Extracellular Vesicles in Preeclampsia: Drivers of Vascular Dysfunction and Inflammation

Abstract

Vascular inflammation, augmented vasoconstriction, reduced vasodilatory capacity, and endothelial dysfunction are cardinal features of the maternal vascular dysfunction phenotype in preeclampsia. Extracellular vesicles (EVs), bioactive molecules loaded with proteins, glycans, lipids, and nucleic acids, facilitate intracellular signaling and cell-to-cell crosstalk. Circulating EV concentrations rise throughout normal pregnancy; however, preeclampsia is characterized by a further increase in multiple types of EVs and a shift toward a pro-inflammatory, vasoactive cargo. Emerging evidence suggests that in preeclampsia, circulating EVs activate maternal endothelial cells, propagate vascular inflammation, and impair vascular tone and endothelial integrity, contributing to the development of hypertension and excess vasoconstriction. This review briefly introduces fundamental knowledge about EV biogenesis, morphology, and cargo selection, then focuses on current evidence on EV-induced endothelial inflammation and vascular dysfunction in pregnancies with preeclampsia versus uncomplicated pregnancies. Finally, we discuss the therapeutic potential of engineered or stem cell-derived EVs to restore maternal vascular health in preeclampsia.

INTRODUCTION

Preeclampsia is a multifactorial pregnancy-specific syndrome with unclear etiology and limited treatment options. It is diagnosed as new-onset hypertension with or without proteinuria with maternal end-organ damage after 20 weeks of gestation (1). Preeclampsia is associated with a high risk of maternal mortality during pregnancy. Moreover, women with a history of preeclampsia have a 3.6-fold increased risk of heart failure, a 2-fold increased risk of coronary heart disease or stroke (2), a 4-fold increased risk of chronic hypertension (3), and a 2-fold increased risk of death from cardiovascular disease (2, 4). Thus, the history of preeclampsia has a significant impact on maternal cardiovascular health during and after pregnancy.

The timing of preeclampsia diagnosis and symptom severity are significant determinants of the magnitude of short-term and long-term cardiovascular disease risk. As such, early onset preeclampsia (i.e., diagnosis at less than 34 weeks of gestation), poor outcomes, and diagnosis of preeclampsia with severe features have the strongest associations with premature and long-term cardiovascular disease (5).

Vascular inflammation, excess vasoconstriction, reduced vasodilatory capacity, and endothelial dysfunction encompass cardinal features of maternal vascular dysfunction in preeclampsia (6, 7). In some cases, vascular dysfunction develops before pregnancy and contributes to poor remodeling of the uteroplacental vascular network and impaired maternal cardiovascular adaptations to the increasing metabolic demands of pregnancy (8–10). In other cases, dysfunction of the maternal vascular system is a response to systemic inflammation and immune system overactivation due to interactions between circulating pro-inflammatory and vasoactive molecules and the maternal vascular system (11–13).

Extracellular vesicles (EVs) are released by most tissues, packaging and transferring a bioactive cargo that facilitates interorgan and intercellular communication (14, 15). Characteristics of EVs, such as size, concentration, and mechanisms of release, differ in preeclampsia compared to healthy pregnancies (16, 17), suggesting a potential contribution of EVs to disease etiology and pathophysiology. Circulating EVs can influence maternal endothelial function and vascular tone either by engaging vascular cells locally or by modulating systemic signaling pathways. The primary objective of this review is to synthesize and critically evaluate the current literature on the role of EVs in maternal vascular dysfunction during preeclampsia. We begin with an overview of EV biology, highlighting their morphology, cargo composition, and mechanisms of biogenesis and release. We then discuss how EVs contribute to vascular inflammation and dysfunction in non-pregnant states, followed by an in-depth examination of their roles in healthy pregnancy and preeclampsia. Finally, we consider emerging evidence on the potential application of EVs in as therapeutic tools in pregnancies complicated by preeclampsia.

OVERVIEW OF EXTRACELLULAR VESICLES

Morphology, cargo, and packaging

EVs are bioactive molecules that contain a diverse cargo of proteins, glycans, lipids, nucleic acids, and amino acids (reviewed in (18)). This cargo can elicit responses in recipient cells, mediating cell-to-cell communication and regulating intracellular processes such as cell proliferation and angiogenesis (19–31). The most frequently used terminology and differentiation for EVs is based on EV size and includes, but not limited to: exosomes (~30–160 nm (32, 33)), microparticles (also known as microvesicles; ~100–1000 nm (34)) and apoptotic bodies (~1000–5000 nm (35, 36)).

Exosomes form intracellularly through the endosomal route, which is initiated by inward invagination of plasma membrane, and they mature into late endosomes or multivesicular bodies (reviewed here (37–39)). Exosomal biogenesis involves two main pathways: an endosomal sorting complex required for transport (ESCRT)-dependent process and an ESCRT-independent pathway (40). The ESCRT pathway is the primary mechanism of exosome formation and is associated with the activation of four protein complexes: ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III (41). ESCRT-0 activation recognizes and captures cargo proteins, followed by membrane invagination and fission mediated by ESCRT-I to ESCRTIII (reviewed in (40)). The ESCRT-independent pathway involves lipid-, tetraspanin-, and RAB31-driven mechanisms (41–43). After formation, exosomes either fuse with lysosomes, leading to their degradation along with their contents, or fuse with the plasma membrane, releasing their contents into the extracellular space (40, 41, 44).

Microparticles (or microvesicles) form differently from exosomes. Their biogenesis involves outward budding of the plasma membrane, followed by shedding from the cell surface and release into the extracellular space (reviewed in detail here (45)). Microparticles arise from cholesterol-rich lipid rafts, and their shedding is reduced when membrane cholesterol is depleted (46). Microparticles can also form through an actomyosin-based membrane abscission mechanism regulated by ARF6-GTP-dependent activation of phospholipase D (47). Key factors affecting their formation and release include hypoxia, calcium, shear stress, high concentrations of extracellular adenosine triphosphate (ATP), ultraviolet B radiation and inflammatory cytokines and growth factors (48–51). Hypoxia-inducible factors activate RAB22A transcription and increase expression of small GTPase RAB22A, a protein that localizes with budding microparticles (49). In addition, depletion of endoplasmic reticulum calcium stores and blockage of store-operated calcium entry inhibit microparticle biogenesis (50), suggesting dependance on a calcium-calpain dependent pathway. ATP stimulation has also been shown to induce microparticles production, and these effects are mediated by functional P2X7 receptor (48).

