A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

Abstract

Plasma‐derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs‐derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean‐matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D‐DIGE)‐based study, and the other one for a label free LC–MS/MS‐based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty‐two and 23 differentially regulated features are detected from 2D‐DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D‐western‐blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma‐derived‐EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…Another example of contaminants are lipoproteins, which are frequently found in EV preparations from plasma. Some research groups usually perform additional washing steps of the exosomal pellet with KBr to solubilise lipoproteins, and remove them from the plasma [ 95 ].…”

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

“…In this context, our group has just carried out a study comparing circulating EVs from obese patient and lean individuals. [166] We applied two proteomic approaches (2D-DIGE and label-free LC-MS/MS) in order to find potential biomarkers in obesity and identified proteins related to the complement and coagulation cascades altered in obese patients. Indeed, we provide novel information on a putative biomarker panel that will be explored to follow cardiovascular risk in obese patients.…”

Extracellular Vesicles in Cardiovascular Diseases: Alternative Biomarker Sources, Therapeutic Agents, and Drug Delivery Carriers

“…Interestingly, a study in patients with manifest arterial disease or CVD risk factors suggests that EV protein levels of cystatin C positively while CD14 negatively correlate with obesity and obesity-induced metabolic complications [81]. In line with this, 40 EV proteins, as demonstrated by 2D-DIGE and MS-based technologies, were altered in obese patients compared with age and gender-matched lean subjects [82]. The upregulation of the complement system (including complement C3, C4, and C9) and coagulation proteins (including fibrinogen, alpha-2-macroglobulin, kininogen-1, and prothrombin) in plasma EVs indicated a pro-inflammatory and pro-thrombotic state in obese patients.…”

“…The upregulation of the complement system (including complement C3, C4, and C9) and coagulation proteins (including fibrinogen, alpha-2-macroglobulin, kininogen-1, and prothrombin) in plasma EVs indicated a pro-inflammatory and pro-thrombotic state in obese patients. On the other hand, EV content of adiponectin, a protective protein hormone in obesity and CVD, was shown to be decreased in obese patients [82,86]. These studies demonstrate that EVs behave as “messengers”, shuffling between obesity and progression to CVD.…”