Zhen, YingHoward, Caitlin2022-09-232022-09-232022-09-232022Howard_washington_0250E_24819.pdfhttp://hdl.handle.net/1773/49260Thesis (Ph.D.)--University of Washington, 2022More than 90% of all malaria mortality is from infection with Plasmodium falciparum. Severe cases of P. falciparum infection can progress to cerebral malaria which is characterized by infected red blood cells sequestering in microvessels in the brain accompanied by endothelial cell dysfunction, vascular leakage, and cerebral inflammation. Microvasculature plays a key role in the progression of cerebral malaria but the specific factors which initiate progression to severe disease are not well understood, in large part because of challenges in studying the disease due to the inaccessibility of the brain and a lack of suitable models. Recent advances in vascular engineering have allowed for the development of models of vascular systems which have great potential to overcome previous challenges in studying brain microvasculature. The body of work presented here reports advances in vascular engineering strategies to create more biologically relevant models and applications of these models to address specific questions in the pathogenesis of cerebral malaria. We first established a method for engineering capillary-scale vasculature using a combination of soft lithography and 2-photon laser-based ablation. Because infected red blood cells sequester primarily in capillaries and post-capillary venules in the brain, replicating this size scale was important and allowed for the study of the spatiotemporal dynamics of normal and infected red blood cells as they travel through a capillary. This study helped to shed light on some of the mechanisms involved in infected red blood cell sequestration and also demonstrated the feasibility of the platform for use to study other hematologic diseases. Next, we expanded on these engineering methods to develop models of complex, organ-specific microvasculature. To do this, we optimized the ablation protocol for use with various extracellular matrix components, endothelial cell and perivascular cell types, and complex 3D geometries at anatomic scale. We verified that ablation is compatible with multiple matrices and cell types and as a proof-of-concept created an endothelialized glomerulus model at anatomic scale. The ability to create perfusable vessels with significant geometric complexity at the capillary scale enables new approaches to the study of vascular biology and can provide more biologically relevant models for probing microvascular questions. We next adapted a microvascular model to use for studying the response of brain endothelial cells to various pathological stimuli implicated in cerebral malaria pathogenesis. From this, we were able to demonstrate that infected red blood cells adhere to brain endothelial cells in the microvessel model and continue to mature in situ throughout the blood stage parasite life cycle. This allowed us to probe the endothelial cell response at various stages of parasite maturation and highlighted that the infected red blood cells induced unique changes to the brain endothelial cells. We also investigated how this response is affected by previous exposure to host pro-inflammatory cytokine TNFα which revealed a strong combinatorial effect from both stimuli. Because the microvessel model is perfusable, we were able to assess an immune function of the endothelial cells by perfusing with leukocytes and determined a strong functional change in the ability of parasite-stimulated endothelial cells to recruit leukocytes. This study provided new mechanistic insights into endothelial cell dysfunction in cerebral malaria. Altogether, this work introduces advances in vascular engineering allowing for more biologically relevant models of vascular disease and facilitating progress towards understanding the mechanisms of cerebral malaria pathogenesis and brain inflammatory processes.application/pdfen-USnoneCapillaryInflammationMalariaVascular engineeringBioengineeringBioengineeringThesis