Causal impact of gut microbiota and associated metabolites on pulmonary arterial hypertension: a bidirectional Mendelian randomization study

Pulmonary arterial hypertension (PAH) is characterised by the chronic elevation of pulmonary arterial pressure and remodelling of the pulmonary arteries, which can ultimately lead to heart failure and mortality [1]. Several factors contributing to the development of PAH have been identified, including genetic susceptibility, underlying cardiovascular diseases, toxic exposure, and inflammation [2]. Recent studies have revealed that gut microbiota and its associated metabolites play vital roles in the pathogenesis of PAH. The gut microbiota, together with its metabolites, participates in various metabolic processes, such as cholesterol accumulation, impaired glucose tolerance, and elevated inflammatory responses, all of which promote the progression of PAH [3, 4]. Previous studies reported that patients with PAH exhibit a distinct gut microbiota profile characterised by a reduction in bacteria that produce short-chain fatty acids (SCFAs), including Coprococcus, Lachnospiraceae, Eubacterium, and Clostridia [5]. Moreover, gut microbiota-derived metabolites, namely trimethylamine N-oxide (TMAO) and its precursors choline, betaine, and carnitine, are elevated in patients with PAH and are associated with an unfavourable prognosis [3‐5].

Despite the growing body of research examining the association between gut microbiota, its derived metabolites, and PAH, the majority of these studies have been cross-sectional or observational in nature and were unable to establish a causal relationship between PAH and gut microbiota. Additionally, the observed association between gut microbiota and PAH in cross-sectional or observational studies might be affected by reverse causation bias or confounders, such as dietary patterns. Moreover, the small sample sizes of these studies limit the robustness and generalisability of the conclusions. Therefore, the causality between gut microbiota, associated metabolites, and PAH warrants further investigation.

Mendelian randomisation, which introduces genetic variants as instrumental variables, is widely used to identify the causal effects of exposure on particular outcomes. Compared to traditional observational or cross-sectional studies, Mendelian randomisation is less prone to confounding because genetic alleles are randomly allocated during gametogenesis. Furthermore, Mendelian randomisation can mitigate the potential for reverse causation bias, as genotypes are determined prior to disease onset [6]. Therefore, in this study, we aimed to use a two-sample Mendelian randomisation method to investigate the causal relationship between gut microbiota, associated metabolites, and PAH. We also explored whether dietary patterns mediated this causal relationship.

Figure 2 demonstrates the study design. The features of the selected SNPs in gut microbiota and its associated metabolites are shown in Supplementary Table S1. A total of 5394 SNPs were selected as instrumental variants for 119 bacterial genera; 311 SNPs for SCFAs, including acetate, indolepropionate, and 3-hydroxybutyrate; and 490 SNPs for TMAO, along with its precursors (Supplementary Table S2).

The current study revealed the intricate interactions between gut microbiota and PAH, contributing to the burgeoning evidence of the influence of the microbiome on cardiovascular pathologies. Our study underscores the potential protective role of specific bacterial genera against PAH, including Coprobacter, Erysipelotrichaceae (UCG003), Lachnospiraceae (UCG008), and Ruminococcaceae (UCG005). In contrast, genera such as Alistipes and Victivallis emerged as potential risk factors, suggesting their contributory roles in PAH pathogenesis. Notably, our findings did not indicate significant mediation by dietary patterns in the microbiota–PAH axis, nor did we observe a causal association between gut microbiota-dependent metabolites and PAH.

The pivotal function of the gut microbiota in maintaining host homeostasis is well documented, with roles in nutrient metabolism, vitamin and hormone synthesis, immune system shaping, and resistance to pathogen colonisation [17]. Dysbiosis has been implicated in various conditions, including diabetes, coronary artery disease, heart failure, and hypertension [18]. Moreover, perturbations in the gut microbial composition have been documented in PH, which is characterised by reduced levels of beneficial bacteria [5], suggesting a possible association with PAH.

