Trophoblast Extracellular Vesicles in Preeclampsia

Overview

A large amount of “debris” is shed from the syncytiotrophoblast (STB) into the maternal circulation; thus, providing a direct, placental-specific contribution to vascular endothelial dysfunction (see Chapter 10). This “debris” ranges in size from large deported multinuclear fragments to subcellular components. An important subset of this material is released as extracellular vesicles (EVs), enclosed within a phospholipid bilayer, that form small “bubble-like” lipid particles. These contain proteins on their surface and within as well as genetic material inside and are categorized as subtypes based on their diameter and biogenesis. The smallest of these are exosomes (<200 nm and sometimes called nanovesicles, originating from intracellular multivesicular bodies (MVBs). Microvesicles (MVs), are somewhat larger, 200–1000nm, originating from the cell membrane surface). The largest are apoptotic bodies (typically >500 nm and released as a terminal cellular event). Syncytiotrophoblast EVs (STBEVs) have been shown to be intercellular signals that contribute to both immune and metabolic changes in normal and pathological pregnancy. It is important to stress that experts still debate which properties define exosomes versus MVs and how to best isolate each class of EVs. It is also evident that several subtypes of exosomes and MVs likely exist. As a result, many studies claiming to specifically study either exosomes or MVs are, probably, isolating a heterogeneous mixture. This has prompted the scientific community to adopt guidelines that use the term EVs, rather than MVs or exosomes, in cases where it is not absolutely clear that a particular class of EVs is being isolated and studied. However, for the purposes of this chapter, we have elected to use the terminology STBEV (i.e., encompassing EVs, MVs, and exosomes).

Definition and Heterogeneity

Biological vesicles are small subcellular structures that may be intracellular or extracellular. They are contained within a phospholipid bilayer derived, directly or indirectly, from the plasmalemma of parent cells. Intracellular vesicles are one of the simplest forms of cell organelle enclosed by a double membrane, which can also include major structures, such as the nucleus, mitochondria, and autophagosomes, or by only a single membrane (endoplasmic reticulum). Intracellular vesicles subsume functions such as intracellular transport between organelles or secretion (at synapses or from endocrine cells). EVs also possess bilamellar phospholipid membranes and crucially, retain characteristics of their parent cells, but their content is regulated to include distinctive features. It is currently believed that all intact cells secrete or shed micro- or nanovesicles. They are evolutionarily conserved from prokaryotes to eukaryotes.

Their importance lies in their ability to promote continuous communication between cells—within a tissue, an organ or organism. They are targeted to responder cells by specific surface molecules and deliver their messages either by classical receptor–ligand interactions or by fusion with the recipient cell, thus deploying their cargo (lipids, miRNA, mRNA, etc.) within the recipient cell. Their relatively recent discovery has revealed a huge new dimension of intercellular coordination and regulation.

Three general classes of EVs were initially defined by their size, namely exosomes, microvesicles (MVs), and apoptotic bodies. Apoptotic bodies overlap in size with MVs and are released from terminally apoptosing cells, but this occurs only once in the life of a cell. Consequently, research into the role of EVs in cell–cell communication has focused on MVs and exosomes.

Both MVs and exosomes are produced by blebbing from a membrane: outward from the plasmalemma (MVs) and inward into the intraluminal space of endosomal multivesicular bodies (exosomes) processed through the endosomal system. They have different modes of production but share common markers. Intracellular calcium stimulates the production of both exosomes and MVs, while the cargo they carry and their interactions with other cells differ in certain ways ( Table 8.1 ).

Extracellular MVs were originally discovered in the blood. They were first described as “platelet dust,” which referred to platelet-like activity retained in plasma supernatant after centrifugation that could then be separated by ultracentrifugation and related to vesicles visualized by electron microscopy. Red blood cell MVs were discovered subsequently and other microvesicles and their dates of discovery are listed in Table 8.2 . Relevant to preeclampsia, trophoblast EVs were first reported, by us in 1998.

Terminology and Size

The term microparticle includes nonvesicular entities such as lipoproteins, chylomicrons, or artifacts generated during sample processing—such as aggregated proteins, immune complexes, calcium-phosphate microprecipitates, and fluorescent antibody aggregates. These are not relevant to this chapter, but are important to consider because they generate false-positive signals in some assays and may cause independent biological effects differing from those produced by EVs.

MVs (also called ectosomes) and exosomes (also called nanovesicles) have become the subject of intense research over the last 20 years. Their sizes are summarized in Table 8.1 but overlap, such that exosomes and MVs cannot be distinguished simply based on size or biomarkers, even though such classifications underlie many reports. MVs are sometimes simply called extracellular MVs, a term that also is applied more generically and may sometimes include exosomes. Here we use the term microvesicles to describe the vesicular products of plasmalemma blebbing. We use the generic, term extracellular vesicles (EVs), to mean all classes.

MVs and exosomes share common markers and tend to be produced together. There are similarities and differences in what stimulates their production, their cargoes, and their interactions with other cells. EVs carry the characteristics of the cells from which they originate, so creating yet further subtypes of all EVs.

In summary, EVs comprised three main classes: apoptotic bodies (>1000 nm), MVs (200–500 nm), and exosomes, also called nanovesicles (50–200 nm). STB generates a fourth unique variety, multinucleate aggregates, which were first detected in the lungs of women dying of eclampsia more than 120 years ago.