Endothelial microvesicles in hypoxic hypoxia diseases

Abstract Hypoxic hypoxia, including abnormally low partial pressure of inhaled oxygen, external respiratory dysfunction‐induced respiratory hypoxia and venous blood flow into the arterial blood, is characterized by decreased arterial oxygen partial pressure, resulting in tissue oxygen deficiency. The specific characteristics include reduced arterial oxygen partial pressure and oxygen content. Hypoxic hypoxia diseases (HHDs) have attracted increased attention due to their high morbidity and mortality and mounting evidence showing that hypoxia‐induced oxidative stress, coagulation, inflammation and angiogenesis play extremely important roles in the physiological and pathological processes of HHDs‐related vascular endothelial injury. Interestingly, endothelial microvesicles (EMVs), which can be induced by hypoxia, hypoxia‐induced oxidative stress, coagulation and inflammation in HHDs, have emerged as key mediators of intercellular communication and cellular functions. EMVs shed from activated or apoptotic endothelial cells (ECs) reflect the degree of ECs damage, and elevated EMVs levels are present in several HHDs, including obstructive sleep apnoea syndrome and chronic obstructive pulmonary disease. Furthermore, EMVs have procoagulant, proinflammatory and angiogenic functions that affect the pathological processes of HHDs. This review summarizes the emerging roles of EMVs in the diagnosis, staging, treatment and clinical prognosis of HHDs.

to left shunt congenital heart disease and pulmonary hypertension).
Endothelial microvesicles are 100-1000 nm anucleated vesicles formed following cytoskeletal and membrane reorganization and are released during apoptosis or activation of ECs into the extracellular milieu. 5,6 EMVs that are released from apoptotic or activated ECs that have been stimulated by hypoxia, oxidative stress, coagulation and inflammation can be used as a marker of EC injury. Coincidentally, elevated EMV levels have been identified in several HHDs, 7 including OSA 8 and pulmonary hypertension (PH). 9 Furthermore, EMVs play a critical role in cell information transmission and exchange, and the procoagulant, proinflammatory and angiogenic properties of EMVs have been confirmed to increase in the occurrence and development of HHDs. All these findings indicate that EMVs have the potential to identify HHD phenotypes, to stratify disease severity, to improve risk stratification for patients who develop HHDs, to better define prophylactic strategies and to ameliorate the prognostic characterization of patients with HHDs. Furthermore, because a pathogenic role for EMVs is clearly emerging in HHDs, EMVs are becoming a novel target for HHD treatment.

| END OTHELIAL MICROVESICLES
Endothelial microvesicles are 100-1000 nm anucleated vesicles that are formed following cytoskeletal and membrane reorganization and can be released into the extracellular milieu following apoptosis or activation of ECs. 5,10 Hypoxia, ischaemia, oxidative stress, inflammation, coagulation and other factors can damage ECs or cause EC activation. 11 Previous studies also found elevated EMV levels in several disease conditions associated with hypoxic hypoxia, 7 such as OSA, 8 chronic obstructive pulmonary disease (COPD) 12 and PH. 9 In addition, EMVs may play important roles in the pathological processes 13 and tissue repair mechanisms 14 of HHDs.

