methods-isolation-purification-extracellular-vesicles-from-different

Methods of Isolation and Purification of Extracellular Vesicles from Different Biological Matrixes: Special Issue at a Glance

ARTICLES DISCUSSED:
Hanine El Itawi, H., et al. Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection. J Vis Exp. ...

Hospital Clínic of Barcelona

Instituto de Salud Carlos III

Circulating microvesicles (cMV) are small phospholipid-rich vesicles that contribute to the atherothrombotic process, and are biomarkers of cardiovascular disease (CVD) burden and progression. Diet is a cornerstone for CVD prevention, but dietary effects on cMV shedding are poorly characterized. We aimed at assessing the long term effects of a Mediterranean diet compared to a low-fat diet (LFD) on MV shedding by cells of the blood and vascular compartments in patients at high cardiovascular risk treated as per guidelines.

The Mediterranean diet decreases prothrombotic microvesicle release in asymptomatic individuals at high cardiovascular risk.

Diabetic subjects are at increased risk of cardiovascular disease. Atherosclerosis, the common soil of most of the cardiovascular complications, is more prevalent and extensive in this population due not only to hyperglycemia, insulin resistance, and dyslipidemia, but also to inflammation and oxidative stress. Lycopenes are bioactive compounds with antioxidant and anti-inflammatory activities mostly supplied by tomato and tomato byproducts. We investigated the association between circulating lycopenes and carotid plaque burden in diabetic patients, in a cross-sectional study in 105 newly diagnosed diabetic subjects. Atheroma plaque (wall thickness ≥ 1.5 mm), number of plaques, and plaque burden (sum of maximum heights of all plaques) were assessed by sonographic evaluation of carotid arteries. Plasma lycopenes (5--, 9--, 13--, and -lycopene) were quantified by high performance liquid chromatography-mass spectrometry HPLC-MS. Atheroma plaque was observed in 75 participants, from which 38 presented one plaque and 37 two or more carotid plaques. No differences were observed in the plasmatic concentrations of lycopenes between subjects with and without atherosclerotic plaque presence. However, plaque burden was inversely associated with 5--lycopene, all -lycopene isomers, -lycopene, and total lycopene isomers (all,  < 0.05). High plasma levels of lycopenes inversely relate to atherosclerotic burden. We provide novel evidence that suggests that the consumption of compounds found in tomato and tomato byproducts might be beneficial for the prevention of atherosclerosis.

5--, - and Total Lycopene Plasma Concentrations Inversely Relate to Atherosclerotic Plaque Burden in Newly Diagnosed Type 2 Diabetes Subjects.

Emerging evidence points to an association between severe clinical presentation of COVID-19 and increased risk of thromboembolism. One-third of patients hospitalized due to severe COVID-19 develops macrovascular thrombotic complications, including venous thromboembolism, myocardial injury/infarction and stroke. Concurrently, the autopsy series indicate multiorgan damage pattern consistent with microvascular injury.

Thrombotic Complications in Patients with COVID-19: Pathophysiological Mechanisms, Diagnosis, and Treatment.

The molecular understanding of the pathophysiological changes elicited by diabetes in platelets may help in further elucidating the involvement of this pseudo-cell in the increased risk of developing cardiovascular disease and thrombosis in diabetic subjects. We aimed to investigate the differential characteristics of platelets from diabetic patients and nondiabetic controls to unveil the molecular mechanisms behind the increased platelet reactivity in diabetes. We compared platelets from diabetic and control subjects by 2 dimensional-electrophoresis followed by mass spectrometry. Changes in selected differential proteins were validated by immunoprecipitation assays and western blot. Platelet aggregation was measured by light transmittance aggregometry induced by collagen and ADP, and dynamic coagulation analysis of whole blood was measured by thromboelastometry. We observed significant differences in proteins related to platelet aggregation, cell migration, and cell homeostasis. Subjects with diabetes showed higher platelet aggregation and thrombogenicity and higher contents of the stress-related protein complex HSPA8/Hsp90/CSK2α than nondiabetic subjects. Changes in the chaperones HSPA8 and Hsp90, and in CSK2α protein contents correlated with changes in platelet aggregation and blood coagulation activity. In conclusion, the complex HSPA8/Hsp90/CSK2α is involved in diabetes-related platelet hyperreactivity. The role of the HSPA8/Hsp90/CSK2α complex may become a molecular target for the development of future preventive and therapeutic strategies for platelet dysfunction associated with diabetes and its complications.

Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets.

Extracellular vesicles (EV, exosomes and microvesicles -MV-) are 30-1000 nm particles surrounded by a phospholipid bilayer membrane that are released from almost all cell types through several pathways. EV encapsulate bioactive molecules, and the molecular cargo is determined by the trigger stimulating its release, reflecting its cell origin and biological functions. This review is primarily focused on the latest evidence of the roles of EV, released from cells involved in the different stages of atherothrombosis. The potential translation of this information to the clinical arena is also discussed. EV can have both pro- and anti-atherothrombotic effects depending on several factors, such as the type of vesicle (MV/exosome), its molecular cargo, its cell of origin, and the context in which are generated, i.e., the stimulus triggering its release. In fact, EV actively participate in every step of atherosclerosis onset and progression, and also in thrombus formation leading to a major adverse cardiovascular event. Moreover, EV have a determinant role in fibrous cap stability, thus determining the propensity of the plaque to rupture. On the other hand, and again, conditioned by the context and stimulus instigating its secretion, some EV may have protective biological functions, perhaps as a compensatory mechanism or even with reparative or regenerative potential. Therefore, the study of the implication of EV in atherothrombosis might be of relevance to unveil new therapeutic targets, vectors and biomarkers of cardiovascular disease (CVD).

Extracellular vesicles in atherothrombosis and cardiovascular disease: Friends and foes.

Scientists on the Spot: A matter of blood flow.

Circulating microvesicles (cMV) are both effectors and biomarkers of cardiovascular disease (CVD), and the effects of omega 3 polyunsaturated fatty acids (n3 PUFA) in MV shedding are not yet well known. Therefore, we aimed to investigate the effects of long-term n3 PUFA supplementation on cMV release from cells of the vascular compartment in elderly subjects at very high risk of CVD.

One year of omega 3 polyunsaturated fatty acid supplementation does not reduce circulating prothrombotic microvesicles in elderly subjects after suffering a myocardial infarction.

The identification of nutritional patterns associated with the development of type 2 diabetes (T2D) might help lead the way to a more efficient and personalized nutritional intervention. Our study is aimed at evaluating the association between fatty acids (FA) in red blood cell (RBC) membranes, as a quantitative biomarker of regular dietary fat intake, and incident type 2 diabetes in a Spanish population. We included 1032 adult Spaniards (57% women, age 49 ± 15 years, 18% prediabetes), without diabetes at study entry, from the Di@bet.es cohort. Incident diabetes was diagnosed at the end of the study follow-up. The FA percentage in RBC was determined at baseline by gas chromatography. Participants were followed on average 7.5 ± 0.6 years. Lower percentages of linoleic acid (LA), α-linolenic (ALA), and eicosapentaenoic acid (EPA), and higher percentages of docosahexaenoic acid (DHA) in RBC membranes were associated, independently of classical risk factors, with worse glucose metabolism at the end of the study follow-up. In addition, higher percentages of ALA and EPA, and moderate percentages of DHA, were associated with lower risk of diabetes. No significant associations were found for LA and diabetes risk. Dietary patterns rich in vegetables are independently associated with lower risk of both deterioration of glucose regulation and incident diabetes, and should be reinforced for the prevention of diabetes.

Low Percentage of Vegetable Fat in Red Blood Cells Is Associated with Worse Glucose Metabolism and Incidence of Type 2 Diabetes.

