Heart pumps blood, in order to supply nutrients and oxygen to all cells, a process conducted by the vascular system. This blood vascular system is also essential for waste removal and immune surveillance. Insufficient formation of vessels, cell degeneration, or dysfunction are linked to multiple pathologies including cardiac diseases or cancer. Targeting the heterogenous vascular beds serves as a promising therapeutic strategy to preserve homeostasis. This Elective course is aimed at students who wish to pursue an experimental doctoral thesis. By providing 4 distinct experimental procedures, the course will train the students on endothelial cell culture and function in response to pro-inflammatory and pro-angiogenic stimuli as well as confocal imaging, and live endothelial cell metabolic mapping. The Elective will run over 2 days, comprising of a Teaching/Seminar component (2hrs) and a Practical component (4-8 hrs in 2 days period). Students may choose any subcategory of interest.
The students will learn about endothelial cell activation by pro-inflammatory stimuli and will get hands-on experience in the handling of human endothelial cells. Each student will perform an experiment where human monocytes adhere to human endothelial cells after treatment with pro-inflammatory cytokines (live cell imaging using IncuCyte). The students will also learn to summarize and analyse the data using GraphPrism.
The students will learn about endothelial cell proliferation and will get hands-on experience in the handling of human endothelial cells and their acute responses to growth factors. Each student will perform an experiment where human endothelial cells with proliferate in response to injury (live cell imaging using IncuCyte) and evaluate the EdU incorporation with confocal microscopy. The students will also learn to summarize and analyse the data using Imaris 9.2 and GraphPrism.
Seeing is believing
The students will learn how to perform confocal fluorescent imaging in heart sections and distinguish different cell types by morphology and specific lineage markers (i.e. cardiomyocytes, endothelial cells and pericytes/ smooth muscle cells). Samples will include healthy and diseased hearts. Each student will perform immunofluoresent stainings, confocal imaging with a Leica Stellaris microscope and data evaluation with Volocity and GraphPad Prism softwares.
Endothelial cell metabolic heterogeneity
Although endothelial cells of lymphatic capillaries have many properties in common with the endothelium of blood vessels, they also have distinct structural and energetic characteristics reflecting their specific functions. Such a metabolic heterogeneity is responsible for each specific cell subtype to support their energetic demands and regulate lineage related transcription. While blood endothelial cells exhibit of preference on glucose consumption, lymphatic endothelial cells import primarily fatty acids. In this setting, students will visualize the difference of the key metabolites imported in growing monolayer blood and lymphatic endothelial cells. Each student will trace the uptake of glucose and palmitate with selective fluorescent probes and results will be visualized with live cell fluorescence activated cell sorting approach. Students will also get familiar with the FlowJo software.