Shear stress potentiates microparticle formation through activation of protein kinase C and binding of von Willebrand factor (vWF) to platelet glycoprotein lb, an event that mediates cleavage of actin binding protein by calpains. In addition, shear stress promotes activation of vWF binding to glycoprotein IIb/IIIa that subsequently induces the shedding of microparticles (52). Low shear stress stimulates microparticle release through increased activation of endothelial Rho kinases and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and reorganization of cytoskeleton. On the other hand, physiological atheroprotective high shear stress prevents the release of microparticles by limiting phosphatidylserine exposure, resulting from release of endogenous nitric oxide (NO) and subsequent downregulation of ATP-binding cassette transporter A1 expression (53).

Numerous cytokines related to inflammation, such as interferon gamma (IFN-γ) and interleukin-4 (IL-4), as well as IL-6, IL-13, IL-23, tumor necrosis factor-α (TNF-α) and transforming growth factor beta (TGF-β) have been shown to mediate microparticle release (48), indicating an association between inflammation and EV formation.

Apoptotic bodies are large vesicles, which are formed in the early stages of apoptosis. Early apoptosis is characterized by cell shrinkage and pyknosis, extensive plasma membrane blebbing, and subsequent packaging of cellular fragments into apoptotic bodies (54, 55). Caspase-3 activates Rho-activated serine/threonine kinase 1, and this process regulates actin-myosin filament assembly, cell contractility, and membrane blebbing through phosphorylation of the myosin light chain. Phosphorylation of myosin light chain then stimulates actomyocin contraction that contributes to delamination of the plasma membrane from the cytoskeleton membrane, leading to plasma membrane blebbing (56). Caspases also activate p21-activated kinase and LIM-kinase 1, which also induce cytoskeletal reorganization and membrane blebbing (reviewed in (56)). Once formed, apoptotic bodies are released into the extracellular space and degraded within phagolysosomes (57). The degradation of apoptotic bodies prevents harmful exposure of pro-inflammatory and immunogenic cargo and plays an important role in the resolution of inflammation (58). It is apparent, therefore, that EV formation and degradation are associated with both the induction, propagation, and resolution of inflammation, underlying the complexity of this relationship and its dependence on EV characteristics, type, and cargo.

Extracellular vesicles: endothelial inflammation and vascular effects

Circulating EVs may contribute to the development of inflammation on the vascular endothelium (59–62). Osada-Oka and colleagues demonstrated increased levels of microRNA (miR)-17, a negative regulator of intercellular adhesion molecule-1 (ICAM-1) expression, in serum-derived exosomes from rats with angiotensin II-induced hypertension. Levels of miR-145–5P, miR-221 and miR-222–5P that target plasminogen activator inhibitor-1 (PAI-1) and endothelial nitric oxide synthase (eNOS) were not affected (59). Co-culture of these exosomes with human coronary artery endothelial cells (HCAECs) upregulated ICAM-1 and PAI-1, thus suggesting that exosomes may regulate ICAM-1 expression via miR-dependent mechanisms. These effects were primarily driven by exosomes derived from macrophages (59). Similarly, Tang et al. showed that small (<100 nm) exosomes from monocytes induced adhesion and proinflammatory factors, such as ICAM-1, C-C motif ligand 2 (CCL2), and IL-6 in human umbilical vein endothelial cells (HUVECs) (61). Exosome-induced expression of ICAM-1 and CCL-2 was, at least in part, driven by activation of toll-like receptor 4 (TLR-4) signaling (61), a key pattern-recognition receptor of the innate immune system (63).

These data suggest that the pro-inflammatory effects of exosomes on the vascular endothelium are linked to innate immune system activation. Indeed, monocyte-derived microparticles that are released from cells stimulated with lipopolysaccharide (LPS), a potent activator of the innate immune system, induce phosphorylation of ERK1/2 and activate the nuclear factor-κB (NF-kB) pathway via downregulation of IκB-α and phosphorylation and nuclear translocation of p65 (a subunit of NF-kB). Also, those microparticles increased expression of NF-κB-dependent genes such as ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HUVECs (60), an effect that was mediated by IL-1 receptor activation. This endothelial cell activation was linked to the presence of IL-1β, elevated levels of ICAM-1 and VCAM-1 and components of the inflammasome (i.e., NLRP3, caspase-1 p10 and inflammasome adaptor molecule ASC) in monocyte-derived microparticles (60).

In addition to miRNAs, several other EV cargo components can contribute to the regulation of inflammatory responses. For example, Hirsova et al. demonstrated that incubation of primary hepatocytes with palmitate or lysophosphatidylcholine increased the release of EVs containing TNF-related apoptosis-inducing ligand, which in turn induced pro-inflammatory il-1β and il6 mRNA expression in macrophages (64). EV-associated lipids also play a role in intercellular communication (reviewed in (65, 66) and have been implicated in the development of inflammation (67). Esser et al. showed that exosomes derived from macrophages and dendritic cells contain enzymes involved in leukotrienes biosynthesis, a well-established proinflammatory lipid mediator (67). Together, these findings highlight that EV proteins and lipids, in addition to nucleic acids, can act as potent modulators of endothelial inflammatory responses.

Microparticle interactions with endothelial cells may be potent inducers of vascular disease. For instance, Gomez et al. previously showed that high-fat diet affects circulating levels and adhesion patterns of microparticles derived from neutrophils, platelets, and monocytes (62). Mice that were fed a Western diet for 6 weeks and treated with neutrophil-derived microparticles (~165 ± 7.5 nm) exhibited high deposition of these vesicles at the atheroprone regions of the aorta (62). These data suggest that neutrophil-derived microparticles adhere preferentially to atheroprone sites within arteries in conditions of hypercholesterolemia (62). In in vitro experiments, Gomez and colleagues modelled flow at atheroprone regions and flow at atheroprotected areas using oscillatory and high shear stress, respectively. Neutrophil-derived microparticles adhered more to HCAECs under oscillatory shear stress compared to high shear stress, (62). The oscillatory shear stress-mediated effect was due to high expression of ICAM-1 on the surface of HCAECs, and thus, application of anti-ICAM-1 antibody inhibited its adhesion. Moreover, under oscillatory shear stress conditions, those microparticles enhanced monocyte adhesion to HCAECs and promoted monocyte transendothelial migration toward the chemokine CCL2 on the surface of microparticles, effects that were mediated by CD18, a component of β2 integrins (62).

