2006	Case-control study (Comparative study)	256	Salivary	Lysozyme	OR with 95% CIs for the association between salivary lysozyme and CHD increased from 1.00 to 1 3.62 (1.60 to 8.16, p < 0.0001)	CAD	Lysozyme could be a marker for the dual contribution of leukocytes to cardiopathogenesis, via infection and elevated AGEs deriving from an unhealthy diet.
Abdul-Salam et al., [143]	2010	Observational Cohort Study	197	Plasma	Lysozyme	A cutoff value of 1.5 µg/mL for Lysozyme, with 86% sensitivity and 93% specificity.	Atherosclerosis (Carotid atheroma)	Increased plasma lysozyme levels may be a prognostic biomarker of atherosclerosis, to distinguish in patients with 1 or more occluded coronary arteries, and response to therapy.

Abbreviations: AGE, Advanced Glycation End Products, CAD, Coronary Artery Disease; CHD, Coronary Heart Disease, CHF, Chronic Heart Failure; CI, Confidence Interval; CV, Cardiovascular; CVD, Cardiovascular Disease; HR, Hazard Ratio; NT-proBNP, N-terminal pro B-type natriuretic peptide; PBMCs, Peripheral Blood Mononuclear Cells

The study by Joseph et al. also demonstrated that higher levels of α-defensin independently predicted all-cause mortality, with a hazard ratio of 1.65 per one standard deviation increase (P = 0.002), even after adjusting for NT-proBNP and other risk factors. Their findings confirmed that α-defensin has greater predictive power for mortality than hsCRP, showing an especially strong association in patients with pre-existing nephropathy following multivariable adjustment [142]. The study highlights α-defensin as a novel prognostic biomarker for cardiovascular outcomes in distinct high-risk populations. This cohort study (n = 389), with 10-year follow-up, employed multivariable Cox regression analysis adjusted for factors including nephropathy, age, blood pressure, lipid levels, HbA1c, smoking status, hsCRP, and baseline CVD. However, the study had several limitations: some confounding factors such as nephropathy and glomerular filtration rate (GFR) should have been more thoroughly adjusted; also, other limitations referred to the wide confidence intervals, the use of an in-house radioimmunoassay (RIA) with questionable validity, limited statistical power in subgroups, and concerns regarding reproducibility [142] (Table 2).

A retrospective cohort study by Shapira et al. also identified α-defensin as a potential biomarker in stable CAD patients, useful for differentiating new lesions from target lesion revascularization (TLR) following percutaneous coronary intervention (PCI). The study suggested α-defensin as a therapeutic target for colchicine, which is known to inhibit neutrophil degranulation. However, the research had several limitations, including a small sample size, being conducted at a single center (which may introduce selection bias), wide confidence intervals, a single-blind design, and the exclusion of high-risk patients with left ventricular ejection fraction (LVEF) below 35% and recent stroke, restricting its applicability to typical CAD populations (real-world CAD). Additionally, the study did not perform multivariable adjustments for important outcomes like mortality [135]. Patients with baseline α-defensin levels (> 9.200 pg/ml) experienced significantly higher mortality rates (12.6% compared to 6.9%) and increased rates of recurrent PCI (17.2% versus 10.3%). Additionally, the incidence of PCI for new (de novo) lesions was substantially greater in the high α-defensin group (15% versus 2.3%, p = 0.005). The combined outcome of death and recurrent PCI for de novo lesions was also significantly associated with α-defensin levels (> 9.200 pg/ml) (log-rank p = 0.02) [135].

The study conducted by Heidi et al. demonstrated that combining elevated levels of α-defensin and NT-proBNP provided more powerful prognostic insight in patients with CHF than using either biomarker individually. This combination effectively identified a high-risk group (NYHA class III-IV) with a mortality rate of 47%, compared to just 6.7% in the low-risk group (NYHA class I-II patients) [141] (Table 2). In this prospective observational study, with complete follow-up and endpoint adjudication (using national registries and blinded physicians) minimize attrition and ascertainment bias. Also, multivariable analyses were performed with adjustment for key confounders, including age, sex, NYHA class, renal function, and NT-proBNP. It is worth noting that α-defensins independently predicted mortality after adjusting for NT-proBNP (hazard ratio, HR 1.65, 95% CI 1.19–2.28, P = 0.002), in which the patients with high α-defensins and high NT-proBNP level had more than three-fold higher mortality risk (P < 0.001). As a study limitation, subgroup analyses (e.g., by NYHA class) lack power, a single-center cohort with predominantly male (72%), older (mean 69 years), systolic HF (LVEF ≤ 45%) patients. Noteworthy, healthy controls had higher baseline creatinine clearance (CrCl 82 vs. 100 µmol/L in CHF), potentially confounding biomarker comparisons. In addition, the exclusion of patients with HFpEF limits applicability to broader CHF phenotypes. In-house radioimmunoassay for α-defensins lacks comparison to commercial kits or multi-lab validation. As a selection bias, patients referred to a specialty clinic may have more advanced HF than community-based cohorts [141].

