**9. Extracellular Vesicles (EVs)**

Extracellular vesicles (EVs) are mediators that can modify the function of target cells by transferring proteins and genomic materials to other cells; thus, EVs have an active participation in cell-to-cell communication [171]. EVs shred from a variety of cells and have a number of important physiological as well as pathological functions as they are capable of tra fficking and transfecting the genetic material from cell to cell. The biogenesis and contents of EVs predominantly depends on the originating cell type and their surrounding microenvironment [172]. Several studies using electron microscopy analysis to characterize the morphology of EVs demonstrate that are spherical with lipid bilayer membrane [173,174]. The correct classification of EVs still a manner of debate and the majority of the information in the literature classify them according to their size and the di fferent biogenesis pathways. Typically, EVs are categorized as exosomes (~40–100 nm), microvesicles (~100–1000 nm) and apoptotic bodies (~1000–5000 nm) based on their size and origin. Microvesicles and apoptotic bodies are formed directly via budding of the plasma membrane, whereas exosomes are produced via an endocytic pathway [174]. Distinction between di fferent EVs subgroups is di fficult, due to the minimal physical and morphological di fferences, to the lack of specific markers, and to the fact that the same cellular source may dynamically produce di fferent class of EVs in response to di fferent conditions [175]. Recently, the international society of extracellular vesicles has recommended classifying the vesicles according to the their size in small EVs (<100–200 nm) and medium/large EVs (>200 nm), or density (low, middle, high, with each range will defined) or their biochemical composition (e.g., CD63+ve) [176]. Currently, there is no single method allowing for accurate characterization and discrimination of the di fferent EVs classes [175]. EVs can be ordinarily isolated from di fferent biological fluids using the di fferential and buoyant density centrifugation methods followed by ultrafiltration/size exclusion chromatography or flow cytometry or precipitation using polymers or antibodies to enrich the pure EVs population [177]. EVs play an important role in cell-to-cell communication and influence a variety of cellular functions, including cytokine production modulation, cell proliferation, apoptosis and metabolism, by transferring their protein, lipid or messenger RNA and micro RNA molecules [173,178]. EVs can be isolated both biological fluids (e.g., plasma/serum, urine, cerebrospinal fluid, saliva, etc.) and in vitro from cell-conditioned media. Moreover, as EVs are natural carriers of bioactive molecules, di fferent research studies are addressing the therapeutic potential of EVs due to their specific genetic material packaging capabilities [175]. Several groups have identified EVs in maternal biofluids during normal and complication of pregnancies and the potential role of EV during pregnancy have been reviewed in details by our group previously [179–182].

#### **10. Extracellular Vesicles**/**Exosomes in Normal and Preeclamptic Pregnancies**

Di fferent research studies utilized a variety of experimental models (i.e., biological fluids, primary placental trophoblasts, trophoblast cell line, placental explant, placental perfusate etc.) to isolate di fferent subpopulations (exosomes, microvesicles) of EVs and studied their role in the context of healthy as well as in pathological pregnancies. Small and large EVs originating from the placenta have been identified in maternal plasma. Concentrations of both total and placenta-derived exosomes present in maternal circulation increase across gestation [183] and are higher in complicated pregnancies (such as those a ffected by PE) as compared to normal pregnancies [184,185]. Interestingly, the global miRNA profile within small vesicles such as exosomes di ffers between normal and PE pregnancies across gestation and it is likely that PE is not only associated with changes in the circulating levels of exosomes, but also in their miRNA content [186]. Recently, Biro et al. identified that hsa-miR-210

level increased in the circulating exosomes isolated from PE pregnancies [187]. Poor placentation is associated with hypoxia and oxidative stress, which are features of PE and a ffects the invasion of extravillous trophoblast (EVT) and the uterine spiral arterial remodeling. Truong et al. studied whether low oxygen tension alters exosome release and the exosomal miRNA profile from HTR-8/SVneo cell line and examined their interaction with endothelial cells [188]. HTR-8/SVneo cells are commonly used as a model for EVT cells, although they are not ideal, as they contain a heterogenous population of trophoblast and stromal cells [189]. In this study, low oxygen tension to exosomes from EVTs cultured under normoxic conditions. Moreover, a specific set of miRNAs within exosomes from EVTs cultured under hypoxia were identified, and these miRNAs are present in circulating exosomes at early gestation from women who develop PE later in pregnancy. This data suggests that aberrant extracellular vesicle signaling is one of the common factors in the development of PE. In normal healthy pregnancy, syncytiotrophoblast derived EVs release into the maternal blood stream where they act upon their target endothelial cells and circulating immune cells [33,190–192]. Placental EVs carry di fferent proteins, lipids and nucleic acids that play a crucial role in feto-maternal communication to maintain pregnancy [193]. Interestingly, concentrations of large EVs gradually increase through pregnancy irrespective of their origin [186] and these EVs convey pro-inflammatory and pro-thrombotic antigens that might contribute to the hypercoagulable state observed in the last trimester of pregnancy [186]. Chang et al. identified that high levels of preeclamptic exosomes contain abundant sFlt-1 and sEng that can induce vascular dysfunction as these proteins were captured by vascular endothelial cells [194]. Tannetta et al. investigated the level of expression of placental protein 13 in syncytiotrophoblast derived extracellular vesicles (STBEVs) isolated from PE and normal pregnancy placental perfusate and found it was low in PE placenta [195]. Tong et al. described a novel mechanism by which placental EVs can attenuate PE pathogenesis in the presence of antiphospholipid antibody (aPL), which can induce the synthesis of toll-like receptors on placental EVs to increase the level of expression of mitochondrial DNA in these vesicles [196]. Thus, placenta-derived EVs are involved in gene regulation, placental homeostasis and cellular function that overall reflect the placental-maternal crosstalk [197]. Placental exosomes were also observed in fetal blood and their concentration correlated with fetal growth [198]. The concentration of placental exosomes in the fetal circulation was higher than that found in the maternal circulation and was also higher in pregnancies a ffected by PE [199]. Interestingly, not only the concentration of circulation exosomes in PE is di fferent compared with normal pregnancies, and specific changes in the protein cargo of exosomes in PE have been identified [200].