Neutrophil-derived microparticles can likewise induce inflammatory activation of HCAECs (62). Static (absent or low shear) and oscillatory (dynamic mechanical bursts) shear stress conditions confer distinct effects on the inflammatory response to neutrophil-derived microparticles. Specifically, under static conditions, neutrophil-derived microparticles favor upregulation of cytokines and adhesion molecules (ICAM-1, VCAM-2) and release of monocyte chemoattractant CCL2 and neutrophil chemoattractant CXCL8 from HCAECs, promoting transient recruitment of monocytes and neutrophils. Conversely, under oscillatory shear stress, neutrophil-derived microparticles significantly increased the release of IL-6 and CXCL8 from HCAECs, while their effects on CCL2 was smaller compared to that observed under static conditions. IL-6 enhances endothelial cell activation and leukocyte adhesion, whereas CXCL8 maintains the recruitment of neutrophils, together orchestrating a chronic inflammatory environment that favors the formation of atherosclerotic plaques (62).

The pro-inflammatory effects of neutrophil-derived microparticles have been attributed, at least in part, to their miRNA cargo. miRNA are single-stranded, non-coding RNAs that regulate gene expression post-transcriptionally (17, 68–72). These vesicles contain several miRNAs, including regulators of inflammation such as miR-9, miR-150, miR-155, miR-186, and miR-223. Once internalized, microparticles can lead to an enhanced endothelial expression of miR-155 that is associated with reduced expression of BCL6 (62), a basal and inducible inhibitor of NF-κB (73). These findings suggest that microparticles may contribute to endothelial inflammation through miRNA-mediated mechanisms.

In addition to their pro-inflammatory effects on the vascular endothelium, EVs impact vascular tone and reactivity and these vasoactive actions vary with the EV cellular source. Densmore et al. showed that endothelium-derived microparticles that were released from HUVECs upon stimulation with PAI diminished endothelium-dependent vasodilation in mouse facialis artery and human colon submucosal arterioles (74). These effects were attributed to reduced eNOS phosphorylation and disrupted interaction with heat shock protein 90 (74), leading to impaired eNOS phosphorylation at Ser1179 and reduced NO production. Free-radical scavengers did not alter these effects, excluding the involvement of reactive oxygen species (ROS). These data demonstrated that endothelium-derived microparticles can affect the NOS pathway without invoking oxidative stress in mouse and human microvessels (74). Similarly, Brodsky et al. observed impaired aortic endothelial function after exposure to microparticles from rat renal microvascular endothelial cells (75). In contrast to the study by Densmore et al. (74), these effects were driven by both increased superoxide production and reduced NO production (75). Even though the parent cells were endothelial in both studies, the distinct mechanistic outcomes may be associated with their distinct organ-specific origin (i.e., kidney vs. umbilicus).

In a subsequent study, Good et al. reported that EVs (size range of <100 nm to 1000 nm) from normotensive Wistar-Kyoto rats reduced endothelium-dependent vasodilation in normotensive resistance arteries but had no effect in arteries from spontaneously hypertensive rats (76). Inhibiting NOS with N(ω)-nitro-L-arginine methyl ester (L-NAME) decreased relaxation in control arteries but not in arteries from hypertensive rats, suggesting diminished involvement of the NO pathway in EV-mediated vascular dysfunction in hypertension. The anti-dilatory effects of EVs that were isolated from rats prior to development of hypertension but not after the establishment of disease led to the conclusion that alterations in EV effects on vascular function occurs during and after the development of hypertension. Thus, EVs may contribute to both the pathogenesis and the establishment of the disease. These findings highlight the importance of the disease environment in altering vesicle cargo.

Inflammatory activation of the endothelium alone is adequate to endow EVs with a distinct proinflammatory profile. Hosseinkhani et al. showed that endothelial cells treated with TNF-α released a mixed population of microparticles and exosomes that exhibit immunomodulatory content (77). Mainly, CD9, CD63 and ICAM-1 were enriched in EVs derived from TNF-α-stimulated HUVECs (compared to unstimulated cells). Also, those EVs had high expression of tumor necrosis factor receptor, TNF-α, granulocyte-macrophage colony-stimulating factor (a growth factor that is important for cytokine production (78)), proinflammatory IL-1β, IL-6 and IL-8, anti-inflammatory cytokines IL-10 and IL-13, ICAM-, and chemokines CXCL-10, CCL-2 and CCL-5 (77). These vesicles mediated immune cell adhesion to HUVECs and promoted transendothelial migration, thereby confirming their role in the transfer of immune inducers across cells. Also, EVs upregulated ICAM-1, IL-6, IL-8, CCL-2, CCL-4, CCL-5 and IL-6 receptors in recipient HUVEC cells, thereby confirming that EVs derived from inflammatory environment can on their own trigger endothelial inflammation.

Complementary work by Chang et al. demonstrated that endothelial cells exposed to TNF-α or oscillatory shear stress became atherogenic (validated by the expressions of ICAM-1 and VCAM-1) and triggered inflammatory responses in macrophages manifested by upregulation of proinflammatory genes such as IL-6, IL-1α, IL-1β, TNF-α, and inducible nitric oxide synthase (iNOS) (79). This response was associated with miR-92a, which was enriched within EVs (79). Increased levels of pri-mi-92a were found in endothelial cells but not in macrophages, while mature miR-92a levels were increased in both cell types. These findings suggested that biogenesis of miR-92a was likely absent in macrophages, and its transport from the atherogenic endothelium to macrophages was facilitated via an EV route. Functionally, EVs increased macrophage expression of TNF-α and IL-6, suppressed macrophage levels of anti-inflammatory CD163, and reduced Krüppel-like factor 4 (KLF4) (79), a critical regulator of macrophage polarization (M1/M2) that suppresses proinflammatory genes in M2 macrophages and inhibits NF-κB transcriptional activity (80). Further analysis revealed that EVs from cytokine-pretreated endothelial cells enhanced macrophage secretion of IL-6, CXCL1, and complement component 5a and IL-1α (79). Together, these findings indicate that endothelial cells in atheroprone environments can package EVs with miR-92a and other inflammatory mediators, thereby indirectly propagating inflammation.

These studies illustrate distinct mechanistic pathways whereby EVs induce vascular dysfunction. The complexity of these molecular interactions reflects EV size, cellular origin, cargo composition, and disease environment. We still know little about how EV cargo changes in different disease states. Clarifying these cargo-specific mechanisms will be critical for developing therapies to counter EV-mediated vascular inflammation and dysfunction.

