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Abstract

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection associated with blood-brain barrier (BBB) breakdown. A hallmark of cerebral malaria is cytoadhesion of P.falciparum-infected red blood cells (iRBCs) to the brain microvasculature, yet the mechanisms behind CM pathogenesis and BBB breakdown remain largely unknown. Dysregulation of the endothelial-protective angiopoietin/Tie-2 axis is a predictive marker of CM-induced fatality and therefore is an attractive causative factor in the promotion of brain endothelial dysfunction observed in CM patients. In the brain microvasculature, the angiopoietin/Tie-2 axis consists of the endothelial receptor Tie-2 and its ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), secreted primarily by brain pericytes and brain endothelial cells respectively. However, previous human in vitro brain endothelial models to study CM have omitted brain pericytes and therefore, do not fully recapitulate this pathway. Here, I developed a minimal 3D microfluidics-based brain microvasculature model that is composed of both primary human brain endothelial cells and brain pericytes, and replicates angiopoietin/Tie-2 axis signaling. I demonstrated that the model reproduces in vivo brain pericyte coverage of the microvessels, cell type-specific marker expression and ultrastructural brain endothelium-pericyte interactions. Perfusion with iRBC egress products increased microvessel permeability, albeit with minor changes in microvessel morphology and brain endothelium-pericyte interaction markers. Strikingly, intact iRBCs and iRBC egress products halted secretion of Ang-1 with the microvessels, revealing for the first time a role of brain pericytes in CM-associated angiopoietin/Tie-2 axis dysregulation. Pre-incubation with recombinant Ang-1 offered partial protection against microvessel barrier breakdown induced by iRBC egress products, highlighting reduction in Ang-1 secretion as a promoter of BBB breakdown during CM. On the contrary, Ang-2 secretion was not increased in the presence of intact iRBCs and their egress products suggesting involvement of additional CM-associated factors in angiopoietin/Tie-2 axis dysregulation. Use of a clinical therapeutic that enhances Tie-2 activity displayed rapid partial protection against the barrier disruptive effects of iRBC egress products, proposing interventions that restore homeostatic function of the angiopoietin/Tie-2 axis as potential adjunctive therapies against CM. Overall, this model provides evidence towards brain pericytes representing a key cell type in angiopoietin/Tie-2 axis dysregulation and BBB breakdown, while offering insight into novel therapeutic avenues in which to counteract CM pathogenesis.