Analysis of urinary exosomal metabolites identifies cardiovascular risk signatures with added value to urine analysis

Background: Subclinical atherosclerosis may result in fatal cardiovascular (CV) events, but the underlying mechanisms and molecular players leading to disease are not entirely understood. Thus, novel approaches capable of identifying the factors involved in pathological progression and providing a better understanding of the subjacent mechanisms are needed. Extracellular vesicles (EVs) have been shown to have numerous biological functions, and their metabolome has recently generated interest as a source of novel biomarkers. The metabolic content of the exosomes has been so far unexplored in cardiovascular disease (CVD), and here, we developed an analytical strategy aimed at probing urinary exosomal metabolite content and its association to CV risk. Results: Direct analysis of the exosomes without metabolite extraction was evaluated by high-resolution magic angle spinning (1 H HR-MAS). Other two methodologies for the analysis of exosomal metabolites by 1 H NMR were set up, based on methanol or organic solvents sequential extraction. The three methods were compared in terms of the number of detected signals and signal to noise ratio (S/N). The methanol method was applied to identify altered metabolites in the urinary exosomes of subjects with programmed coronary artery by-pass grafting (CABG) versus a control group. Target mass spectrometry (MS) was also performed for differential analysis. The clinical performance of exosomal metabolites of interest in CVD was investigated, and the added value of the exosomes compared to urine analysis was evaluated. Based on S/N ratio, simplicity, reproducibility, and quality of the spectrum, the methanol method was chosen for the study in CVD. A cardiometabolic signature composed by 4-aminohippuric acid, N-1-methylnicotinamide, and citric acid was identified in urinary exosomes. Directly in urine, 4-aminohippuric acid and citric acid do not show variation between groups and changes in N-1-methylnicotinamide are less pronounced, proving the added value of exosomes. Conclusions: We set up a novel methodology to analyze metabolic alterations in urinary exosomes and identified a cardiometabolic signature in these microvesicles. This study constitutes the first evidence of a role for the exosomal metabolism in CVD and demonstrates the possibility to evaluate the urinary exosomal metabolic content by NMR and MS

Analysis of Vascular Smooth Muscle Cells from Thoracic Aortic Aneurysms Reveals DNA Damage and Cell Cycle Arrest as Hallmarks in Bicuspid Aortic Valve Patients.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.This study was supported by the Instituto de Salud Carlos III with cofunding from the ERDF (PI20/01103, CP22/00100, FI21/00128, IF08/3667-1, PI21/00158, PRB3[IPT17/0019- ISCIII-SGEFI/ERDF], RD16/0009, RD21/0005/0001), the Spanish Ministry of Science, Innovation and Universities (PGC2018-097019-B-I00 and PID2021-122348NB-I00), Getinge Group Spain SL, CAM (PEJ-2020-AI/BMD-17899, PEJD-2019-PRE/BMD-16992, 2018-T2/BMD-11561, PIPF2022/SAL-GL-25760), Fundacioń SENEFRO/SEN, Fundacioń Mutua Madrileña and Fundacioń Conchita Rabago ́ de Jimenez ́ Dí az, and “la Caixa” Banking Foundation (project codes HR17-00247 and HR22-00253). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacioń (MCIN), and the Pro CNIC Foundation) and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/ AEI/10.13039/501100011033).S

Metabolic Alterations Identified in Urine, Plasma and Aortic Smooth Muscle Cells Reflect Cardiovascular Risk in Patients with Programmed Coronary Artery Bypass Grafting

Atherosclerosis is the predominant pathology associated to premature deaths due to cardiovascular disease. However, early intervention based on a personalized diagnosis of cardiovascular risk is very limited. We have previously identified metabolic alterations during atherosclerosis development in a rabbit model and in subjects suffering from an acute coronary syndrome. Here we aim to identify specific metabolic signatures which may set the basis for novel tools aiding cardiovascular risk diagnosis in clinical practice. In a cohort of subjects with programmed coronary artery bypass grafting (CABG), we have performed liquid chromatography and targeted mass spectrometry analysis in urine and plasma. The role of vascular smooth muscle cells from human aorta (HA-VSMCs) was also investigated by analyzing the intra and extracellular metabolites in response to a pro-atherosclerotic stimulus. Statistically significant variation was considered if p value < 0.05 (Mann-Whitney test). Urinary trimethylamine N-oxide (TMAO), arabitol and spermidine showed higher levels in the CVrisk group compared with a control group; while glutamine and pantothenate showed lower levels. The same trend was found for plasma TMAO and glutamine. Plasma choline, acetylcholine and valine were also decreased in CVrisk group, while pyruvate was found increased. In the secretome of HA-VSMCs, TMAO, pantothenate, glycerophosphocholine, glutathion, spermidine and acetylcholine increased after pro-atherosclerotic stimulus, while secreted glutamine decreased. At intracellular level, TMAO, pantothenate and glycerophosphocholine increased with stimulation. Observed metabolic deregulations pointed to an inflammatory response together with a deregulation of oxidative stress counteraction

Unusual Heart Involvement of Wegener’s Granulomatosis and Literature Review

A female patient, 60 years of age, was presented to our hospital with chest pain and monomorphic ventricular tachycardia (VT). She was transferred to the Coronary Care Unit and amiodarone perfusion restored basal rhythm in atrial fibrillation. She has not sign of heart failure. A transtoracic echocardiogram (TTE) was performed and an one mitral mass was found at atrioventricular junction with displacement of the posterior mitral leaflet A transesophageal echocardiogram (TEE) demonstrated a mass at atrioventricular junction level with severe mitral regurgitation. Cardiac Magnetic Resonance (CMR) confirmed the mass and anterolateral papillary muscle was thickening and hypertrophied with hyperenhancement consistent with fibrosis. Moreover, T2-weighted imaging demonstrated hyperintense mass with respect to the surrounding myocardium in relation of inflammatory mass. She had saddle nose by destruction of the septum, bilateral hearing loss, sinusitis and scleritis and renal involvement as well. This patient was diagnosed of Wegener's Granulomatosis (WG) and she was treated with methylprednisolone during 3 days, continued with prednisolone and cyclophosphamide. An 8 days later echocardiogram did not find the mass. However, the patient developed symptomatology of heart failure and in the context of severe mitral regurgitation, mitral valve replacement was decided in multi-disciplinary Cardiology-Cardiothoracic meeting.