EXTRACELLULAR VESICLES IN HEALTHY PREGNANCY AND PREECLAMPSIA

Changes in EV concentration and cargo

Circulating EVs are essential for normal pregnancy progression due to their involvement in embryo implantation and development (81–84), immunomodulation (85, 86), maternal-fetal communication (87, 88), regulation of maternal vascular reactivity (89–97), and remodeling (98).

The concentration of circulating EVs is at least 3-fold greater in pregnant compared to the non-pregnant state and increases with the progression of gestation (99). Specifically, the concentrations of both total and placental alkaline phosphatase-positive (PLAP⁺) EVs rise at later stages of pregnancy, while in the first trimester, the concentrations of placenta-derived EVs are very low (100, 101). In addition, the EV cargo changes during healthy pregnancy, supporting immunosuppression. For example, exosome-like EVs (~145 nm) at 12 and 28 weeks of gestation carry higher amounts of the immunosuppressive cytokines IL-10 and transforming growth factor β1 compared to EVs from non-pregnant controls (102). These EVs are taken up by B cells and natural killer (NK) cells (102). Compared with EVs from non-pregnant controls, EVs from pregnant women enhanced caspase-3 activity in a cytotoxic subset of NK cells (102) known as CD56^dim NK cells (103), promoting apoptosis. Because NK cell functionality is vital for maternal immune system adaptation, these findings suggest that EVs contribute to the immunosuppressive state of pregnancy via caspase-3-dependent apoptosis of cytotoxic CD56^dim NK cells (102).

Circulating EV concentrations fluctuate in response to physiological stress, and such changes may reveal important differences between normal and preeclamptic pregnancies. Exercise provides a unique model to probe these responses. Abolbaghaei et al. found that moderate-intensity exercise reduces circulating levels of large EVs in non-pregnant participants, whereas these changes are absent in pregnant women (104). Conversely, focusing on small EVs (~ 10–120 nm), Mohammad et al. observed higher post-exercise levels of small EVs in pregnant women compared to non-pregnant controls (105). Although the functional importance of exercise-induced EV adaptations during pregnancy remains to be determined, exercise-derived EVs can exert antioxidant and cardioprotective effects in other contexts. For example, EVs (~70–200 nm) released after a single bout of endurance exercise increased glutathione peroxidase and glucose 6-phosphate dehydrogenase activities, decreased ROS and lipid peroxidation, and improved cell viability in young healthy men (106). Similarly, in mice, exosomes released after exercise increased endothelial cell survival under high glucose and hypoxic conditions via upregulation of miR-126 (107). These findings raise the possibility that pregnancy-related differences in exercise-induced EV release may influence maternal vascular resilience, and that such mechanisms could be dysregulated in preeclampsia.

Compared to normotensive pregnancies, pregnancies with preeclampsia are characterized by greater abundance of multilayer vesicles containing small vesicles, with a prevalent content of placenta-derived EVs (PLAP⁺-EVs (108)). EVs from pregnancies with preeclampsia are of similar size to EVs from normotensive pregnancies (99), while only one study has reported bigger diameter of exosomes in preeclampsia (107.5 nm) compared to healthy pregnancy (84.9 nm) (71). In contrast to similarities in size, the concentration of both small and large EV fractions and their protein content are elevated in preeclampsia (99, 108). Also, in preeclampsia, the biggest increase in EV concentration occurs in the first trimester, while dropping on the second and third trimesters (99). Nevertheless, EV concentrations remain higher in pregnancies with preeclampsia compared to healthy pregnancies throughout gestation (99).

Preeclamptic sera causes greater release of micro- and nano-EVs from placental explants compared to healthy controls (109), indicating dissimilar mechanisms of placental release of EVs in preeclampsia and healthy pregnancy. In addition, there are cargo differences in EVs from preeclamptic and healthy pregnancies. Compared to small EVs from healthy pregnancies, those from preeclamptic pregnancies (size < 200 nm) contain higher levels of miR-517c-3p and miR-519d-3p, and incubation of macrophages with these EVs increases macrophage miR-517c-3p levels (108). Moreover, Nejad et al. reported increased circulating cell-free levels of miR-517c-3p and miR-210–3p in preeclampsia (110); these miRNAs were linked to reduced cell proliferation and impaired trophoblast invasion (111, 112).

In addition to distinct cargo content, preeclamptic EVs display differential downstream effects when compared to EVs from normal pregnancies. Liu et al. reported that trophoblast-derived EVs from women with preeclampsia upregulated gene markers (i.e., IL-1β, TNF-α, and IL-6) in proinflammatory M1 macrophages and downregulated the anti-inflammatory endocytic receptor CD163 (113) compared with EVs from normal pregnancies (114). Interestingly, this effect was attributed to alterations in 37 lipid species in preeclamptic EVs that modulated classical inflammatory pathways in these macrophages (114).

Placenta-derived EVs have been proposed as biomarkers of preeclampsia. Jiang et al. reported that first-trimester serum levels of CD10-PLAP⁺ and CD63-PLAP⁺ small EVs were higher in women who later developed early-onset preeclampsia compared to those with normal pregnancies (115). These findings support that preeclampsia is associated with elevated EV levels and altered cargo, which may contribute to differential effects on the maternal immune and cardiovascular systems (Figure 1). Thus, EVs may serve as valuable early biomarkers for identifying women at risk for preeclampsia.

Figure 1.

Circulating extracellular vesicles mediate maternal vascular dysfunction in preeclampsia

Preeclampsia-associated stressors, such as hypoxia, oxidative stress, and inflammation, act on placental villi, triggering the release of extracellular vesicles (EVs) enriched with proinflammatory and vasoactive cargo (i.e., miRNA, DNA, cytokines). These EVs enter the maternal circulation, where they bind to and are internalized by vascular endothelial cells, leading to increased adhesion molecule expression, reactive oxygen species (ROS) production and decreased nitric oxide (NO) bioavailability. Collectively, these effects promote endothelial inflammation, disrupt endothelial integrity, and impair maternal vascular function. EVs from the maternal vascular endothelium, other maternal organs, and circulating cells (i.e., immune cells; not shown in schematic) may also contribute to the circulating EV pool in preeclampsia. Additionally, EVs can potentiate the interactions between oxidized low-density lipoprotein (oxLDL) and lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), as well as angiotensin II (Ang II) and its receptor (AT1-R), further amplifying EV release, reducing vasodilation, and enhancing vasoconstriction. HMBG1, High Mobility Group Box 1; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1. Created in BioRender. Goulopoulou, S. (2025) https://BioRender.com/ ewfykfy.

