APPswe mutation causes functional deficits in endothelial cells generated by transient ETV2 overexpression in human iPSCs

Background Brain endothelial cells (ECs) lining blood vessels are essential for the normal function of the brain. They form the first layer of the blood-brain barrier (BBB) and regulate nutrient exchange, immune responses, and angiogenesis. Numerous studies have reported the disruption of the BBB in neurodegenerative diseases, including Alzheimer’s disease (AD). However, the impact of cell-intrinsic amyloid pathology on EC function remains to be clarified. Methods To optimize the method for producing functional ECs from human induced pluripotent stem cells (hiPSCs), we compared two different protocols. The first, a widely used method, relies on spontaneous differentiation after mesoderm specification. The second method involves transient overexpression of ETV2 to guide EC differentiation. To study the impact of beta-amyloid overproduction on EC function, we generated ECs from hiPSC lines carrying the APP Swedish mutation (APPswe), which causes AD. We assessed the functionality of both control and APPswe ECs using in vitro permeability assays, 2D and 3D vessel formation assays, and adhesion assays. Results Our results show that ECs generated using transient ETV2 overexpression exhibit higher levels of classic EC markers, tight junction proteins, transporters, leukocyte adhesion molecules, and angiogenesis-associated receptors than ECs derived by spontaneous differentiation. This suggests that ETV2-ECs more closely resemble ECs in vivo. Additionally, ETV2-ECs responded to inflammatory and angiogenic stimuli, displaying functional and transcriptional changes, whereas spontaneously differentiated ECs did not. This indicates that ETV2-ECs are a better model for investigating functional impairments. Furthermore, ETV2-ECs carrying the APPswe mutation displayed a reduced angiogenic potential following exposure to the sprouting mix and higher levels of leukocyte adhesion molecules following inflammatory stimulation, leading to increased adhesion of monocyte-like cells. Conclusions Overall, our study suggests that APPswe mutation in ECs impairs their response to inflammatory and angiogenic stimuli, potentially contributing to AD progression. Additionally, we confirmed that ETV2 overexpression during a critical window effectively guides hiPSCs toward the EC lineage, resulting in a stable and pure population of ECs suitable for disease modeling and drug screening.