Previously, in a cohort study, it was found that elevated plasma lysozyme in patients with 3-vessel CAD may serve as a useful biomarker for atherosclerotic disease compared to those without CAD (with a cutoff value of 1.5 µg/mL, 86% sensitivity and 93% specificity) [143].

The evidence presented in the study by Abdul-Salam et al. could be strengthened by the use of multiple independent cohorts and adjustment for confounders. The reproducibility of the findings across cohorts and the specificity of the biomarker are remarkable strengths. However, potential biases related to patient selection and limited geographic diversity should be considered when interpreting the results [143].

Another study conducted by Janket et al. investigated the relationship between salivary lysozyme levels and the progression of CHD, using an age- and sex-matched control group with a sufficiently large sample size (n = 500). Elevated salivary lysozyme levels, divided into higher quartiles, were linked to progressively greater odds of developing CHD. Lysozyme appears to represent two pathways linking oral health to CHD: oral infections and diet-related advanced glycation end products (AGEs). Notably, lysozyme demonstrated a stronger association with CHD than the asymptotic dental score (ADS), which measures oral infection burden [144]. Although, the analysis included multivariable adjustments for factors such as age, sex, smoking status, BMI, diabetes, lipid profiles, hypertension, C-reactive protein (CRP) levels, and oral health assessed by the ADS; nevertheless, the study had some limitations related to the specifics of sample collection, the lack of detailed dietary diet, and the absence of pathogen-specific analyses [144] (Table 2).

PCs and angiogenesis

To our knowledge, the gut microbiota plays a key role in the development of a microvascular network in the intestinal villus [145]. According to available evidence, beyond the antimicrobial effect of substrates secreted by PCs, the new blood vessel generation (neovascularization) potential in the GIT and subsequent increased blood pressure in the portal vein, in response to the microbial signal, has drawn significant attention. Portal vein hypertension, determined by increased portal pressure gradient more than 6 mmHg, is defined as a pathologic condition in the portal venous system, mainly caused by cirrhosis, and finally leads to liver transplantation and death [146]. However, a pre-clinical study also showed the high potential of PCs in the regulation of enteric pro-angiogenic molecules under the control of gut microbiota signals [147]. Furthermore, a significant reduction in portal hypertension was observed following tamoxifen-induced complete depletion of PCs. Additionally, the findings of this study interestingly revealed the potential impact of PCs on the density of blood vessels located in the mesentery and intestinal wall [147]. Also, the expression of genes involved in intestinal and mesenteric angiogenesis, such as VEGF-A, VEGF-C, VEGF-D, Tie-1, Tie-2, and ANGPT-2 genes, as well as vascular proliferation, was markedly decreased in the PCs depletion model (Fig. 4) [147]. Interestingly, Hassan et al. showed that PCs also have the potential to develop lymphatic vessels and regulate specific gene expression involved in intestinal and mesenteric lymphangiogenesis, i.e., PROX-1, FOXC2, VEGFR2, and VEGFR3 in an experimental portal hypertension induced by partial portal vein ligation [148]. Correspondingly, PCs can also be considered key regulators of the intestinal microvasculature, which is stimulated by microbiota signals; despite the PCs’ potential in the intestinal microvasculature regulation, the correlation with the extra-intestinal vascular system, with special focus on the cardiovascular system, remains to be determined.

Fig. 4.