An interplay between placental- and maternal-derived EVs, along with their molecular cargo, has been implicated in the initiation and progression of the pathogenesis of preeclampsia (116). Elevated concentrations of endothelium-derived EVs have been linked to the development of systemic pro-inflammatory and pro-coagulant states in preeclampsia. Similarly, erythrocyte-derived EVs, which are reported to be elevated in preeclampsia (117), exert potent pro-coagulant effects (reviewed in (118)) and may contribute to thrombosis. In addition, syncytiotrophoblast-derived EVs have been shown to enhance platelet activation (119), underscoring the systemic vascular consequences of placental EV release. Collectively, these findings demonstrate that multiple cellular sources of EVs contribute to the complex vascular pathology of preeclampsia.

Extracellular vesicles and endothelial cell inflammation

Previous studies in preeclampsia have reported an upregulation of proinflammatory cytokines: maternal plasma and choriodecidual blood IFN-γ, plasma IL-8 and IL-6 and C-reactive protein (CRP), and villous TNF-α, indicating systemic activation of the maternal immune system (120–124). Conversely, preeclampsia is associated with diminished anti-inflammatory signaling. Maternal IL-4 is reduced in plasma, while IL-10 is decreased in villous trophoblast cells, serum and plasma (121–123). Blood mononuclear cells from preeclamptic pregnancies secrete less IL-5, and maternal serum contains high levels of IL-17 (125, 126). Dong et al. further reported higher placental IL-2/IL-10 and TNF-α/IL-10 ratios in preeclamptic compared to uncomplicated pregnancies (127), indicating a T helper 1 (Th1)-dominant, proinflammatory shift in the maternal immune milieu, which characterizes preeclampsia. This systemic Th1 bias is mirrored, and is possibly amplified, by changes in circulating EVs.

Indeed, EV-mediated endothelial inflammation has been documented in pregnancies with preeclampsia. For instance, EVs from first-trimester placental explants that were exposed to preeclamptic serum upregulated ICAM-1 expression in endothelial cells and increased levels of the proinflammatory alarmin high-mobility group box 1 (109). Complementing these results, Ramos et al. demonstrated that microvascular endothelial cells exposed to sera supplemented with preeclamptic EVs exhibited greater ROS production, as well as increased expression of vWF and VCAM-1 compared to non-supplemented sera (128).

In non-pregnant states, the pro-inflammatory properties of monocyte-derived exosomes have been attributed, in part, to activation of TLR-4 signaling. This may also be relevant to preeclampsia, since TLR-4 has been involved in the induction of sterile inflammation and the development of pregnancy complications. Specifically, Mulla et al. reported that the TLR-4/MyD88 pathway mediates placental inflammatory responses in women with antiphospholipid antibodies, a population with high risk for pregnancy loss and late gestational complications, including preeclampsia (129). These effects were accompanied by increased secretion of IL-8, MCP-1, CXCL1α, and IL-1β (129). Furthermore, the same group in their later study demonstrated that TLR-4 is involved in the production of uric acid in human trophoblast that activates the Nalp3/ASC inflammasome and leads to IL-1β processing and secretion (130). Alterations in abundance and function of TLR-4 are associated with the development of preeclampsia. For instance, single nucleotide polymorphism in TLR-4 aggregate with early-onset preeclampsia (131). Moreover, Litang et al. have shown high serum levels of TLR-4 and NF-κB in patients with preeclampsia compared to normal pregnant group, thus suggesting it as a potential biomarker for predicting preeclampsia (132). Those results were also shown in monocytes and neutrophils from women with preeclampsia (133, 134). As such, by activating TLR-4, EVs potentially may induce endothelial inflammation in preeclampsia, however, further research is needed to confirm this hypothesis.

Collectively, these findings demonstrate that EVs originating, or conditioned by a preeclamptic environment drive a prooxidant, proinflammatory state in endothelial cells, with potential consequences for maternal vascular function and health.

Extracellular vesicles and vascular function

EVs released from normal and preeclamptic placentas have divergent effects on vascular tone, which may arise from engagement of a common molecular pathway that becomes differentially regulated in preeclampsia, leading to distinct vascular outcomes. Syncytiotrophoblast-derived EVs (STBEVs) from term, uncomplicated pregnancies reduce endothelium-dependent dilation in arteries from human subcutaneous adipose tissue and this effect has been attributed to endothelial disruption by deported microvilli (135). Spaans et al. confirmed a similar impairment of endothelium-dependent vasodilation in late-pregnant mouse uterine arteries exposed to STBEVs isolated from normal human placenta (136). Authors linked the STBEV-induced impaired vasodilation to activation of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and a reduced contribution of endothelial NO. Neutralizing LOX-1 with a blocking antibody preserved vasodilation and increased eNOS expression (136).

Independent evidence implicates LOX-1 in the pathophysiology of preeclampsia (137, 138). Maternal plasma from preeclamptic women upregulates LOX-1 expression and enhances uptake of oxidized, small-dense low-density lipoprotein (LDL) in HUVECs (138), effects reproduced in the reduced uteroplacental perfusion pressure (RUPP) rat model of preeclampsia (139). Plasma from these patients also elevated NADPH-oxidase activity and increased levels of superoxide and peroxynitrite in HUVECs, highlighting a redox-dependent component (138). It is noteworthy that oxidized LDL, which is increased in pregnancies with preeclampsia (140), can trigger the release of EVs, potentially perpetuating a vicious cycle (141). Together, these findings indicate that STBEV-driven vascular dysfunction in normal pregnancy converges on the LOX-1/eNOS pathway, which is impaired once pregnancy is complicated by preeclampsia and an environment of increased concentrations of small lipoprotein subfractions.

A previous study by Kao et al. reported vascular dysfunction caused by circulating factors in the plasma of patients with preeclampsia (142). Specifically, incubation of uterine arteries from pregnant rats with preeclamptic plasma resulted in increased superoxide production, impaired endothelial function, increased eNOS expression, and decreased iNOS expression (142). These effects were vascular bed-specific, as no functional alterations were observed in the mesenteric arteries, which represents the systemic circulation.

STEVBs, including microvesicles (323.2 ± 7.1 nm) and exosomes (89.3 ± 9.7 nm), from normal pregnancies express eNOS (but not iNOS) and can produce NO (143). In contrast, syncytiotrophoblast-derived exosomes from preeclamptic pregnancies exhibit reduced NOS activity, suggesting a contribution to the overall decrease in NO bioavailability (143). Supporting this, Murugesan et al. demonstrated that 48-hour incubation of mouse thoracic aortas with EVs derived from patients with preeclampsia reduced total and phosphorylated eNOS levels and impaired endothelium-dependent vasodilation compared to EVs from normotensive pregnant women (90).

