Please note that this presentation aims at summarizing currently available data only without the claim of representing established knowledge. Thus, information presented in this course may be subject to change in the future due to the rapid evolvement of the field of extracellular vesicles. Hello I am [inaudible] from the University of Helsinki and a member of the International Society of Extracellular Vesicles. I will narrate this presentation on the behalf of ISF educational board. This presentation has been created by Chantal Boulanger from Paris Cardiovascular Research Center Paris, France. Over the past decades most cell types found in the heart, blood vessels, and in the circulating blood have been shown the release extracellular vesicles in vitro. These observations have been made either on the best of conditions or following cell activation or apoptosis. Earlier findings in the cardiovascular field have identified these vesicles as microparticles or membrane shed particles which harbor membrane markers from the mother cell. They have also been shown to exhibit protocol gland activity which is associated with the externalization of the anionic phospholipid phosphatidylserine during the process of membrane budding. Later, together with the increase in sensitivity of the different detection techniques also more heterogeneous populations of vesicles and vesicles of smaller sizes have been observed. Initial studies quantified levels of medium or large circulating EVs using flow cytometry or capture assay from the blood of healthy individuals and from the blood of patients with cardiovascular diseases. These vesicles express membrane markers of the parent cell and they appear as a promising surrogate marker of cell activation or dysfunction in cardiovascular diseases. However, identification and analyzing the numbers of circulating small EVs is more complex due to the presence of lipoproteins of similar diameters in the blood. The first part of this MOOC will be devoted to extracellular vesicles or EVs circulating in the blood in the context of cardiovascular diseases. We will discuss general considerations, the presence of EVs in the blood and plasma of healthy individuals and in the general population. Then we will address EVs in patients with cardiovascular diseases. Finally the potential prognostic value in particular for EVs which originate from endothelial cells. It is difficult to quantify and characterize small-sized EVs in numerous plasma samples such as in clinical studies, as the current technologies separating small EVs from lipoproteins are time-consuming. In addition purification of EVs from plasma samples of patients with hyperlipidemia may not be complete. EVs circulating in the blood have a short half-life. They appear to be rapidly taken up by different organs such as the spleen, the liver, and also by the lungs, the bone marrow and other tissues. EV biodistribution appears to depend upon EVs cellular origin and composition and the route of administration when infusion of exogenously generated EVs has been investigated. In addition labeling EVs with fluorescent probes have also been shown to generate artifacts. Which is why proper controls need to be included in the experimental protocols. It is important to be aware that most biodistribution studies of large EVs from platelets, red blood cells, or of endothelial origin were performed in healthy experimental animals. These studies identified a role for phosphatidylserine in recognizing ligands such as Gas6, Del1, and Lactadherin. It is important to evaluate in vivo EV biodistribution in experimental models of cardiovascular diseases. Recent findings show that endothelial EVs can mobilize splenic monocytes after myocardial infarction. EVs have also been shown to transfer specific biological information such as microRNA126 to the vascular wall. Large and medium size EVs from endothelial origin circulate in the blood of healthy individuals and their levels increase transiently upon acute chemical aggression as shown in young study subjects exposed secondhand smoking which impairs endothelial vasodilatory functions and concomitantly increases endothelial EVs in plasma. The release of platelet and endothelial medium and large EV's is also regulated in vitro and in vivo by mechanical shear forces generated by the flowing bad blood. In the general non-diabetic population, plasma levels of endothelium-derived medium or large EVs associate with markers of metabolic syndrome. Plasma levels of medium and large EVs originating from platelets, from leukocytes, or from the endothelium increase in patients with cardiovascular diseases and in patients presenting cardiovascular risk factors, such as hypertension, smoking, hypercholesterolemia, and diabetes. However, we cannot conclude at the present whether these augmented EV levels reflect an increased formation or an impaired clearance of these EVs in these patients. Plasma levels of lymphocyte-derived medium and large EVs are increased in patients with high cardiovascular risk and in patients with familial hypercholesterolemia. In humans, circulating levels of leukocyte-derived medium and large EVs also associate with the presence of unstable atherosclerotic plaques. The general consensus is that levels of medium and large EVs originating from platelets reflect platelet activation. This is particularly important when preparing plasma from clinical blood samples. Residue platelets present in the platelet poor plasma preparations may generate artefactual EVs which do not reflect