A correlation between Paneth cells regulation and cardiovascular health. Following the gut dysbiosis, serum levels of toxic metabolite, TMAO, which plays a crucial role in atherosclerotic plaque formation, increase. Positive impacts likely refer to the pro-angiogenic factors secreted by PCs contributing to the development and maintenance of the intricate vascular network and lymphoid system (right side). However, negative impacts have also been reported following PCs’ dysregulation and excessive release of AMPs in the serum of patients with CVD, such as heart failure (left side). Abbreviations: ANGPT2, angiopoietin 2; FMO3, flavin-containing dimethylaniline monooxygenases 3; FOXC2, Forkhead box protein C2; PROX-1, Prospero homeobox protein 1; TMA, Trimethylamine; TMAO, Trimethylamine N-oxide; VEGF, vascular endothelial growth factor

Protein-protein interaction (PPI) between PCs, microbiota, and the cardiovascular system

The Fig. 5 presents an intricate network of the downstream hub proteins, revealing how PC dysfunction is linked to CVD via several signaling pathways. In detail, there is a dynamic and interconnected network involving inflammatory, angiogenic, fibrotic, and oxidative stress pathways, with multiple overlaps and feedback between these pathways.

Fig. 5.

Protein–protein interaction network obtained using STRING software for prediction of Hub genes and signaling pathways in cardiovascular pathology and Paneth cells dysfunction. The images show the confidence view (http://string-db.org/). Stronger associations are represented by thicker lines 10

Notably, the crosstalk between core microbiota and PCs may activate other downstream pathways and protein networks that were predicted using the STRING Database v. 10.5 (https://www.string-db.org).

STRING analysis

Using the STRING database, the list of input genes or proteins was searched to identify both direct (physical) and indirect (functional) connections. Each interaction was assigned a confidence score indicating the probability of a true association based on the combined evidence [149]. The resulting network of interactions was then visualized and analyzed for functional enrichment using Gene Ontology and KEGG pathway annotations, with statistical significance adjusted for multiple comparisons using the Benjamini–Hochberg method [149]. According to our analysis, the main hub signaling, consisting of known and predicted interactions, that may be linked to the pathophysiology of cardiovascular events in gut-heart axis is listed as follows:

Inflammatory signaling pathways

NLR family pyrin domain containing 3 (NLRP3) is the central pathway to inflammasome activation in CAD and MI [150]. In this regard, multiple pre-clinical studies have been designed to establish a correlation between NLRP3 modulation and IRI in cardiac microvascular endothelial cells and myocardium [151–153]. On the other hand, according to the previous publication, early activation of the NLRP3-related pathway has a potential protective effect on intestinal epithelial cells by limiting pathogen colonization in inflammatory diseases like IBD [154].

The other hub gene that must be mentioned is IL-17-related signaling. It is a key pathway in CAD and MI pathogenesis, alongside TNF and C-type lectin receptor pathways. An experimental study by Han et al. also elucidated the crucial role of intestinal TLR-9 deficiency (TLR9^−/−) in mice with hyperplastic PCs, which in turn, leads to IRI due to impaired degranulation process (large size granules). Therefore, IRI can be exacerbated in mice with intestinal TLR-9 deficiency because of a robust inflammatory response mediated by increased levels of cytokines (e.g., crypt IL-17 A) and macrophage infiltration, leading to cell death. Together, intestinal TLR-9, via PCs and IL-17 A regulation, can be encountered as a therapeutic option to protect against IRI and vital organs’ failure.

Several inflammatory mediators have also been identified based on this analysis. In this regard, stimulator of interferon-1 (STING1) and RIPK2 are importantly labeled as triggers of inflammatory responses, specifically following MI and HF [155]. In detail, the structural and functional cardiac remodeling [156] has been investigated following treatment with small-molecule STING inhibitors in an experimental MI model in mice [157, 158]. The results interestingly showed that after three weeks of treatment with pharmacologic blocking of the cGAS-STING pathway, the left ventricle ejection fraction, cardiac hypertrophy, and infarct size were improved. Given the recent correlation found between PCs and ISC dysfunction and the cGAS-STING pathway in transgenic mice [159], it could be considered an imperative signaling pathway involved in the cross-link between PC dysfunction and CVD.

