PLAC-S-19–00704 / Placental Small Extracellular Vesicles: Current Questions and Investigative Opportunities

Abstract

The discovery of regulated trafficking of extracellular vesicles (EVs) has added a new dimension to our understanding of local and distant communication among cells and tissues. Notwithstanding the expanded landscape of EV subtypes, the majority of research in the field centers on small and large EVs that are commonly termed exosomes, microvesicles and apoptotic cell-derived vesicles. In the context of pregnancy, EV-based communication has a special role in the crosstalk among the placenta, maternal and fetal compartments, with most studies focusing on trophoblastic EVs and their effect on other placental cell types, endothelial cells, and distant tissues. Many unanswered questions in the field of EV biology center on the mechanisms of vesicle biogenesis, loading of cargo molecules, EV release and trafficking, the interaction of EVs with target cells and the endocytic pathways underlying their uptake, and the intracellular processing of EVs inside target cells. These questions are directly relevant to EV-based placental-maternal-fetal communication and have unique implications in the context of interaction between two organisms. Despite rapid progress in the field, the number of speculative, unsubstantiated assumptions about placental EVs is concerning. Here we attempt to delineate existing knowledge in the field, focusing primarily on placental small EVs (exosomes). We define central questions that require investigative attention in order to advance the field.

Introduction

Placental trophoblasts exquisitely regulate gas exchange, supply of nutrients, removal of waste products, hormonal support, and immunological defense, all crucial for the development and growth of the eutherian fetus. These functions are orchestrated through communication of molecular cues that are essential for pregnancy health. Until recently, the repertoire of regulatory signals was comprised of hormones (such as hCG, placental lactogens, and steroids) or growth factors (such as epidermal growth factor or and placental growth factor) [1]. The recent identification of a spectrum of extracellular vesicles (EVs), their presence in virtually all biological fluids, and their trafficking among diverse cell types [2] has offered a new dimension of signal communications, and established these EVs as a means of local or distant communication among cells and tissues.

With the recent accelerated research on EV biology, it should be noted that the concept of extracellular vesicles was introduced in the 1860’s by Charles Darwin, who proposed that every cell in the body can produce “gemmules,” which are particles of minute size that contain diverse molecules and that are communicated to other bodily cell types [3,4]. Directly relevant to pregnancy, Georg Schmorl in 1893 detailed the first evidence of placental-maternal particle trafficking in the form of fetal cells, syncytialized fragments and syncytial knots that were found in the lungs of pregnant women [5]. Exosomes were initially considered a means to remove waste cargo (“cellular debris”) from reticulocytes during their differentiation into red blood cells [6,7]. This has been suggested again, but not fully pursued, in the context of the high prevalence of tRNAs and vault RNAs within EVs [7]. Considering EVs’ ability to exploit the cell’s innate exocytosis and endocytosis pathways for transferring cellular cargo to other cells, tissues, and even organisms, they were termed “Trojan horses” [8].

The EV sphere is made of a various non-replicating cellular phospholipid membranes, and naturally forms a globular structure in aqueous solutions, optimized to minimize the water-lipid interface. EV cargos, including nucleic acids, proteins, carbohydrates, lipids, and other metabolites, are highly diverse, and depend on their cell lineage, stage of differentiation, activation, neoplastic transformation, injury or infection of the cell of origin. Similarly, their sizes and other biophysical characteristics are heterogeneous and largely underexplored. Based on thermodynamic considerations related to membrane bending, the smallest vesicles would likely measure 10–20 nm in diameter [9]. In addition to the most commonly explored EVs (commonly termed exosomes, microvesicles, and apoptotic cell-derived vesicles), the recent deployment of asymmetric flow field-flow fractionation technology [10] has revealed distinct populations of larger and smaller EVs, including the 35-nm-in-diameter exomeres. Large EVs, including apoptotic bodies and oncosomes [11], measure several microns in diameter. Stromal extracellular macrovesicles, also measuring several microns in diameter, where recently observed within the placental villous stroma [12]. EV properties also depend on the type of membrane phospholipids and the presence of membrane proteins. It is therefore clear that the commonly employed size-based definition of EVs, using nanoparticle tracking analysis (NTA) or similar systems, is overly simplistic, and comprehensive analysis of EV characteristics related to vesicle biogenesis, cargo, trafficking, and targeting properties should be contemplated in order to fully appreciate their diverse functionality. Additional considerations of EV definitions and their isolation technologies, which are not the subject of this text, can be found in recent publications by the International Society for Extracellular Vesicles (ISEV) and the Extracellular RNA Communication Consortium (ERCC1) [13–15]. As recommended by ISEV [13–15], herein we use the term EVs as a general term that defines released vesicles, and small EVs to specifically denote exosomes.

