ABSTRACT
EVs are cleared from the circulation by not only the liver and the spleen but also the lung.28-31 These studies have also identified the major role of lactadherin and Del-1 in the clearance of MVs by macrophages and endothelial cells, respectively.28,30,32 So far, there is no information on exosome in vivo clearance by specific organs, but macropinocytosis appears to be one way of exosome integration within cells.33 Alternatively, the lactadherin pathway may also contribute to exosome clearance, although this has not been proven in vivo yet. Despite knowing the exact contribution of each process on plasma vesicle levels, modification in the plasma levels remains important information about the severity of the diseases in patients suffering from CVDs. 16.2.2 Mechanisms ImplicatedSeveral factors involved in the development of atherosclerotic lesions, such as lipoproteins, cytokines, oxidative stress, or shear stress level, increase in vitro the release of EVs from vascular and/or circulating cells (Table 16.1). On the basis of the results obtained from experiments on platelets, EV formation at the plasma membrane of the cell appears to require some specific modifications. First, intracellular calcium-and caspase-dependent mechanisms are major determinants of the loss of membrane asymmetry.34 Intracellular calcium levels are modulated by mitochondrial permeability and plasma membrane calcium channel activities. This leads to calcium-dependent up-regulation of scramblase or floppase-/adenosine triphosphate (ATP)-binding cassette A1 and inhibition of translocase/flippase activity-induced exposure of phosphatidylserine (PS) on the outer leaflet.34 Storeoperated calcium entry (SOCE) and P2X1 activation by calcium also contributes to PS exposure. Second, blebbing requires cytoskeletal reorganization. Calcium could contribute to this reorganization in activating calpains and proteases. During apoptosis, bleb formation depends on the actin cytoskeleton and actin-myosin contraction, which is regulated by caspase-3-dependent Rho kinase I and II activation.35,36 Interestingly, cytoskeletal reorganization and PS exposure could interact as RhoA could modulate SOCE activity and in turn increase PS exposure.34 A recent study identified TMEM16F (a calcium-dependent scramblase) as a potent regulator of PS exposure in Scott syndrome patients,37 as these patients have a
defect in platelet activation and release of EVs, suggesting a potential role of TMEM16F. Endothelial vesicle formation and release have received significant attention over the past recent years, and different signaling pathways have been identified depending on the stimuli (Table 16.1).38 Endothelial vesicle shedding can occur independently of endothelial apoptosis.39 Curtis et al.40 identified p38 mitogen-activated protein kinase as a key factor for the shedding of endothelial cells under tumor necrosis factor-a (TNFa) stimulation. In opposition, thrombin stimulation of endothelial cells induces a complex biphasic release of endothelial vesicles. Several different mechanisms concur to vesiculation. First, thrombin binds to its protease-activated receptor-1, followed by Rho kinase II activation. Second, a later pathway involves TRAIL/Apo2L, a cytokine that belongs to the TNFa superfamily,41 followed by interleukin (IL)-1 release and IL-1 receptor activation.42 The second phase is characterized by an amplification loop based on the release by endothelial cells stimulated by thrombin of soluble forms of TRAIL and of IL-1 that act in an autocrine or paracrine manner on endothelial cells and stimulate EV shedding. A recent study also demonstrated that costimulation of endothelial cells with CD40 ligand (CD40L) and thrombin leads to an increase in endothelial vesicles 10+ EVs via p38 activation.43 Interestingly, these findings demonstrate that thrombin-induced activation of endothelial cells leads to the release of EVs of different compositions. Angiotensin II stimulation in vitro increase endothelial vesicle release and this is mediated by Rho kinase activation and involves a cholesterol-rich domain such as lipid rafts and caveolaes.44 Endogenous nitric oxide (NO) appears to play a protective role against endothelial vesicle formation by a mechanism involving tetrahydro-biopterin, as observed after C-reactive protein (CRP) endothelial activation.45So far, no other study has addressed the potential effects of NO on endothelial vesicle formation and release. Monocyte macrophages also release MVs under activation (Table 16.1). Endotoxin activates macrophage MV formation via a pathway requiring inducible nitric oxide synthase (NOS) activation.46 Furthermore, tobacco smoke stimulates the generation of highly procoagulant monocytic MVs in a process requiring extracellular signal-regulated kinase (ERK1/2) activation and caspase-3-dependent apoptosis.47