To our best, MyD88/TLR axis, especially TLR-4, are responsible for various inflammatory process during myocarditis and cardiomyopathy [160–163]. Noteworthy, it has also been established that enteric bacteria are directly sensed by CD74 + PCs, which are crucial mediators in relation to inflammatory signaling during IBD and MyD88−/− deficiency in vivo. Hence, it could be inferred a possible link between PC dysfunction and cardiovascular outcomes through MyD88/TLR-mediated inflammatory signaling [18, 164].

As another proposed pathway, previous preclinical studies highlight that autophagy regulation affected by stimuli such as various stress conditions and pharmacological modulations is vital for cardiovascular pathophysiology conditions [165–169]. Importantly, it has been shown that PCs employ the autophagy pathway (such as ATG16L1) to support antimicrobial defense and maintain intestinal balance by regulating AMPs, particularly lysozyme secretion, in conditions like IBD and Crohn’s disease in vivo [170, 171].

As mentioned earlier, PCs are a source of lysozyme production, an enzyme that breaks down bacterial cell walls, primarily playing a key role in regulating gut microbiota and intestinal immune responses. Although the direct role of PC-derived lysozyme in CVD is not explicitly elucidated, lysozyme deficiency or dysregulation has the potential to affect the intestinal immune signaling pathways, such as NOD-like receptor (NLR) signaling, inflammation, and gut microbiota composition can be linked to systemic effects relevant to CVD. Since long-term inflammation and gut microbiota dysbiosis contribute to the development of CVD, PC-derived lysozyme may indirectly play a role in modulating cardiovascular risk through modulation of gut immunity and inflammation.

Fibrosis and remodeling

Based on hub-signaling analysis, DLG5 and RhoA are found as key mediators of cardiac fibrosis and post-MI remodeling, more likely mediated by PC dysfunction as a potential upstream trigger. Gut-microbiome-associated proteins like LYZ further link intestinal inflammation/fibrosis to cardiovascular outcomes. Together, RhoA (a GTPase regulating epithelial cytoskeleton/junctions), DLG5 (a member of the membrane-associated guanylate kinase family), and LYZ (a key AMP) may work together to impair PC-mediated epithelial integrity and immune functions, thereby worsening CVD.

Notably, RhoA has a dual and intricate role in cardiac fibrosis, functioning as both a protective factor and a contributor to pathology in mice with cardiomyocyte-specific genetic modifications of RhoA. In better words, the activation of RhoA within cardiomyocytes is crucial for sustaining cardiac contractility and enabling compensatory hypertrophy in response to stressful conditions such as pressure overload, ischemia, and aging. While RhoA signaling also facilitates cardiac fibrosis by triggering downstream molecules like ROCK, MRTF-A, and serum response factor (SRF), which promote the expression of profibrotic genes and the activation of myofibroblasts [172, 173].

Angiogenesis and endothelial function

The right side of the network is addressed to angiogenesis and endothelial function. In this regard, various vascular growth factors, including VEGFD, VEGFB, and VEGFC, form a cluster of angiogenic regulators. In addition, FLT4 (VEGFR3) and Tie1 appear to be key receptors involved in vascular development and maintenance. According to Hassan et al., findings in the transgenic mouse model of PC dysfunction [147, 148], these angiogenic factors make a strong connection between PC dysfunction and vascular disorders in CVD, especially CAD.

Conclusion and future direction

Considering the gut-heart axis, the PC dysfunction emerges as a potential contributor to CVD pathogenesis. Although the direct PC-CVD relationship remains underexplored, accumulating evidence points to a significant indirect effect mediated by excessive AMP secretion and subsequent systemic inflammation. A better understanding of these gut-heart axis interactions, particularly the role of PCs, could open new avenues for novel therapeutic strategies in cardiovascular risk management. To further clarify the underlying mechanisms, we specifically recommend designing: (1) longitudinal studies to track PC-derived AMP profiles and their correlation with CVD progression; and (2) Clinical trials evaluating the therapeutic potential of SCFA supplementation in mitigating PC-driven inflammation and CVD risk. In addition, further investigation regarding the possible impact of aging and environmental modulators on this pathway is also warranted.

Author contributions

AR prepared the first draft; GP, FA, and AB conceived and designed the study; SH and RP revised the manuscript; AR depicted the figures; AB performed the bioinformatics analysis. All authors read and approved the final version of the manuscript.

Funding

This study did not receive any financial support.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

N/A.

Consent for publication

N/A.

Competing interests

The authors declare no competing interests.