Similarly, Villalobos-Labra et al. showed that STEVBs from pregnancies with preeclampsia reduced the NO-mediated component of vasodilation in mesenteric arteries from late pregnant rats (89). Notably, inhibition of LOX-1 restored NO bioavailability, while treatment with superoxide dismutase or the NADPH-oxidase inhibitor apocynin rescued endothelium-dependent vasodilation (89). These findings further support the involvement of the LOX-1/NO/ROS signaling pathway in EV-induced reduction in vasodilatory capacity in preeclampsia.

While dysregulation of the LOX-1/eNOS pathway is a common theme, EVs also affect other vasoactive signaling pathways, sometimes enhancing and other times dampening contractile responses. Erlandsson et al. showed that preeclamptic STBEVs (50–450 nm) increased angiotensin II (Ang II)-mediated contraction and induced structural damage on human subcutaneous arteries via (AT1-R) (93). These effects were prevented by blocking vesicle uptake with chlorpromazine or neutralizing LOX-1, implicating LOX-1 and clathrin-mediated endocytosis in STBEV-AT1R interactions and their vascular effects (93).

In contrast, Tesse et al. observed that preeclamptic microparticles reduced vascular contraction to high potassium, phenylephrine (PE) and serotonin (5-HT) in late pregnant mouse aortas. This effect was accompanied by ~2-fold increase in NO production, elevated prostaglandin E₂, and increased 8-isoprostane levels (144). Similarly, studies by Meziani et al. and Boisrame-Helms et al. reported reduced PE- and 5HT-induced vasoconstriction in human omental arteries and mouse aorta in response to EVs from patients with preeclampsia. These effects were associated with elevated iNOS expression, enhanced synthesis of 8-isoprostane and NF-κB p65 expression, increased oxidative stress (145, 146) and nitrotyrosilation, and decreased production of thromboxane A₂ (146) and upregulation of cyclooxygenase-2 (145).

Supporting this multifaceted dysfunction, Sandoval et al. demonstrated that EVs from patients with preeclampsia downregulated the tight junction protein claudin 5 (CLDN5) in cultured endothelial cells. In vivo, RUPP dams exhibited posterior blood-brain-barrier disruption attributable to CLDN5 loss (147), likely mediated by CLDN5-targeting microRNAs (miRNAs) present within the vesicles. Collectively, these studies illustrate that EV-mediated vascular dysfunction in preeclampsia varies with vessel type, stimulus, and EV subtype.

Beyond vessel and stimulus-specific influences, the clinical subtype of preeclampsia further determines how circulating EVs affect the maternal vasculature. The time of diagnosis (early-onset vs. late-onset) is a significant determinant of maternal and fetal outcomes. Early-onset preeclampsia carries the greatest severity and worst perinatal outcomes, including fetal growth restriction, hemolysis, neurological complications, and severe cardiorespiratory, hematological, and fetoplacental complications (148). EVs isolated from placentas of early- vs. late-onset preeclamptic pregnancies exhibit distinct vasoactive profiles. Specifically, mesenteric resistance arteries from mice injected with EVs from early-onset preeclampsia displayed increased contractile responses to PE, Ang II, and endothelin-1 (ET-1), and reduced vasodilatory responses to acetylcholine (ACh) compared to arteries from mice treated with EVs from normotensive pregnancies (94). In contrast, EVs from late-onset preeclampsia elicited less pronounced pro-contractile effects. These functional differences mirror the hemodynamic profiles of the two preeclampsia subtypes. Early-onset preeclampsia is characterized by higher systemic vascular resistance and reduced cardiac output (10, 149), whereas late onset preeclampsia presents with lower systemic vascular resistance and increased cardiac output (10, 150). Placenta-derived EVs may therefore contribute to these divergent hemodynamic and vascular phenotypes of early and late-onset preeclampsia.

Although strong evidence links EVs to reduced vasodilation, impaired vasoconstriction, and barrier disruption in preeclampsia, direct mechanistic connections between EV-induced endothelial inflammation and overt vascular dysfunction remain to be elucidated.

EXTRACELLULAR VESICLES AS A POTENTIAL THERAPEUTIC INTERVENTION IN COMPLICATED PREGNANCIES

Although EVs contribute to the development and progression of vascular dysfunction, they can also serve as therapeutic agents in preeclampsia. For instance, preeclampsia has been associated with lower levels of transcription factor cellular promoter 2 (TFCP2) (151) which has diverse functions, including embryonic development (152). Yang et al. showed that EVs derived from human umbilical cord mesenchymal stem cells (HUCMSC-EVs, 103 ± 30 nm) loaded with TFCP2, decreased apoptosis, activated the Wnt/β-catenin pathway in extravillous trophoblast cells, enhancing their proliferation, migration, and invasion (151). These findings indicated that TFCP2-enriched HUCMC-EVs could target preeclampsia-associated trophoblast deficits.

Building on this proof-of-concept, recent preclinical studies have tested HUCMSC-EVs in animal models. Yu and colleagues reported that HUCMSC-EV (100–200 nm) promoted cell proliferation and reduced the expression of inflammatory markers (ICAM-1, VCAM-1, IL-6) and endothelial cell injury markers (ET-1, tissue-type plasminogen activator and soluble FMS-like tyrosine kinase-1 (sFlt-1) in TNF-α or LPS-treated HUVECs, a cellular model of inflammation-induced endothelia dysfunction, a key contributor to preeclampsia (95). Moreover, administration of HUCMSC-EVs in a mouse model of preeclampsia reduced maternal hypertension and proteinuria, alleviated renal injury, and promoted placental vascularization (95).These benefits were correlated with increased arginine levels, higher NO content and NO synthase activity, and activation of the arginine metabolic pathway that indicates improved endothelial function. Endothelium-dependent vasodilation was not tested in this study (95).

