Role of endothelial cells in severe malaria pathogenesis: effects of parasites and antimalarial drugs

Endothelium plays a crucial role in the regulation of vascular homeostasis and permeability. Its adhesion proprieties are highly regulated to allow physiologic functions, such as the migration of leucocytes into tissue, and to avoid pathologic degenerations. In particular, the adhesion of red blood cells (RBC) to endothelial cells (ECs) is involved in pathologies such as sickle cell anaemia, diabetes and malaria. In severe malaria RBC infected with Plasmodium falciparum (Pf) undergo membrane alterations which cause a receptor-mediated adhesion to endothelium. This phenomenon is called cytoadherence and is responsible for the microvascular occlusions and consequent tissue hypoxia, which are associated with severe malaria. The mechanisms and the molecular pathways activated by cytoadherence are extensively studied due to the high necessity of new adjuvant therapies for severe malaria. The apoptosis of ECs by PRBC is one of the processes described in vitro and in vivo. It is considered critical for the development of the disease but its mechanisms and molecular consequences have not been clarified yet. Among the drugs used to treat malaria, including the severe forms, the derivatives of artemisinin, an active principle extracted from the plant Artemisia annua, are very important due to their high efficacy and tolerability. Besides their antimalarial activity, this class of molecules is studied for the antiangiogenic and antitumor effect. In a previous work from our laboratory we investigated the antiangiogenic effect of artemisone, one of the newest artemisinin derivatives. In particular, the inhibition of ECs growth and the production of pro-angiogenic cytokines have been demonstrated in different in vitro and ex vivo models. The aim of this work was to study the role of ECs in malaria considering two aspects: the effect of parasitized RBC (PRBC) and the effect of dihydroartemisinin (DHA), the active metabolite of most artemisinins. The first part of the project was performed at INSERM/UPMC UMR S511 Faculté de Médecine Pierre et Marie Curie, Paris, under the supervision of Dominique Mazier. We characterised two genes of Pf with putative pro-apoptotic function on pulmonary ECs. Using sera from immunised mice, we demonstrated the nuclear localisation of the proteins encoded by these genes. Therefore, it was not possible to proceed with the functional studies, which consisted in using the antibodies to inhibit the pro-apoptotic function. However, the information obtained is important to further investigate the function of these proteins. The second part of the thesis reports the studies on the effect of DHA on ECs in hypoxia, which is a condition common to the microvessels of severe malaria patients and to the tumour microenvironment. In the first case hypoxia may contribute to inflammation and endothelial damage, whereas in tumours it is a potent stimulus for the production of new vessels, fundamental to bring nutrients to the tumour mass. We demonstrated that the inhibition of ECs growth by DHA is strongly influenced by the oxygen tension: at low doses (similar to those found in the plasma of malaria patients) DHA is more active in hypoxia, whereas at high doses (more similar to the doses proposed as antiangiogenic and antitumor) it is more effective in normoxia. We verified that DHA induces apoptosis only at high doses and affects the cell cycle via induction of oxidative stress. Further studies are needed to understand the mechanisms of growth inhibition in hypoxia, evidenced at the low doses of DHA, and to envisage the possible therapeutic application of this phenomenon.