Introduction: Extracellular vesicles (EV) are a heterogeneous group of membranous vesicles that differ in size, structure, mechanism of biogenesis, and function. They are part of a complex cellular communication pathway and are produced inside or on the cell`s surface with release in high concentration into body fluids (in blood from 106 up to 1013/ml of blood plasma, depending on the used methodology). Most of the research so far has been focused on the study of EV as biomarkers of pathological conditions, but there is a lack of knowledge of the characteristics of blood EV in a normal, physiological state. Knowledge of EV` physiological characteristics would make it possible to determine reference values that would provide the framework for numerous EV studies concerning pathology.
In the doctoral dissertation, we defined EV's biophysical (concentration, size) and molecular (surface proteins) characteristics isolated from blood cells, and assessed their association with certain demographic, clinical, and laboratory factors of test subjects (sex, age, BMI, blood pressure, smoking status, and hemogram values). Based on our results, we will propose reference values for the concentration, size, and surface protein composition for physiological EV` values in the blood plasma of a healthy adult Slovenian population.
Hypotheses: (1) The concentration and size of extracellular vesicles isolated from blood plasma are related to age, body mass index, smoking, and/or blood pressure in the healthy adult population. (2) The ratio of blood cell subtype concentrations affects the concentration and/or surface protein composition of extracellular vesicles isolated from blood plasma in healthy adults.
Methodology: The study was conducted in two parts, namely in the first part, we collected clinical samples and basic demographic, clinical, and laboratory data of the subjects and established a collection of blood plasma. We conducted the study on blood donors who are considered to be healthy adults. In the second part, we isolated EV from the collected blood plasma samples and performed biophysical (NTA analysis to determine the concentration and size of ZV) and biochemical (detection of surface proteins of EV by imaging flow cytometry and detection of tetraspanin-positive EV surface proteins with bead-based flow cytometry) analysis. In addition, we checked the quality of the performed procedures (measurement of residual cells in plasma for EV isolation, measurement of lipoprotein concentration in EV isolate, measurement of platelet activation, etc.) to more precisely define the study results. Statistical analysis was then used to verify the association between the measured concentration, size, and presence of surface proteins with general clinical factors (e.g. gender, age, blood pressure, BMI, smoking, and haemogram).
Results: The concentration of nanoparticles in a cohort of healthy adults [/ml plasma] was in the range of 109, measured using NTA, while the average of median concentrations was in the range of 107, measured by imaging flow cytometry. Based on the surface protein profile of EVs, imaging flow cytometry revealed the highest concentrations for EV subpopulations originating from activated platelets and erythrocytes, while the lowest concentration was observed for endothelial-derived EVs. According to surface cell markers, platelet- and leukocyte-derived EVs predominated among tetraspanin-positive EVs. The degree of inter-individual variability was high, with tetraspanin-positive EVs showing lower variability compared to EV concentrations measured by imaging flow cytometry. In our study, sex, age, smoking, ABO blood group system, and menopausal status influenced specific EV subpopulations.
Conclusions: A general effect on the properties of EV has not been demonstrated, but we have demonstrated an effect on certain subpopulations of EV, thus only partially confirming the first hypothesis. The second hypothesis was not confirmed because we proved that haemogram values do not affect the properties of blood EV.
Contribution to Science: We were the first to precisely describe the physiological population of EVs in blood. We demonstrated that EVs released from the same cell type exhibit highly diverse and unrelated profiles, making it necessary to measure multiple distinct EV subpopulations for each cell type (using different cell markers for the same cell type). For the first time, we established the influence of the ABO blood group system on EV characteristics. In the study of EVs in physiological states, it is essential to ensure the quality of plasma and the procedures performed, allowing for more accurate evaluation and contextualization of the results.
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