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The blood-brain barrier (BBB) is a specialized vascular interface that protects the central nervous system (CNS) by tightly regulating molecular exchange between the blood and brain parenchyma. Despite its importance, the mechanisms underlying BBB development, maintenance, and dysfunction remain incompletely understood. In this thesis, we investigated the effects of Angiopoietin 2 (ANGPT2) deficiency during development and Claudin 5 (CLDN5) loss in adulthood on BBB integrity, developed a refined method for isolating brain vascular cells, and performed cross-species transcriptomic comparisons to identify gene expression similarities and differences between mouse and human brain pericytes.

Our results show that constitutive Angpt2 knockout leads to spatially localized vascular malformations in the adult brain, particularly in the basal ganglia and somatosensory cortex, accompanied by increased permeability, angiogenesis, mural cell alterations, ECM accumulation, and glial reactivity. Single-cell RNA sequencing identified a distinct endothelial population in Angpt2 knockout mice enriched for genes involved in angiogenesis and matrix remodeling. In contrast, inducible, endothelial-specific deletion of Cldn5 in adult mice caused size-selective BBB leakage, neuroinflammation, and widespread transcriptional changes in both CLDN5-positive and -negative endothelial cells, indicating indirect effects. Additionally, to facilitate high-quality single-cell analysis, we developed a magnetic bead-based protocol for isolating brain microvascular fragments, which enriched for vascular and perivascular cell types while preserving mRNA and protein integrity. Finally, we compared gene expression profiles of mouse and human brain pericytes using public datasets, revealing species-specific transcriptional differences.

Together, these studies provide new insights into the molecular regulation of the BBB in both developmental and mature contexts. By integrating genetic models, imaging, single-cell technologies, and cross-species analysis, our work advances understanding of neurovascular biology and offers valuable tools and knowledge to enhance the translational relevance of preclinical models in neurological research.