the EVA levels in the patient plasma. Plasma levels of platelet EVs appear to be increased in patients with cardiovascular risk. Platelet derived EVs contribute to thrombosis under the endothelial recruitment of inflammatory cells, therefore, they appear to have a deleterious effect. Circulating levels of endothelial medium and large EVs strongly correlate with in vivo endothelial dysfunction in patients with coronary artery disease with or without diabetes and in patients with end-stage renal failure. Therefore, plasma endothelial medium and large EV levels may serve as a surrogate marker of endothelial function and as an early marker of the development of cardiovascular diseases. Plasma levels of large EVs of endothelial origin may also serve as a prognostic marker of major cardiovascular events and death in patients with myocardial infarction or with end stage renal diseases. Circulating medium and large EVs from patients with myocardial infarction have been shown to have a functional effect on the vascular wall. They impair endothelial nitric oxide release and augment endothelial senescence apoptosis and tissue factor expression. These effects appear to be mediated by increased expression of angiotensin-converting enzyme and angiotensin AT R1 receptor expression and activation of MAP-kinase and PI3 kinase Akt pathways in target cells. Taken together, these effects could contribute to the systemic endothelial dysfunction observed in patients with myocardial infarction. This schematic picture summarizes the potential harmful or beneficial effects of EVs in the development of atherosclerosis which have been observed in in vitro studies. For the sake of clarity, the schematic focuses mainly on the roles of EVs in inflammation, thrombosis, and endothelial function. The used abbreviations in the schematic are: microvesicles, MVs, microvesicles of endothelial origin, EMVs, extracellular vesicles, EVs, nitric oxide, NO, reactive oxygen species, ROS, interleukin, IL, intercellular adhesion molecule 1, ICAM-1, regulated on activation normal T-cell expressed and secreted, RANTES or chemokines CCL5, cyclooxygenase type 2, COX-2, microRNA, miR, P-selecting glycoprotein ligand-1, PSGL-1, and low density lipoprotein, LDL. Microvesicles can exert both inflammatory and anti-inflammatory effects in various situations. It is therefore important to examine whether or not these vesicles can affect the development of cardiovascular diseases. So far, available information is extrapolated from studies using microvesicles generated in vitro and for concentrations that are not always comparable to those in pathophysiological conditions. Most of these in vitro studies summarized on this schematic report that microvesicles increase the release of pro-inflammatory cytokines from endothelial cells and leukocytes. In particular, interleukin-6 and interleukin-8, which in turn will promote monocyte adhesion to the endothelium and favor monocyte migration towards the plaque. EVs of endothelial origin can also regulate monocyte activation by transferring miRNA-10a and targeting components of the NF-kB inflammatory pathway. In vitro findings of nitric oxide suggests that microvesicles of several cellular origins interact with the vascular endothelium or may favor underlying cellular dysfunction, hence, endothelial microvesicles can decrease production by endothelial cells potentially through modifications in nitric oxides in the dephosphorylation or in local oxidative stress, leading to alterations of vascular tone and endothelial athero protective properties. These effects were observed for endothelial microvesicles isolated ex vivo from patients with associated diseases, such as chronic renal failure and metabolic syndrome, or in those which were produced in vitro in non-physiological conditions. In contrast, microvesicles isolated from healthy subjects or those released under normal conditions had no effects. These data suggests that microvesicles effects are largely influenced by their composition, which in turn results from the cellular origin to local environment and the stimuli initiating the vesiclization process. Furthermore, endothelial and large-diameter platelet microvesicles may also increase endothelial permeability and promote leukocyte adhesion to the endothelial cells. The increase of endothelial permeability could be mediated by local enhanced apoptotic process through delivery of Caspase-3 and raw kinase enzymes by the microvesicles. Several in vitro experiments indicate that microvesicles could promote adhesion of inflammatory cells to the endothelium. First, microvesicles enhance endothelial expression of adhesion molecules particularly ICAM-1 and of their counter receptors such as CD11a on the surface of monocytes. ICAM-1 expression can also be regulated by MiR-222 content in microvesicles. Moreover, microvesicles isolated from human atherosclerotic plaques can directly transfer functional ICAM-1 to target cells. Accordingly, microvesicles increase adhesion of monocytes to endothelial cells, which is a crucial step for the subsequent leukocyte diabetes. Arachidonic acid, an oxidized phospholipids carried by microvesicles also contribute to this increased adhesiveness. Second, the transfer of the proatherogenic chemokine, RANTES or CCL-5 from platelet microvesicles to the endothelial cells has also been implicated in this enhanced adhesion. Third, platelet microvesicles expressing P-selecting may promote monocyte infiltration by favoring leukocyte-leukocyte interaction under unfavorable