| EMV phenotypes
As the surface of EMVs contains a variety of membrane glycoprotein antigens (such as CD31, CD144 and others), EMVs can be defined and detected using membrane glycoprotein antigens. These membrane glycoprotein antigens also make it easier to identify, distinguish and study EMVs.
First, membrane glycoprotein antigens help us to identify EMVs.
We can use membrane glycoprotein antigens to detect and identify EMVs. However, a major issue is that most surface markers are not unique to EMVs. In the blood, platelets, red blood cells and other cells can release microvesicles, and many different cell-derived microvesicles have common surface markers. To identify EMVs and distinguish EMVs from other vesicles, researchers have proposed a combination of marker proteins derived from different vesicles to resolve these difficulties. For example, we can use positive EC markers (eg CD31 and CD144) in combination with the absence of platelet markers (CD41 or CD42b) to distinguish between EMV and platelet microvesicles. 15 As shown in our previous report 16   Second, classification via membrane glycoprotein antigens will facilitate the identification of stimulatory factors that cause EC activation or apoptosis. As shown in Figure 1, 10,11,[17][18][19][20][21] Ang II and other cell activation factors can increase EC release of CD62E + EMVs, CD54 + EMVs, and CD106 + EMVs. In contrast, when the endothelium becomes functionally apoptotic, CD31 + , CD51 + and CD144 + EMV levels are increased. The relative proportion of CD62E + /CD31 + EMVs, rather than absolute levels, distinguishes EMVs released by activated ECs from those derived from apoptotic ECs. A level of CD62E + /CD31 + EMVs less than 1% indicates that most EMVs originated from apoptotic ECs, whereas 10% or more reflects an activated origin. 20 Third, different surface markers likely play different roles in the pathogenesis of diseases. 22 As shown in Table 1, different EMV subtypes may play a role in different HHDs. The level of CD31 + EMVs is related to the apnoea-hypopnea index (AHI) in OSA patients 23-25 ; mild COPD and emphysema in COPD patients 26,27 ; and hemodynamic severity, risk stratification and treatment effects in PH patients. 9,28,29 In addition, CD31 + EMVs may play diverse roles in vascular biology by regulating platelet function, angiogenesis, T-cell and B-cell activation, EC permeability and transmigration across the endothelium. [30][31][32][33][34][35] Therefore, the released CD31 + EMVs likely reflect the apoptosis of injured ECs. CD51 is present on ECs, B lymphocytes, monocytes, macrophages and platelets. 36 CD51 + EMVs may increase leucocyte homing and rolling and angiogenesis. 35,37 The LFA-1/ICAM-1 interaction is critical to the firm adhesion of T cells to the vascular endothelium of inflamed tissues and influences the diapedesis and migration of these adhered lymphocytes out of the vasculature directly into adjacent tissues on the ocular surface. 38 CD62E is rapidly induced on activated ECs a few hours after inflammatory stimulation, and CD62E + EMVs may increase the recruitment of leucocytes to the site of injury during inflammation. [30][31][32][33][34][35] In OSA patients, the level of CD62E + EMVs was related to treatment effects of continuous positive airway pressure (CPAP) and AHI. 39 In COPD patients, the level of CD62E + EMVs was a good predictor of rapid forced expiratory volume in 1 second (FEV1) decline, severe COPD and hyperinflation. 12,27,40 In PH patients, the level of CD62E + EMVs was related to adverse clinical events and thromboembolic complications. 41,42 In venous thromboembolism (VTE) patients, the level of CD62E + EMVs was related to inherited thrombophilia. 43 Thus, CD62E + EMV levels may reflect the degree of ongoing endothelial inflammation. Endoglin EMVs are CD105 + EMVs. The level of CD105 + EMVs is related to severity, EC survival and angiogenesis in PH patients. 44,45 The level of CD144 + EMVs is related to hemodynamic severity, pulmonary artery intima media thickness and right DENG ET AL. | 3709 ventricular function. 9,46,47 The release of CD144 + EMVs may reflect the structural destruction of the endothelium rather than an inflamed lung. CD146 + EMVs, another indicator of EC injury, may be closely related to signal transduction, endothelial permeability, cell migration, angiogenesis and immune response. 48

| EMVs as messengers
As shown in Figure 1, 10,11,[17][18][19][20][21] EMVs can be used as an indicator to reflect stimulatory factors, the disease state and prognosis. In contrast, EMVs also play a role in pathological processes and disease development. EMVs have important functions in the inflammatory response, coagulation and angiogenesis. As shown in Figure 2, 17,49 we identified the signal pathways involved in thrombin-induced EMV formation and potential mechanisms involving in circulating EMVs in the interrelationship among inflammation, angiogenesis, and coagulation. We previously published an article detailing these features of EMVs; thus, for more detailed information, please refer to our previously published paper. 16

Endothelial microvesicles have been investigated in several human
HHDs, which include very low partial pressure of inhaled oxygen, external respiratory dysfunction-induced respiratory hypoxia and venous blood flow into the arterial blood, as possible pathogenic elements, prognostic markers, and therapeutic targets. The data reported in the following sections are summarized in Table 2.

| EMVs and atmospheric hypoxia
When people are at an altitude of 3000 metres above sea level or at a high altitude, are in poor ventilation tunnels or inhale a hypoxic mixture, the amount of oxygen in the body first depends on the oxygen partial pressure of the inhaled gas. In a plateau, as the altitude increases, the atmospheric pressure decreases, and the partial pressure of oxygen of the inhaled gas decreases correspondingly, which results in a decrease in the partial pressure of oxygen and oxygen in the alveoli. Then, the oxygen diffused into the blood decreases, and the arterial oxygen saturation decreases. Lichtenauer et al. found that circulating CD31 + /Annexin V (AV) + EMVs were correlated with an increase in simulated sea level and decline in oxygen saturation in young healthy volunteers. Concomitantly, we found a significant decrease in CD62 + /AV À EMVs when the partial pressure of oxygen declined with increasing sea level. 50 We believe that this difference is attributable to CD31 + EMVs (primarily released from apoptotic ECs and reflecting hypoxia-induced acute injury, while CD62E + EMVs are primarily released from activated ECs). As Table 2 shows, however, the results of different groups are not consistent or even F I G U R E 1 Differences in the release mechanism and antigen expression of EMVs derived from activation vs apoptosis of ECs. Activated stimuli cause a cytosolic calcium increase, which leads to EC membrane disruption. Apoptotic stimuli activate caspases and cause membrane disruption in ECs. Activation inducers and apoptosis inducers can both lead to vesiculation and EMV generation, but the levels of EMVs surface antigen markers are not the same. The surface of activated EMVs contains a higher level of E-selectin, ICAM-1, and VCAM-1, while PS, PECAM-1, VE-cad, TF, and endoglin antigen markers show higher expression on apoptotic EMVs. ICAM, intercellular adhesion molecule; TF, tissue factor; VCAM, vascular cell adhesion molecule; PECAM, platelet endothelial cell adhesion molecule; VE-cad, VE-cadherin; PS, phospholipid; vWF, von Willebrand factor 10,11,[17][18][19][20][21] contradictory. This difference may be closely related to the altitude selected by the researchers. 51-54

