Identification of novel early biomarkers of cardiovascular disease risk and their relationships with bioactive dietary compoundsmetabolomic and gut metagenomic approaches

Resumen

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, reaching 18.6 million deaths in 2019. Hypertension and hypercholesterolemia are the primary causal and modifiable metabolic risk factors that directly contribute to CVD risk. These risk factors have traditionally led to the development of risk assessment models. However, they are restricted by their limited predictive value because of their delayed response once the disease progresses and pharmacological treatment is required. Therefore, it is essential to identify novel risk biomarkers that assist in recognizing the preliminary stages of the CVD pathophysiological processes. The integration of ‘multiomics’ approaches, such as metabolomics, lipidomics, and gut metagenomics, allows for the combination of different molecular biomarkers, including gut microbiota- and metabolite-based biomarkers, that may help to improve the accuracy in risk detection. Moreover, knowledge of early biomarker interactions with dietary compounds would benefit the implementation of preventive nutritional strategies to avoid the continuous advancement of the main causal and modifiable CVD risk factors. OBJECTIVE The present doctoral thesis was aimed to identify novel early gut microbiota- and metabolite-based CVD risk biomarkers suitable for the preliminary stages of human hypertension, hypercholesterolemia and other main causal and modifiable CVD risk factors through the integration of multiomics approaches and to assess their relationships with bioactive dietary compounds to speculate about the involved pathways and their possible effects in CVD development. METHODS AND MAIN RESULTS Two cross-sectional studies involving healthy, grade 1 hypertensive (HT) (the Cardiogut study), and moderate to high hypercholesterolemic (the Bioclaims study) individuals were performed. In the Cardiogut study (Article 1), faecal microbiota composition and bacterial functions were determined by 16S rRNA gene sequencing and metagenomic analysis in 29 nontreated HT and 32 normotensive (NT) individuals (systolic blood pressure (SBP) ≥ 140-159 mmHg and < 120 mmHg, respectively). Moreover, microbial metabolites, including faecal and plasma short-chain fatty acids (SCFAs) and plasma trimethylamine-N-oxide (TMAO), were detected by targeted metabolomics. The results showed that a set of bacterial taxa, mainly Bacteroides spp., Intestimonas and genera of Lachnospiraceae, were enriched in HT individuals, whereas Faecalibacterium spp., Roseburia spp., and other genera of Ruminococcaceae and Christensenellaceae families were depleted. These findings suggested a strong association with hypertension. Additionally, in HT individuals, lower plasma SCFA levels and increased faecal SCFA levels suggested less efficient intestinal absorption, which is probably due to imbalanced host-microbiome crosstalk. Furthermore, in Article 2, the relationships between dietary phenolic compounds (PCs) and targeted bacterial taxa in HT and NT individuals were examined. A multiple-way interaction was found for coffee PCs with SBP, diastolic blood pressure (DBP), faecal SCFAs and HT-enriched Bacteroides spp. However, inverse associations were noted for these PCs with NT-enriched bacterial taxa, indicating a negative impact of coffee PCs on the gut microbiota preceding blood pressure (BP) alteration. In NT individuals, olive fruit PCs were positively associated with SCFA-producing bacteria, suggesting a protective role against the onset of hypertension. In the Bioclaims study (Article 3), plasma TMAO and serum lysophosphatidylcholines (lyso-PC) and ysophosphatidylethanolamines (lyso-PE) were identified through targeted metabolomics and lipidomics approaches in 70 individuals with low LDL cholesterol (L-LDL-c) and 48 with moderate to high (MH-LDL-c) LDL cholesterol (LDL cholesterol ≤ 115 mg/dL and 116-190 mg/dL, respectively). Predictive models were applied to discern accurate biomarkers for the onset of hypercholesterolemia. The results revealed that lyso-PC 15:0 and lyso-PE 18:2 were suitable susceptibility/risk biomarkers of hypercholesterolemia progression. In addition, in MH-LDL-c subjects, a positive correlation was found between lyso-PE 18:2, liver GGT and dietary omega (n)-polyunsaturated fatty acids (PUFAs). Then, the human lipidomic results were validated in an in vivo study involving 8 high-fat diet (HFD)- and 8 low-fat diet (LFD)-fed hamsters. In the HFD-fed group, the intrahepatic accumulation of polyunsaturated lyso-PLs, particularly lyso-PE 18:2, revealed a possible hepatic adaptive mechanism to counteract hypercholesterolemia progression in humans. Finally, a systematic review and meta-analysis of randomized clinical trials was performed to clarify the effects of dietary fatty acids (FAs) on circulating bioactive lipidome and the enzymatic precursor lipoprotein-associated phospholipase A2 (Lp-PLA2) mass in healthy subjects, with CVD and with CVD risk factors (Article 4). The PRISMA 2020 guidelines and PICOS criteria were followed for study selection. A total of 27 randomized clinical trials were included in the systematic review, and 10 controlled trials were selected for meta-analysis. As a result, from the meta-analysis, marine omega-3 (n-3) PUFAs, provided as supplements, increased plasma pro-inflammatory lyso-PC(16:0 and 18:0) in obese subjects, while decreased plasma Lp-PLA2 precursor mass in healthy, dyslipidemic and coronary artery disease subjects. Therefore, daily n-3 PUFA supplementation, provided as EPA+DHA, consumed from 1 to 6 months, exhibit positive effects on the plasma Lp-PLA2 mass in healthy subjects, with CVD and with CVD risk factors, suggesting an anti-inflammatory effect. However, an impaired saturated lyso-PC response to n-3 is manifested in obese subjects. CONCLUSIONS The identification of particular gut bacterial signatures, such as B. coprocola, B. plebeius, Intestimonas and genera of Lachnospiraceae, and higher faecal levels of acetate, propionate, butyrate and valerate provide novel biomarkers of the gut microbiota ecosystem that could be useful to discriminate HT individuals from NT individuals. In addition, knowledge of these biomarker relationships with BP, coffee and olive fruit PCs highlights the complex dietary pathways involved in the pathogenesis or prevention of hypertension. Additionally, increased serum lyso-PC 15:0 or decreased lyso-PE 18:2, together with intrahepatic accumulation of lyso-PE 18:2, could represent promising susceptibility/risk biomarkers of hypercholesterolemia progression. Dietary FAs, particularly n-3 PUFAs, provided as supplements or oily fish ED, modulate circulating lyso-PC moieties and decrease plasma Lp-PLA2 mass. These findings favour the management of the main causal and modifiable CVD risk factors. Overall, our results offer novel promising health/disease biomarkers that may help more accurately detect the main causal and modifiable CVD risk factors in their preliminary stages. Nevertheless, further prospective studies are required for successful predictions of CVD risk.