**9. EV release by various stimuli**

EV release can be triggered by several types of stimuli. Many of these stimuli alter exosome release in an ADAM dependent manner. For example, EV release by p53 activation in tumor cells during radiation treatment has been reported [44]. Bacterial toxins including lipopolysaccharide have been shown to enhance the release of exosomes enriched in ADAM17 [24]. Calcium mobilization by treatment with calcium ionophores such as ionomycin has been reported to activate ADAM10 and trigger the secretion of EVs [24]. On the other hand, PMA has been reported to activate ADAM17 and induce its enrichment in exosomes [24]. Hypoxia has been reported to induce metalloproteinases and the release of EVs [24].

Akuthota et al. showed human eosinophils which are known to secrete chemokines, cytokines, and cationic proteins also secrete MVs, and the secretion of these vesicles increase in response to inflammatory stimuli such as tumor necrosis factor alpha (TNF-α) stimulation and eotaxin-1 (CCL11) [45]. In another study, Hunter et al. showed MVs are released from human brain microvascular endothelial cells in response to either thrombotic or inflammatory stimuli in a sex dependent manner [46]. Experimental evidence suggests nitric oxide (NO) negatively regulates EV release [47].

Numerous studies have demonstrated oxidative stress regulates the release of EVs from various cell types. One study showed oxidative stress triggers the release of microparticles by human alveolar cells and human bronchial epithelial cells [48]. Another study showed oxidative stress induces the release of membrane complement regulatory protein positive microparticles and this was blocked by the thiol antioxidant N-acetylcysteine amide [49]. Another study showed cigarette smoke extract induces exosome release by airway epithelial cells by depleting cell surface thiols but this is prevented by N-acetyl-L-cysteine and glutathione [50]. In another study, ATP mediated signaling through the purinergic P2X7 receptor was found to trigger macrophage activation of tissue factor activation procoagulant EVs and this was blocked by the ROS inhibitor N-acetyl cysteine or the flavoenzyme inhibitor diphenyleneiodonium but not nitric oxide synthase inhibitors [51]. In another study, elevated levels of carbon dioxide were reported to activate mitochondrial ROS in neutrophils resulting in an increase in microparticles release [52].