Microvesicles (MVs) are actively secreted by cells. The NLRP3-inflammasome and the interleukin 6 (IL-6)-pathways are central in cardiovascular disease. Knowledge of how the inflammasome influences the MVs is limited. In a cross-sectional study, we assessed whether MVs in plasma associate with genes encoding inflammasome signalling in coronary thrombi. Moreover, any relationships between inflammasome activation and phosphatidylserine (PS) externalization, determined through Annexin V (AV) labelling, and myocardial injury, assessed by cardiac troponin T (cTnT), were analysed. Intracoronary thrombi and blood samples from STEMI patients ( = 33) were investigated. mRNA of NLRP3, caspase-1, interleukin-1β (IL-1β), interleukin-18 (IL-18), IL-6, soluble IL-6-receptor (sIL-6R), and glycoprotein-130 (gp130) were isolated from the thrombi and relatively quantified by RT-PCR. MVs were analysed by flow cytometry. Total AV MVs, mainly reflecting hypercoagulability, correlated positively to NLRP3 gene expression (r = 0.545,  = 0.009). A similar pattern was seen for platelet, endothelial and leukocyte derived MVs, separately. The majority of the MVs were AV (96%). Total and AV MVs correlated inversely with IL-1β (r = -0.399 and -0.438, respectively,  &lt; 0.05, both) and gp130 (r = -0.457 and -0.502, respectively,  &lt; 0.05, both). No correlations between MVs and cTnT were observed. Our findings indicate an association between NLRP3-inflammasome in coronary thrombi and procoagulant AV MVs in STEMI patients. The inverse relationships between AV MVs and the gene expression of inflammasome activation may indicate an immuno-dampening role of this subpopulation.

Circulating Microvesicles in Association with the NLRP3 Inflammasome in Coronary Thrombi from STEMI Patients.

Sex-biased analyses still remain as one of the biggest limitations to obtain universal conclusions. In biomedicine, the majority of experimental analyses and a significant amount of patient-derived cohort studies exclusively included males. In nutritional and molecular medicine, sex-influence is also frequently underrated, even considering maternal-inherited organelles such as mitochondria. We herein illustrate with in-house original data examples of how sex influences mitochondrial homeostasis, review these topics and highlight the consequences of biasing scientific analyses excluding females as differentiated entities from males.

Article	Year
The Mediterranean diet decreases prothrombotic microvesicle release in asymptomatic individuals at high cardiovascular risk. Clinical nutrition (Edinburgh, Scotland)\| PubMed ID: 32147198	2020
5--, - and Total Lycopene Plasma Concentrations Inversely Relate to Atherosclerotic Plaque Burden in Newly Diagnosed Type 2 Diabetes Subjects. Nutrients\| PubMed ID: 32517202	2020
Thrombotic Complications in Patients with COVID-19: Pathophysiological Mechanisms, Diagnosis, and Treatment. Cardiovascular drugs and therapy\| PubMed ID: 33074525	2021
Molecular mapping of platelet hyperreactivity in diabetes: the stress proteins complex HSPA8/Hsp90/CSK2α and platelet aggregation in diabetic and normal platelets. Translational research : the journal of laboratory and clinical medicine\| PubMed ID: 33887528	2021
Extracellular vesicles in atherothrombosis and cardiovascular disease: Friends and foes. Atherosclerosis\| PubMed ID: 34256307	2021
Scientists on the Spot: A matter of blood flow. Cardiovascular research\| PubMed ID: 34718452	2021
One year of omega 3 polyunsaturated fatty acid supplementation does not reduce circulating prothrombotic microvesicles in elderly subjects after suffering a myocardial infarction. Clinical nutrition (Edinburgh, Scotland)\| PubMed ID: 34742136	2021
Low Percentage of Vegetable Fat in Red Blood Cells Is Associated with Worse Glucose Metabolism and Incidence of Type 2 Diabetes. Nutrients\| PubMed ID: 35405981	2022
Circulating Microvesicles in Association with the NLRP3 Inflammasome in Coronary Thrombi from STEMI Patients. Biomedicines\| PubMed ID: 36140297	2022
Relevance of sex-differenced analyses in bioenergetics and nutritional studies. Frontiers in nutrition\| PubMed ID: 36245509	2022

Gemma Chiva-Blanch

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