| EMVs and OSA
Obstructive sleep apnoea, including intermittent hypoxia and intrathoracic pressure changes and arousals, can result in endothelial dysfunction and ultimately arterial disease. 39,55,56 There is growing evidence that EMVs can be used as a biomarker for diagnosis, treatment and prognosis in patients with OSA. Significantly, higher levels of EMVs have been reported, both in adult and childhood OSA, compared with matched controls. In addition, EMVs were correlated with OSA severity and the AHI. [23][24][25]39 Moreover, injection of EMVs from OSA patients into mice impaired the endothelium-dependent relaxation in the aorta and FMD in small mesenteric arteries. 8 Therefore, circulating EMVs are considered not only a marker of vascular damage but also a cause of vascular disease in patients with OSA.
This relationship is one mechanism that links OSA and accelerated atherosclerosis, and increased levels of circulating EMVs may underlie the altered endothelial function and coagulation homeostasis in OSA. 24,57 However, 2 research groups found no increase in CD31 + / CD41 À or CD146 + EMVs in OSA patients, which may be due to differences in EMV subpopulations and population differences. 8,58 Continuous positive airway pressure treatment improves endothelial function in OSA patients. 57,59,60 Following CPAP treatment, CD62E + EMVs (but not CD31 + /CD42 À EMVs) were significantly reduced. 25 These findings were also confirmed by Jelic et al. 24 , who reported elevated levels of EMVs in OSA patients and a trend towards decreased levels following CPAP in a treatment uncontrolled study. In addition, CPAP withdrawal leads to OSA recurrence, and CD62E + and CD31 + /CD42b -EMV levels were increased significantly in the CPAP withdrawal group compared with patients administered continuous therapy. 39 In addition, the ratio of CD62E + EMVs to CD31 + EMVs, which contributes to distinguishing between activation and apoptosis ECs, suggests that EMVs in children and adults with OSA are primarily the apoptotic subtype. As markers of EC apoptosis, CD31 + /CD42b -EMV levels were strongly correlated with OSA severity, endothelial dysfunction and carotid intima media thickness 23,25 and may reflect the chronic vascular damage induced by long-term exposure to repeated apnoeas. In contrast, CD62E + EMV levels (reflecting EC activation) are not correlated with OSA or the severity of vascular damage but are correlated with CPAP initiation or withdrawal. 15,25,39

| EMVs and Asthma
Asthma is usually characterized by different patterns of airway inflammation with a complex network of cellular and molecular mediators. 61 In contrast to OSA, very little is known about the possible role of EMVs in asthma. The scarce available data are preliminary, and further research in the field with properly designed studies is needed.

| EMVs and COPD
Chronic obstructive pulmonary disease is a lung disease characterized by nearly irreversible lung destruction, which results in airflow limitation. Accumulating evidence suggests that EC injury in lung tissues is closely related to disease progression in COPD. 62

| EMVs and PH
Vascular remodelling and endothelial dysfunction-related EMVs are involved in PH. 64 Three research groups have demonstrated that EMV levels were significantly higher in PH patients than controls.
The levels of CD31 + EMVs and CD144 + EMVs, but not those of CD62E + EMVs, predict hemodynamic severity in PH patients. 9 Elevated levels of circulating CD62E + EMVs in PH patients prior to treatment were associated with adverse clinical events. 41 The levels of CD144 + EMVs were positively associated with pulmonary artery intima media thickness but not CD146 + EMVs. 46 70 Finally, in a murine model, the injection of EMVs into mice induced a significant release of the proinflammatory cytokines TNFa and IL-1b and a subsequent recruitment of neutrophils. 70 These effects were further increased by the concomitant or sequential administration of bacterial LPS, indicating that EMVs might represent a signal that primes the lung for the following inflammatory response to an external injury. 71 Again, from this point of view, EMVs could represent a potential therapeutic target for ALI/ARDS. EMVs could be a link between alveolar inflammation and coagulation (2 key steps in the pathobiology of ALI/ARDS) and may be a target for future treatment.

| EMVs and venous thromboembolism/ pulmonary embolism
The pathophysiology of VTE involves endothelial damage, blood stasis and hypercoagulability. 72 EMVs may be used as diagnostic and differential diagnostic criteria in VTE and PE. EMV levels were significantly higher in patients with VTE, 43,73 a previous VTE history, 74,75 and the prothrombin gene mutation G20210A (PTM) compared with controls. 74   miR-92 levels increased, but did not reach the level of significance. 79 Serban et al. found that circulating EMVs are emerging as biomarkers of COPD in individuals exposed to cigarette smoke and the EMVs were significantly enriched in let-7d, miR-191, miR-126 and miR125a, which reciprocally decreased intracellular in cigarette smoke-exposed endothelium. 80

CONF LICT OF I NTEREST
Authors declare that they have no conflict of interest.