Another recent study measured the level of di fferent biomarkers including copeptin, annexin V and placental growth factor in maternal serum derived microparticles at 10–14 gestational weeks in women with PE and compared with that of normal healthy pregnancy [201]. Interestingly, the levels of nitric oxide synthase enzyme in the STBEVs were lower in STBEVs from PE compared to normal pregnancies [202]. In this regard, in a similar study, the levels of the protein neprilysin were increased in EVs of PE placenta [203].

Kohli et al. identified a novel pathway by which the placental EVs interact and causes release of EVs from endothelial cells and platelets that further activate the inflammasome in the trophoblast resulting in the development of PE [204]. The role of EVs in relation to PE pathophysiology including their di fferent contents has been summarized in Table 2.


**Table 2.** Updated Research Studies on EVs in PE Pathophysiology.


**Table 2.** *Cont.*



A number of drugs that can be used to treat PE appear to modulate EV expression. Some studies also addressed the mode of action of different antihypertensives, including thiazide diuretics that are used to treat the hypertension in PE. Hu et al. identified some changes in the sodium transporters in the renal tubule that were incorporated in the urinary exosomes isolated from PE women [206]. Another very interesting study by Chamley L. et al. identified melatonin as an effective agen<sup>t</sup> that can reduce the endothelial cell activating placental EVs release in PE [209]. In a similar study, transthyretin which is the thyroxin binding protein, was found in aggregated form and packaged in the small placental EVs in PE [210]. Xu et al. identified potential molecular mechanisms by which vitamin-D can reduce oxidative-stress induced PE [213]. Among the different therapeutic agents, the efficacy of aspirin was evaluated due to its availability and cost-effectiveness. However, there is lack of understanding in the mechanism of action of aspirin in the context of EV secretion regulation.

#### **11. E**ff**ects of LDA on Exosomal Secretion**

Up until now, there has been very limited evidence on the potential effect of aspirin on the release and content of EVs. Goetzl E.J. et al., discovered that in the presence of some coagulation factors (e.g., thrombin/ collagen) induces changes in the plasmatic levels of platelet-derived exosomes and their protein content (i.e., α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1)) [214]. Incubation of normal platelets with aspirin significantly inhibits arachidonic acid (AA)-induced platelet reactivity, EV formation and pro-coagulant activity [215]. Interestingly, aspirin therapy can significantly reduce microparticle (MP) shredding from erythrocytes, monocytes and vascular smooth muscle cells, reversing the effects of diabetes-induced stress on these cells [216]. Other studies have identified that aspirin changes the miRNA profile and EV release from platelet [217]. Syncytiotropholast-derived extracellular vesicles that are placental alkaline phosphatase (PLAP) positive inhibit the aggregation of platelets that were treated with aspirin [218]. Tannetta et al. observed that STBEV that are placental alkaline phosphatase (PLAP) positive inhibit the aggregation of platelets that were treated with aspirin [218]. On the contrary, platelets were activated and thrombus formation was increased by the STBEV isolated from preeclamptic placentas. Another study observed the effect of anticoagulant therapy (treatment with either unfractionated heparin (UFH) or low molecular weight heparin (LMWH), and/or LDA) on cell derived microparticles and outcome of pregnancy [219]. These findings indicate that placenta-derived extracellular vesicles may provide understanding in their potential role in low dose aspirin induced placental functions. Although there is significant evidence for dysregulation of both concentrations and bioactivity of circulating placental EVs in PE compared to normal pregnancies, no studies have described the potential effect of aspirin on EVs released from placental cells and their bioactivity.
