Extracellular vesicles (EVs) have been established as a means of cellular communication and are involved in physiological and pathophysiological processes through the transfer of bioactive molecules such as proteins, RNAs and lipids [1]. EVs are released by all cell types and can be found in all body fluids. The subtype and composition of EVs are dependent on the route of EV generation and parental origin, respectively [2]. The content of EVs can be packaged based on signals received from other cells or environmental factors such as oxygen, glucose concentration and sheath stress. In pregnant women, trophoblast-derived EVs are found in the blood [3], amniotic fluid [4], and urine [5]. These EVs have placenta-specific markers such as HLA-G [6], syncytin-1 [7] and placental-type alkaline phosphatase (PLAP-1) [8]. Due to their small size, EVs can cross the placental barrier, thereby enabling feto–maternal communication throughout the pregnancy [9] (Figure 1). Given that EVs are involved in a wide range of processes, it is no surprise that they play an important role in pregnancy. EVs regulate various normal physiological processes during pregnancy, including implantation of the embryo by regulation of the endometrium, trophoblast invasion, immune regulation of maternal responses and spiral artery remodeling [10]. Changes in the total number, content, and bioactivity of EVs have been reported in pregnancy complications such as preeclampsia (PE) [11].

Angiogenesis, the process by which blood vessels form for the delivery of nutrients to the body, is necessary both prior to and during pregnancy. A balance between pro and anti-angiogenic factors during pregnancy is key to a successful pregnancy. Imbalance of angiogenic factors, and subsequently, widespread endothelial dysfunction is considered the hallmark of pregnancy-related diseases such as preeclampsia (PE) [12]. The clinical manifestation of PE includes the development of hypertension after 20 weeks of gestation [13] and the coexistence of either proteinuria or other maternal organ dysfunction such as renal insufficiency, liver dysfunction, neurological features including headache or visual disturbances, hemolysis or thrombocytopenia, pulmonary edema ^[13][14][15][13,14,15]. Moreover, PE increases the risk of maternal and perinatal mortality and morbidity, and is associated with future cardiovascular disease risk [16]. Preeclampsia is classified as a new onset hypertensive pregnancy disorder. Depending on the time of delivery, early-onset preeclampsia manifests before the 34th week of gestation and is described as a fetal disorder that is associated with placental dysfunction and adverse fetal outcomes. Late-onset preeclampsia requires delivery at or after the 34th week of gestation and is considered as a maternal disorder associated with endothelial dysfunction and end organ damage [13].

Despite great scientific advances, PE is still defined as a “disease of theories”. Insufficient placenta development including abnormal spiral artery remodeling, placental hypoxia, oxidative stress, impaired angiogenesis and insufficient placental perfusion contribute to the development of preeclampsia ^[17][18][19][17,18,19]. Over the last two decades, increasing evidence suggests that angiogenic factors imbalance plays a pivotal role in PE pathogenesis ^[20][21][20,21]. Soluble fms-like tyrosine kinase 1 (sFlt-1), a soluble form of vascular endothelial growth factor receptor 1 (VEGFR-1) extracellular ligand-binding domain, is the key anti-angiogenic factor released by placenta during pregnancy. This soluble form of VEGFR-1 can bind to all isoforms of VEGF ^[22][23][22,23], as well as to the PlGF. Indeed, sFlt- 1 may act as a decoy receptor and hinder VEGF signaling through binding to its cognate receptors, thus inhibiting VEGF-mediated pro-angiogenic effects [24]. During normal pregnancy, the levels of sFlt-1 increase throughout gestation, providing a limiting and protective barrier for potentially unhealthy VEGF-dependent over signaling ^[25][26][25,26]. However, maternal circulating sFlt-1 levels are significantly increased in PE compared to normal pregnancy prior to the onset of the disease [27]. Furthermore, in vivo studies have showed that exogenous administration of sFlt-1 led to typical PE-like symptoms such as hypertension and glomerular endotheliosis in pregnant rats [20], whereas reduced circulating levels of free sFlt-1 below critical threshold rescued the damaging effects of sFlt-1 ^{[21][28][29][30]}[21,28,29,30]. Another anti-angiogenic factor released by placenta is soluble endoglin (sEng), a soluble form of the coreceptor of transforming growth factor-β (TGF-β), which acts as a decoy receptor of TGF-β leading to a decrease in angiogenesis [31]. Not surprisingly, harmful molecules released from the placenta may reach the fetal circulation, causing endothelial dysfunction in the fetus. Indeed, many reports ^[32][33][32,33] have described fetoplacental endothelial dysfunction associated with preeclamptic pregnancies (Figure 1). A vicious cycle is therefore created, which is to the detriment of the mother and the fetus.

2. Pro-Angiogenic Functions of EVs in Healthy Pregnancy

The influence of EVs on the angiogenic process has been reported in various in vitro and in vivo studies [12]. EVs are considered to modulate the angiogenic process through the transfer of several molecules including small RNA species and proteins. In particular, small RNA species, such as microRNA, are transferred by EVs and are considered pivotal for the reprogramming of target cells ^[34][35]. Lombardo et al. reported that miRNA-126 and p-STAT5 in endothelial-derived EVs are responsible for IL-3-mediated paracrine pro-angiogenic signals ^[35][36]. Similarly, endothelial-derived exosomes require miRNA-214 to simulate angiogenesis in recipient cells ^[36][37]. In vivo, injection of EPC-derived EVs containing the angiogenic miRNAs miRNA-126 and miRNA-296 in a mouse hind limb ligation model significantly increased capillary density and blood perfusion ^[37][38]. Several other microRNAs present in the EV cargo have been reported to be involved in the induction of angiogenesis, including miRNA-125a ^[38][39], miR31 ^[39][40] and miRNA-150 ^[40][41]. In parallel, several proteins released by EVs have also been found to be involved in the modulation of angiogenesis, including VEGF ^[41][42][42,43], FGF-2 ^[43][44], PDGF ^[42][44][45][43,45,46], c-kit ^[46][47], sphingosine-1-phosphate ^[47][48], regulated on activation of normal T cell expression and secretion ^[47][48], CD40L ^[48][49], C-reactive protein ^[49][50][51][50,51,52], metalloproteases ^[48][52][49,53], stem cell factor ^[53][54], and urokinase-type plasminogen activator. Furthermore, EVs can promote angiogenesis through the transfer of key lipids and proteins involved in activation of PI3K ^[54][55], extracellular signal-regulated kinase 1 and 2 ^[55][56][57][56,57,58], Wnt4/βcatenin ^[58][59], and nuclear factor-κB ^[59][60][60,61] pathways. In pregnancy, a large number of studies focus on the activity of EVs released by maternal and fetal cells, including endothelial cells, immune cells, trophoblast and stem cells with the latter being present in the umbilical cord, placenta, amniotic fluid and amniotic membranes (Figure 1). These EVs are capable of inducing tissue regeneration and angiogenesis and can be found in maternal circulation starting from 7 weeks of gestation ^[61][62], in amniotic fluid [4], and urine [5]. The heterogeneity of EVs produced in this environment results in a plethora of pro-angiogenic factors being delivered to support the physiological development of normal pregnancy and the maintenance of endothelial homeostasis (Table 1). The following paragraphs will discuss the role of healthy pregnancy-related EVs in angiogenesis, according to their different tissue types of origin.