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Page 4 of 15 Cheng et al. Vessel Plus 22020;4:17 I http://dx.doi.org/10.20517/2574-1209.2020.08
[36]
group in 2004 in Malawian children with CM , where an elevation in the number of MVs of endothelial
origin was described. MVs released by RBCs were later found to be increased in both P. falciparum and
P. vivax malaria [37,38] . EVs have been shown to be involved throughout the entire life-cycle of malaria
infection and at different stages, the parasite can affect various immune and vascular cell types in different
[32]
ways, ultimately altering the endothelium and BBB function .

A pan-vascular, cell-derived MV release was also observed in children with CM in Cameroon. Of these
[39]
MVs, an increase in platelet MVs was most significantly correlated with disease severity . Exosomes were
[40]
first explored in 2011 in a murine model of malaria and will be discussed in a later section. While mostly
descriptive, these clinical studies were essential for suggesting a role for these EVs as either markers of CM
severity, or as players in the pathogenesis of CM infection, and paved the way for subsequent work on the
composition and functional potential of EVs during malaria infection.

IN VITRO MODELS OF MALARIA - INTERACTIONS BETWEEN HOST CELLS AND
EXTRACELLULAR VESICLES
Most in vitro models of CM simulate the interactions between microvascular endothelial cells and
circulating vascular cells (e.g., iRBCs, nRBCs, platelets, and leucocytes) in either static or shear stress
environments [41,21] . The brain endothelial cells used can be of human, simian or murine origin (primary or
immortalised), and co-cultured with one or more other cell types in two-dimensional systems [42-45] . The
recent introduction of more complex three-dimensional models will help to examine and understand the
pathogenesis of this disease better [46-48] .

Very much like their cells of origin, EVs interact with their target cells and modulate their responses. In
vitro, platelet MVs behave in a similar fashion as platelets by increasing the adherence of iRBCs to human
[49]
brain endothelial cells (HBECs) by providing iRBCs with surface receptors such as CD31 and CD36
such that platelet MVs act as a bridge between HBECs and iRBCs. The internalisation of platelet MVs by
vascular endothelial cells is also associated with an alteration of their phenotype such that ultimately, their
inflammatory effects and subsequent activation can be potentiated .
[50]

RBCs release increased levels of EVs when infected with a Plasmodium parasite and late-stage infections
[38]
are associated with even greater release of EVs . This is mainly due to membrane changes occurring
within iRBCs during parasite maturation. The composition of EVs derived from iRBCs is also dependent
on the parasite’s stage of development. Indeed, specific parasite proteins, considered as virulence factors,
were present in EVs only at specific developmental stages and PfEMP1 was only detected in EVs from
iRBCs with parasites at early stages. Potentially, such developments would allow EVs to bind and prime
[51]
endothelial cells for later adherence and sequestration of late-stage iRBCs .
EVs from iRBCs have also been shown to contain a functional microRNA-argonaute 2 complex that
can modulate gene expression and alter barrier function [52,53] when transferred to endothelial cells after
vesicle uptake. Such EVs do not only affect endothelial cells but are also able to induce pro-inflammatory
responses, particularly the activation of macrophages, monocytes as well as other immune cells through the
upregulation of cytokines [54,55] . Interestingly, when these EVs were compared to their mother cells, they were
able to activate inflammation and immune activation to a greater degree [54,55] . EVs from iRBCs also contain
small RNAs and genomic DNA. After internalisation by monocytes, they can induce the innate immune
cytosolic adaptor-dependent DNA sensing pathway (STING), leading to downstream alterations of DNA
sensing pathways in target cells . Activation of these pathways has been shown to correlate with parasite
[56]
[57]
[57]
survival . Thus, this could possibly be used as a decoy method for immune escape by the parasites .
Similarly, the release of PfEMP1-containing EVs as previously mentioned, has also been suggested as a