The production and release of placental EVs had been suggested nearly 15 years ago [16,17], with the subsequent isolation of placental small EVs in 2006 using size exclusion chromatography followed by purification with anti-placental-type alkaline phosphatase (PLAP) antibody by Sabapatha et al [18]. This was followed by small EV isolation directly from placental explants, cultured human trophoblasts, or trophoblast cell lines [19–23]. Interestingly, EVs are found in uterine secretions even prior to implantation [24,25]. Termed uterosomes, they have been functionally implicated in fertilization, blastocyst intercellular communication, and implantation [26–29]. Based on differential ultracentrifugation and PLAP-purification, it is estimated that the concentration of small EVs in pregnant women’s plasma in early pregnancy is on the order of 1–2 × 10¹¹ small EVs /ml plasma, with 10–20% contributed by PLAP-positive placental small EVs [30]. These levels gradually rise until the third trimester and are largely unaffected by fetal sex and maternal BMI [31–33]. Interestingly, similar levels of EVs were recently reported in the circulation of pregnant mice [34]. A similar concentration of small EVs was also reported in fetal circulation, with approximately 45% emanating from the placenta and with no significant effects due to fetal sex or growth restriction [33].

In this text, we will summarize key questions and research opportunities related to EV biology and their role in communication between trophoblasts and other cell types (Fig. 1). In accordance with the explicable ambiguity in the EV field, we focus mainly on small EVs in the broader context of EV biology. Recent reviews on exosomes as biomarkers can be found elsewhere [35–38].

Fig. 1. From biogenesis to target cell function: Key nodes in the biology of placental EVs.

Shown are key processes that should be pursued in order to elucidate the biology of trophoblastic extracellular vesicles.

Extracellular vesicle biogenesis, cargo loading, and release

Like many other cell types, placental trophoblasts produce EVs of diverse sizes [12,39,40]. While the precise mechanism of EV production by trophoblasts has not been fully elucidated, synthesis pathways implicated in other cell types, including production of small EVs by ESCRT protein–based pathways of intraluminal vesicles within multivesicular bodies, production of larger EVs by pinching off the plasma membrane, and apoptosis with surface membrane budding and cell body fragmentation [41,42], are likely to take place in trophoblasts. Whether trophoblast syncytialization affects EV biogenesis remains to be elucidated.

Recently, our understanding of the types of molecules packaged within EV cargo has been expanded and revised [43]. The cargo of trophoblastic small, medium and large EVs is variable [39], yet largely influenced by the cells of origin [44,45]. Placental small EVs are known to carry pro-apoptotic proteins such as TRAIL and Fas-ligand, cytokines, eicosanoids, growth factors, and their receptors [23,46–48]. As expected, unique placental proteins, such as PLAP, syncytin, and HLA-G, are also expressed in placental small EVs [49,50]. Akin to other tissues, placenta-derived EVs have a high concentration tRNAs [51] and microRNAs (miRNAs), including miRNAs expressed from the chromosome 19 miRNA cluster (C19MC) [22,40]. The repertoire of trophoblastic lipids has also been detailed [39]. Interestingly, discrete placental EV proteins may play a role in disease pathogenesis. For example, placental EVs from women with preeclampsia contain transthyretin, a thyroxine- and retinol-binding protein transporter, which is enriched in placental small EVs and implicated in the pathogenesis of preeclampsia [52].

Apart from the fact that some proteins are unique to certain EV subtypes, reflecting their biosynthetic pathways, an important question in the field of EV biology centers on the possibility that certain cell-specific proteins, RNA, or lipid molecules are selectively sorted to distinct EV sub-types. The presence of such selectivity in trophoblast EVs is currently unknown. For example, several recent publications illuminated the role of RNA-binding proteins, such as YBX1, hnRNPA2B1, and HuR, in selective sorting of miRNAs into small EVs [53–57]. In contrast, using primary human villous trophoblasts, we were not able to recapitulate selective C19MC miRNA sorting into small EVs [22]. Selective sorting of proteins (for example, cytokines) has also been documented in EVs from other cell systems [58]. Whereas some of these proteins may function in their target cells, others are implicated in protecting EV cargo (such as RNA) against degradation [59]. The presence of enzymes, such as the RNA-processing DICER [60], within EVs may also suggest that processing of certain cargo molecules is ongoing during the trafficking and delivery process [60]. These exciting possibilities remain to be explored in the context of placental EVs.