Studies have revealed that Lachnospiraceae and Ruminococcaceae, which are predominant in healthy individuals, are integral to gut integrity and metabolic processes [19]. Their associations with cardiovascular health indicators, such as arterial stiffness [20] and heart rate variability [21] further suggest that these bacteria contribute to cardiovascular homeostasis. A decline in the abundance of these genera in coronary artery disease [22], chronic heart failure [23], and HIV [24] highlights their beneficial roles.

Concurrently, an increase in Alistipes is associated with a higher risk of PAH. Alistipes, a potential pathogen, is associated with colorectal cancer [25], depression [25], diabetic nephropathy [26], and inflammation [25]. Its influence on hypertension pathogenesis is suggested by its positive correlation with systolic blood pressure in hypertensive individuals [27]. Similarly, the involvement of Victivallis, a strictly anaerobic bacterium, in the risk of PAH points to metabolic interplay, although the literature on its role is scant, warranting further investigation.

This study explored the controversial relationships between TMAO, its precursors, and PAH. Although some studies have indicated a correlation between elevated TMAO levels and adverse cardiovascular outcomes [28] [29], our MR analysis did not confirm a causal association. This is consistent with other MR studies that have questioned the causality of TMAO in cardiometabolic diseases [30, 31]. The inconclusive nature of the role of TMAO in PAH, coupled with the mixed prognostic implications [32, 33], indicates that observational associations may be confounded, highlighting the need for more detailed investigations.

However, the role of SCFAs in the development of PAH remains unclear. Despite their well-recognised systemic effects [34, 35], our findings do not support a direct causal relationship with PAH, pointing to a gap that future research should address.

The methodological strength of this study lies in the systematic evaluation of causal relationships using MR analysis, which is supported by extensive summary-level microbiota data. This approach mitigated reverse causation and confounding biases. Additionally, we investigated potential dietary confounders and validated our findings using multiple MR methods to ensure robustness.

However, this study had some limitations. Firstly, we selected SNPs for metabolites and bacteria at a suggestive locus-wide significance threshold of P < 5 × 10⁻⁵ [36], and our results might have been influenced by weak instrument bias. We performed a sensitivity analysis at the study-wide significance level of P < 5 × 10⁻⁸ and found that no SNP remained for metabolites and bacteria. Secondly, in the analysis of microbiota influences on PAH, MR only examines the impact of genetic factors that predispose individuals to specific abundances of particular microbial genera and how these genetic components influence the development of PAH. However, the impact of the environment and acquired risk factors on the causal association between bacteria and PAH could not be evaluated, which might contribute to the development of PAH. Thirdly, PAH may exhibit an association with specific microbial ‘types’ rather than taxonomic genera, and alpha diversity may also connect with PAH [37, 38]. Unfortunately, owing to the limitations of the MR method employed in our study, we were unable to evaluate the association between permatypes or enterotypes and the alpha diversity of microbiomes with PAH. We aim to investigate these issues in future studies. Fourthly, different ethnicities with PAH may have hereditary discrepancies. The current Mendelian randomisation analysis is primarily based on the European population, which limits the generalisability of the findings to other ethnic populations. However, limiting the European population may reduce the bias introduced by population stratification. Fifthly, this study explored the association between PAH and gut microbiota at the genus level. However, different species in the same genus may have discrepancies in their effects on metabolism and health, varying in the host and exposed environment. Some species may be protective, whereas others may exert detrimental effects on health. Studies at the species level can shed light on the precise causal associations between bacteria and diseases, along with the underlying pathological mechanisms. However, in the MiBioGen database, the lowest available level of bacteria was the genus level due to the limitations of 16S sequencing. Whole-genome shotgun metagenomics can address this limitation and provide insights into species-level resolution, which should be considered in future studies.

Genetically predicted gut microbiota has a suggestive causal effect on PAH, and the underlying mechanism may be attributed to alternative mechanisms rather than the production of SCFAs and TMAO.