Abstract
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:
ECs generated using transient ETV2 overexpression exhibited higher levels of canonical EC markers, tight junction proteins, transporters, leukocyte adhesion molecules, and angiogenesis-associated receptors than ECs derived by spontaneous differentiation. Additionally, ETV2-ECs responded robustly to inflammatory and angiogenic stimuli, displaying functional and transcriptional changes, whereas spontaneously differentiated ECs did not. Consequently, we chose the ETV2 overexpression protocol to study the impact of APPswe mutation on endothelial function. We found that ETV2-ECs carrying the APPswe mutation displayed a reduced angiogenic potential following exposure to the sprouting mix and elevated expression 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.
Supplementary Information:
The online version contains supplementary material available at 10.1186/s12987-025-00728-8.
Keywords:
Alzheimer’s disease (AD); Angiogenesis; Blood-brain barrier (BBB); Brain endothelial cells (ECs).