EV Trafficking to specific targets

The extremely small volume for cargo within EVs implies that the mobilization of bioactive molecules to discrete target cells would depend on a most efficient trafficking and EV-specific delivery system [42,61]. This might be a smaller hurdle when EV trafficking occurs among neighboring cells but becomes a major challenge in the signal traffic to cells of distant organs. The introduction of human small EVs into mice has been used to study small EVs distribution and systemic effect in the context of physiology or diseases. An important example of the contribution of guided trafficking to disease pathogenesis is provided through the work of Lyden’s group, who demonstrated the role of various integrins in guiding small EVs trafficking, which in turn directs tumor metastatic spread [62]. Tracking small EVs in vivo usually requires their labeling with fluorescent or luminescent tracers, using lipophilic dyes or genetic tags, such as the small, (20kDa) ATP-independent nanoluciferase (NanoLuc) [63], carboxyfluorescin succinimidyl ester (CFSE) [64], or other technologies, with subsequent real-time localization using an in vivo imaging system. Several groups have used different methods to trace mouse placental small EVs or their cargo to distant maternal tissues [65,66]. Trafficking of maternal small EVs into the placenta, and even into the fetal compartment, has also been reported [66]. Within that context, it is easier to envision the trafficking of EVs from trophoblasts, which, in the hemochorial placenta, are directly bathed in the maternal blood, into the maternal circulation. Identifying pathways used by maternal small EVs to cross the placental barrier and reach the fetal compartment may be more challenging, as the basement membrane and additional cell types need to be engaged and crossed to enable that trafficking route. Deciphering these pathways is necessary in order to elucidate maternal-fetal patterns of placental EV trafficking.

EV targeting to distant “addresses” may be guided by the presence of a unique “barcode,” which may be located on the surface of small EVs or their target cells. Such a barcode may be generated by a combination of particular surface molecules, such as integrins [62] or cytokines [58], and can be recognized by a “barcode reader.” While this remains a central question in EV biology [67,68], it is likely that molecules unique to the EV donor cells may be required for specific recognition by target cells. Within that context, it has been shown that the expression of syncytin-1 and -2 is essential for the entry of trophoblastic small EVs into target cells [69]. The potential role of syncytin in EV fusion with target cells has been reviewed [70]. Deeper research into the interaction of EVs with target cells and the mechanism underlying barcode recognition will be essential for deciphering the mechanisms of EV trafficking and targeting and for EV-based therapeutics.

EV entry and impact on target cell physiology

Discrete mechanisms, such as endocytosis, fusion of EVs with the plasma membrane, and other processes, promote EV entry into target cells [42,68,71]. Some of these mechanisms may also be intertwined with cell-specific recognition systems, trigger a surface molecule “kiss and run” interaction, or interact through activation of target cell receptors [72]. The mechanisms utilized by target cells to uptake trophoblastic small EVs are largely underexplored. Limited data point to the role of phagocytosis and clathrin-mediated endocytosis in the interaction of placental small EVs with endothelial cells in vitro [65].

It is generally believed that EV uptake into target cells may impact their physiology. Several recent examples in the fields of cancer, metabolism, and aging support this notion [62,73–76]. In pregnancy, trophoblastic EVs can be taken up by endothelial cells or circulating monocytes, B cells, or platelets and possibly affect their function [31,77–79]. Trophoblastic small EVs harboring C19MC miRNA attenuate viral replication in target cells via autophagy-mediated pathways [75]. Placental small EVs also attenuate mesenteric artery vasodilatory response [65].

