Aims & Methods: Extracellular Vesicles (EVs) are functional sub-cellular fragments capable of transferring surface markers, proteins, and nucleic acids to cells that internalise them. Recent studies have suggested that EVs participate in the development of the vascular lesion during Cerebral Malaria (CM). Using an in vitro Blood-Brain barrier (BBB) model, we aim to investigate the effects EVs have on the modulation of endothelial integrity by measuring the expression of junctional protein, VE-cadherin and the activation status of the endothelial monolayer. EVs released by both normal and Plasmodium falciparum-parasitised red blood cells (nRBCs, pRBCs) after 48h culture were purified from the supernatant by sequential centrifugation.
Primary human brain endothelial cells (HBECs) were incubated with nRBCs, pRBCs, nRBC-/pRBCs-EVs (nEVs, pEVs), or a combination of them. Modulation in VE-cadherin expression both local and in relation to the presence of PRBCs and/or EVs was assessed by a combination of high-content, high-resolution and OMX super-resolution microscopy. Expression of cell adhesion molecules (CAMs) was measured by flow cytometry.
Results: Quantitative image analysis showed that non-parasitised conditions triggered up-regulation of VE-cadherin expression, whereas parasitised conditions resulted in a significant down-regulation. We also observed that p-EVs were taken up by HBEC at twice the rate of n-EVs. Expression of CAMs was increased in the presence of p-MVs and PRBC-Mix.
Conclusion: These results suggest that interactions between EVs and their cells of origin have similar effect of target cells. Further studies are currently ongoing in order to determine which molecular pathways are involved in the changes observed.