Similarly, Xiong et al. found that exosomes from HUCMSCs (with diameters of 30–100  nm) decreased blood pressure and urinary protein concentrations, increased litter size, preserved placental architecture and organelle function, increased microvascular density and serum levels of vascular endothelial growth factor, and reduced apoptosis and circulating levels of sFlt-1 in an L-NAME-induced rat model of preeclampsia (153). In this study, placentas from rats with preeclampsia-like phenotype exhibited marked ultrastructural abnormalities, including swollen mitochondria within the syncytiotrophoblast, increased mitochondrial vacuoles, and dilated endoplasmic reticulum. These changes were ameliorated following treatment with HUCMSC-derived exosomes. It is important to note, however, that these cellular observations were based on a small number of animals and were not validated using complimentary techniques. Collectively, while these findings suggest that HUCMSC-EVs may help preserve mitochondrial and endoplasmic reticulum integrity and represent promising candidates for preeclampsia therapy, they also underscore the need for confirmation across multiple models and methods.

Investigations are now leveraging vesicular cargo, particularly dysregulated miRNAs, to refine EV-based therapies. Cui and colleagues showed that EV-encapsulated miR-101, which is downregulated in preeclampsia, suppressed its targets, bromodomain-containing protein 4 and C-X-C motif chemokine ligand 11 (CXCL11), promoted trophoblast proliferation and migration via inhibition of the NF-κB/CXCL11 axis, and reduced blood pressure and proteinuria in vivo (154).

In parallel, Yang et al. demonstrated that restoring miR-18b, which is markedly reduced in placental tissue and HUCMSC-EVs from patients with preeclampsia enhanced trophoblast migration and proliferation in vitro, potentially through repression of Notch2 and downstream inhibition of the TIM3/mTORC1 signaling pathway (155). miR-18b-enriched HUCMSC‐EVs also reduced blood pressure, proteinuria, and placental apoptosis in L-NAME-treated rats with preeclampsia-like symptoms (155).

Collectively, these studies demonstrate that EV-based delivery, whether of intact vesicles or vesicles loaded with specific miRNAs, such as miR-101 and miR-18b, restores trophoblast function, reduces endothelial inflammation, and ameliorates preeclampsia-like symptoms in in vitro and in vivo preclinical studies. However, there are several safety concerns and limitations regarding the implementation of MSC-EV-based delivery into clinical practice. MSCs are well known for their reparative ability (156–158) and, as mentioned above, are proposed as a potential therapeutic intervention in complicated pregnancies in pre-clinical setting. However, safety concerns arise in its implementation in practice. For instance, cells can be transformed in the cell culture setting and express different morphological, physiological and functional properties (reviewed in (158)). Despite routine screening for certain types of viruses, MSCs may contain genetic material of other types of viruses and, in this way, induce an immune response. A number of studies show unpredictable side effects (159, 160) or no beneficial effects upon implementation of MSCs therapies (161, 162). Regarding side effects, Kuriyan et al. reported a case study where patients lost their vision after intravitreal injections of autologous adipose tissue-derived “stem cells” (159). Another study by Saraf et al. reported retinal detachment after bilateral intravitreal injections of “stem cells” in a patient with exudative macular degeneration (160). These studies raise concern about clinical significance of MSC therapies. Moll et al. raised concern regarding the implementation of MSC therapies in COVID-19 patients (163). Mainly, MSCs products express pro-coagulant tissue products, and in combination with COVID-19-mediated hypercoagulable state, those types of therapies may put patients at risk for the development of intravascular coagulation and other accompanying health issues. As such, the authors emphasize the importance of precise characterization of products with a robust manufacturing procedure. This is in line with work by Takakura et al., which raised the urgency for establishment and characterization of cell banks (164). This step is essential to confirm the identity of each cell type, and to test for the presence of specific marker molecules and expression of surface markers, as it is essential to maintain reproducibility and effectiveness of EVs production.

OPPORTUNITIES, METHODOLOGICAL CONSIDERATIONS, AND PERSPECTIVES

Circulating EV concentrations rise with advancing gestational age in healthy pregnancies and increase even further in preeclampsia. Overall, EVs released during normal pregnancy have anti-inflammatory effects, whereas EVs from pregnancies with preeclampsia are enriched with proinflammatory, vasoconstrictive, and oxidative mediators that facilitate endothelial dysfunction, vascular inflammation, and aberrant trophoblast function.

High-resolution characterization of EV cargo is highly promising in the development of new therapeutics for preeclampsia. Molecular “fingerprinting” can offer a minimally invasive readout of placental health and yield prognostic markers of evolving maternal cardiovascular dysfunction. A few studies have focused on the regulatory role of EV-contained miRNAs; yet other nucleic acids, proteins, lipids, and metabolites could also contribute to preeclampsia pathogenesis and warrant further investigation. In a previous study, we demonstrated that vesicle-encapsulated cell-free mitochondrial DNA, a pro-inflammatory damage-associated molecular pattern, is approximately 400-fold more abundant than the vesicle-free fraction in healthy pregnancy and greater than 1,000-fold more abundant in preeclampsia (165). Furthermore, gestational hypoxia selectively increased mitochondrial DNA loading into exosome-like EVs without altering vesicle size (166). Such cargo-specific signatures could enable early risk stratification and inspire cargo-editing strategies to restore maternal endothelial function.

To fully harness these opportunities, however, several methodological limitations must be addressed. First, there is a lack of uniform EV characterization across studies, with heterogeneity in isolation and analytical methods (see Table 1), which complicates cross-study comparisons. International guidelines on EV nomenclature and standardization provide an important framework that should be consistently implemented (167). Second, species differences remain a challenge: while bioassays using human biological fluids (e.g., plasma) as the trigger and animal tissues as the target provide mechanistic insights, the compatibility of EV effects across species—particularly in immune responses—requires careful interpretation. Third, some studies stimulate healthy tissues (e.g., vessels from healthy pregnant animals) with plasma EVs from patients with preeclampsia, a strategy that, although highly informative, may not fully recapitulate human pathophysiology.

Table 1.

Methods of characterization and tissue sources for extracellular vesicles.