Several immune functions are modulated by placental small EVs [80]. Maternal adaptive immunity, including impaired T-cell signaling, cytotoxicity and apoptosis are impacted by placenta-derived small EVs [81]. Placental small EVs modulate CD3, Janus kinase 3 (JAK3), and activation of caspase 3 in T-cells [18]. Human NKG2D ligands, expressed on the surface of trophoblastic small EVs, downregulate the NKG2D receptor on NK cells and CD8 T lymphocytes and modulate their cytotoxicity [20]. The miRNA miR-517a, within trophoblastic small EVs, regulates the serine/threonine kinase PRKG1 in Jurkat T-cells. [82]. Endoplasmic reticulum stress stimulates the production of EVs that contain damage-associated molecular pattern (DAMP) molecules [83]. Other immunological targets of placental EVs have recently been reviewed [84].

Placental EVs and diseases during pregnancy

In general, a disease may stem from abnormal EV production and packaging by the donor cells, trafficking miscommunication, or anomalous uptake or processing by target cells. There are several examples that mechanistically link small EVs to non-gestational disease processes [85,86]. These include the role of small EVs in cancer metastasis [62,87,88], embryonic stem cell–derived small EVs in cardiac healing after myocardial infarction [85], and neurological disorders [89,90].

Placental small EVs have been recently implicated in a number of gestational disorders, largely based on the association of a disease with abnormalities in small EV number or cargo molecules. Abnormal EV number, communication, and function have been implicated in preeclampsia [91,92]. Specifically, EVs from preeclamptic placentas exhibit altered levels of Flt-1, endoglin, and syncytin-2 [69,93]; the activity of endothelial nitric oxide synthase (eNOS) has been reported to be reduced [94]; small EVs from the serum of preeclamptic patients reduce eNOS expression in target endothelial cells [95] and differentially influence platelet function [78]. The membrane-bound metalloprotease neprilysin is higher in EVs from preeclampsia [96]. Finally, EVs from injured placentas can induce a preeclampsia-like phenotype in mice [97].

In the context of diabetes in pregnancy, hyperglycemia increases small EV release by trophoblasts [98,99]. Trophoblastic EVs from patients with gestational diabetes contain more dipeptidyl peptidase IV, implicated in the pathogenesis of type 2 diabetes by degradation of GLP-1 [100], and miRNA in placental small EVs of patients with gestational diabetes have been implicated in reduced insulin sensitivity and glucose uptake by striated muscle cells [101].

Final thoughts

Deeper understanding of EV biology has led to an unequivocal acceptance of the presence of diverse EV types, which has been validated not only in vitro, but also in vivo, in virtually all bodily fluids. Moreover, changes in EV concentration and composition and their various effects on target cells have been associated with altered physiological and disease states. In addition, the ability of EVs to deliver bioactive molecules to target cells has been validated and explored for the development of new drug delivery approaches. The recent deployment of effective tools that allow better EV isolation, size characterization, and definition of cargo composition has helped in untangling the complexity of EV biology, enabling a deeper level of research in the field and overcoming some of the challenges related to data reproducibility.

Notwithstanding the rapid progress in the field, our understanding of EV biology, particularly as pertaining to placental EVs, is severely lacking. Key aspects of placental EVs remain enigmatic and await a more profound mechanistic investigation. First, we need to define the biological implications of trophoblastic EV diversity and the changes in EV characteristics during pregnancy and in response to gestational disorders. Second, we must define the key determinants of EV biogenesis pathways and processes underlying cargo loading and assess them during placental development and through the differentiation of cytotrophoblasts to syncytiotrophoblasts. Are changes in EV cargo relevant to placental physiology, pregnancy homeostasis or disease state, and does cargo modification underlie altered EV function? Along the same line, do trophoblasts also produce small EVs that are negative for PLAP or other markers currently known to be present in small EVs? Third, we should interrogate signals that guide EV trafficking and targeting. If selective EV targeting is proven, which surface molecules may signal an interaction between the “barcode” and the “barcode reader”? Fourth, we should define the mechanisms underlying the uptake of placental EVs by target cells, cargo processing by target cells, and the implications of these processes to the biology of pregnancy. Which cellular pathways link EV effectors (protein, RNA, and the like) to their functional machinery? Finally, better tools and animal models should be developed to define the role of EVs in health and disease during pregnancy. Can we harness knowledge derived from placental EVs to develop better biomarkers, drug delivery systems, or new therapeutics that target pregnancy-related diseases?

The field of placental EV biology should move from unproven hypotheses and weak biomarker screens to innovative mechanistic pursuits that can propel the field to the forefront of research in early development and perinatal biology.