flow conditions. Altogether, these results suggest that microvesicles released in pathological conditions could influence leukocytes and LDL infiltration towards sub endothelial space, which favors early atherosclerotic lesion development. We will now move on to the EVs in the diseased vascular wall and summarize their presence and their potential role in human atherosclerotic plaque, and in calcified lesions. Investigating endogenous EV release from disease to tissues has a definite interest in the cardiovascular field and provides additional information in addition to studies performed in vitro. However, extreme caution should be taken during the isolation procedures to avoid artifact findings. First, the presence of EVs should be documented using electron microscopy and compared between control or sham treated samples, and the samples from the diseased tissue. Second, the isolation method should be chosen bearing in mind that the same isolation procedure applied to control and sham treated tissue should not generate EVs. Third, the isolated EVs should be characterized in depth and their cellular origin identified. Fourth, the functional effects of the EVs isolated from the diseased tissue should be compared to those caused by the EVs isolated from control or sham treated tissues. Medium or large and small EVs accumulate in a human atherosclerotic lesions. Atherosclerotic plaque derived medium or large EVs which accumulate in human atherosclerotic lesions could contribute to plaque progression due to the pro-angiogenic effect mediated by the expression of CD40 ligand at their surface. In addition, plaque EVs cause a pro-inflammatory effect by functional transfer of adhesion molecule ICAM-1, promoting monocyte recruitment by endothelial cells. Plaque drive large EVs could also contribute to thrombus formation at the time of plaque rupture which will lead to the occlusion of the vascular lumen in the coronary arteries, this leads to myocardial infarction. So far there is no in vivo evidence that EVs participate in atherosclerotic plaque initiation. EVs are found in vascular calcified lesions. Smooth muscle cell derived EVs are involved in arterial micro-calcification, which is the characteristic of vulnerable atherosclerotic plaque which is prone to rupture. Valvular intertitial cells and macrophages also can release pro-calcifying EVs. The formation of calcifying EV's is controlled by sortilin which regulates the loading of tissue non-specific alkaline phosphatase, TNAP into EVs, conferring their calcification potential. We will now move on to the EVs in heart diseases. We will discuss EV released from cardiac cells, EV transfer between cardiac cells, and the potential role of cardiac EVs following myocardial infarction. Cardiomyocytes and cardiac cells produce medium or large EVs and small EVs which were identified in vitro from cultures of cardiomyocytes and cultures of fibroblasts. Several in vitro studies demonstrate the transfer of EVs between cardiac cells. Evs are transferred from hypoxic to normoxic cardiomyocytes. This leads to increased hypoxia-reperfusion injury mediated by microRNA 208 a and b. EVs are also transferred from macrophages to fibroblasts where they promote inflammation by transferring microRNA-155. EVs are also transferred from fibroblasts to cardiomyocytes. This might cause cardio protective effects by transferring microRNA 423-3p. Alternatively, other studies have reported that fibroblast EVs are pro-hypertrophic due to the transfer of microRNA 21 or the activation of the renin angiotensin system. Endothelial EVs are transferred to cardiomyocytes in which they cause apoptosis and oxidative stress. Finally, cardiomyocyte EVs are taken up by endothelial cells, and transfer GLUT transporters and glycolytic activity. Cardiomyocyte EVs might have conflicting effects on endothelial cells as they could either promote angiogenesis following the transfer of microRNA 222 and microRNA 143 or they may inhibit angiogenesis in a microRNA 320 dependent manner. There is experimental evidence for the transfer of cardiac EVs in heart diseases. For instance, the functional transfer of ATR1 from cardiac EVs to resistance arteries confers blood pressure responsiveness to angiotensin II infusion after cardiac pressure overload. In addition, cardiac EVs of cardiomyocyte and endothelial origin which are released after myocardial infarction stimulate a local proinflammatory response in infiltrating monocytes. There is also experimental evidence for the transfer of circulating EVs in heart diseases. Endothelial EVs can mobilize and transcriptionally activate splenic monocytes after myocardial infarction. Plasma EVs protect the heart from ischemia reperfusion injury by activating HSP27 toll-like receptor 4 dependent prosurvival pathways in cardiomyocytes. Finally, plasma EVs appear to contribute to toll-like receptor 9 Nf kappa beta dependant inflammation in chronic heart failure patients. In conclusion, several future challenges still need to be addressed in the cardiovascular field. First, we need to improve in vivo EV detection and characterization, which will require further significant progress also in vitro. We also need to better identify molecular mechanisms controlling EV release and clearance. Finally, a large number of studies in the cardiovascular field are performed in vitro, and the patho-physiological relevance of these observations remains unclear at the moment. Thank you for listening. We hope that you enjoyed this presentation on EVs and cardiovascular diseases.