Citation	NTA	WB	TEM	FC	Tissue source
Li et al., 2023 (101)	✓	CD63, TSG101			Plasma
Nardi et al., 2016 (102)	✓	Hsp70, ICAM-I, CD63, TSG101, CD9, CD81 Negative control: CYC1			Serum
Mohammad et al., 2021 (105)	✓	TSG-101, flottilin-1; Negative control: calnexin	✓		Plasma
Lisi et al., 2023 (106)*	✓	ALIX, TSG101, Syntenin-1, APO A1	✓		Plasma
Ma et al., 2018 (107)	✓	CD63, TSG101, CD34, VEGFR2 Negative control: Isotype-matched (IgG) nonspecific antibodies			Plasma
Winter et al., (108)**	✓	ALIX, TSG 101, CD63, PLAP, GAPDH,	✓	✓	Plasma
Li et al., 2020 (71)	✓	CD63, CD9, TSG 101, PLAP	✓	✓	Plasma
Xiao et al., 2017 (109)	✓				Placenta
Liu et al., 2022 (114)	✓	CD9, TG101, PLAP	✓	✓	Placenta
Ramos et al., 2024 (128)	✓		✓	✓	Serum
Spaans et al., 2017 (136)	✓			✓	Placenta
Maaninka et al., 2023 (141)	✓	CD41, CD63, CD9, TSG101 Negative control: calnexin	✓	✓	Platelets
Motta-Mejia et al., 2017 (143)	✓	PLAP, ALIX, syntenin, CD9		✓	Placenta
Murugesan et al., 2022 (90)	✓	CD63, flotillin 1, GM130, APO A1	✓		Plasma
Villalobos-Labra et al., 2023 (89)	✓	PLAP, TSG101 Negative control: CYC1		✓	Placenta
Erlandsson et al., 2023 (93)	✓	PLAP			Placenta
Tesse et al., 2007 (144)		CD11, CD31			Plasma
Boisrame-Helms et al., 2015 (145) ***					Plasma
Sandoval et al., 2025 (147)	✓	HSP70, CD81, CD63, Alix, TSG101, PLAP	✓		Placenta
Lau et al., 2024 (94) bioRxiv.	✓	CD63, cytokeratin 7, vimentin Negative control: calnexin	✓		Placenta

EVs, extracellular vesicles; NTA, nanoparticle tracking analysis; WB, western blotting; TEM, transmission electron microscopy; FC, flow cytometry; Hsp70, heat Shock Protein 70; ICAM-1, intercellular adhesion molecule 1; CYC1, mitochondrial protein cytochrome C; VE-Cadherin, vascular endothelial-cadherin; ALIX, ALG-2-interacting protein X; TSG101, Tumor Susceptibility Gene 101; APOA1, Apolipoprotein A1; VEGFR2, vascular endothelial growth factor receptor 2; PLAP, placental alkaline phosphatase.

Characterization of EVs was also performed with

^(*)surface plasmon resonance imaging analysis

^(**)micro bicinchoninic acid assay

^(***)prothrombinase assay and a covalently coated streptavidin multiwell plate.

Fourth, the methodology of EV extraction and preparation can also impact their biological effects and represent a major limitation in data assessment and interpretation. For instance, Tannetta et al. evaluated differences in preparations of STBVs (i.e., placental perfusion vs. mechanical disruption) and their biological characteristics (168). They reported that PLAP⁺ mean fluorescence intensity was lower in mechanically prepared STBVs compared to placental perfusion preparations (168). Endoglin (CD105), a protein highly abundant on endothelial cells and placental syncytiotrophoblast and a prominent marker of preeclampsia (169–171), was not altered in mechanically prepared STBVs, whereas the perfusion method revealed decreased levels in preeclampsia (168). The authors speculated that placental perfusion more closely mimics in vivo conditions, as it maintains the integrity of the syncytiotrophoblast layer, whereas mechanical disruption introduces damage of the villous tissue. Thus, methodological choices in EV preparation should be carefully considered in experimental design and interpretation of results.

An additional consideration for therapeutic translation is whether EVs offer advantages over other drug delivery systems, such as liposomes, polymeric nanoparticles, and conventional intravenous infusion of drugs, miRNAs, or other bioactive molecules. EVs provide a natural delivery platform with the ability to protect their cargo from enzymatic degradation, extend circulation time, and facilitate uptake via receptor-mediated endocytosis. Compared to conventional approaches, EVs exhibit lower immunogenicity and toxicity while mimicking natural cell-to-cell communication. The potential use of patient-derived EVs further minimizes immunogenicity and reduces off-target toxicity (172). Nonetheless, challenges remain in controlling EV biodistribution, ensuring scalable production, and standardizing cargo loading methods. Importantly, the biodistribution of EVs to the placenta and fetus must also be taken into consideration. Addressing these issues will be critical to determine whether EV-based delivery truly surpasses traditional systemic administration in efficacy and safety for preeclampsia therapies.

Future work should focus on combining longitudinal clinical cohorts with preclinical studies that a) identify the tissue and cellular sources of EVs in preeclampsia, b) map temporal changes in EV cargo, c) test bioengineered or stem-cell-derived therapeutic EVs in preclinical models of preeclampsia. Transforming EVs from pathophysiological transporters into therapeutic tools could lead to biomedical applications of clinical-grade EVs as new therapeutic tools for preeclampsia, improving pregnancy outcomes and reducing maternal and offspring cardiovascular risk later in life.

ABBREVIATIONS

ACh

Acetylcholine

Ang II

Angiotensin II

AT1-

Angiotensin II receptor type 1

ATP

Adenosine triphosphate

CCL2

C-C motif ligand 2

CRP

C-reactive protein

CLDN5

Claudin 5

CXCL11

C-X-C motif chemokine ligand 11

eNOS

Endothelial nitric oxide synthase

ESCRT

Endosomal sorting complex required for transport

ET-1

Endothelin-1

ERK1/2

Extracellular signal-regulated kinases 1 and 2

EVs

Extracellular vesicles

HCAECs

Human coronary artery endothelial cells

HUCMSC(s)

Human umbilical cord mesenchymal stem cell(s)

HUVECs

Human Umbilical Vein Endothelial Cells

iNOS

Inducible nitric oxide synthase

ICAM-1

Intercellular adhesion molecule-1

IFN-γ

Interferon gamma

IL

Interleukin

KLF4

Krüppel-like factor 4

LOX-1

Lectin-like oxidized low-density lipoprotein receptor-1

LDL

Low-density lipoprotein

LPS

Lipopolysaccharide

miRNA

MicroRNA

NK

Natural Killer

NO

Nitric oxide

L-NAME

N(ω)-nitro-L-arginine methyl ester

NF-kB

Nuclear factor-κB

PE

Phenylephrine

PLAP

Placental alkaline phosphatase

PAI-1

Plasminogen activator inhibitor-1

ROS

Reactive oxygen species

RUPP

Reduced uteroplacental perfusion pressure

sFlt-1

Soluble fms-like tyrosine kinase-1

STBEVs

Syncytiotrophoblast extracellular vesicles

Th1

T helper 1

TFCP2

Transcription factor CP2

TGF-β

Transforming growth factor beta

VCAM-1

Vascular cell adhesion molecule-1

vWF

von Willebrand factor