Elisa Vázquez-Liébanas: Cellular and Molecular Studies on the Blood-Brain Barrier

Abstract

The Blood–Brain Barrier (BBB) is a highly selective interface that regulates molecular exchange between the blood and the brain, maintaining the ionic and metabolic environment essential for brain function. Although BBB dysfunction is a feature of many neurological diseases, the cellular and molecular mechanisms that preserve or compromise barrier integrity remain incompletely defined. This thesis investigates three major regulators of BBB function in the adult brain: the tight-junction protein Claudin-5 (CLDN5), pericyte–endothelial interactions mediated by PDGFB/PDGFRβ signalling, and the context-dependent TEK antagonist Angiopoietin-2 (ANGPT2). To examine the role of CLDN5, we generated an inducible, endothelial-specific CLDN5 loss-of-function mouse model. Adult deletion of CLDN5 resulted in BBB leakage to tracers up to 10 kDa, accompanied by endothelial and glial inflammation, seizures, and lethality. Single-cell RNA sequencing and immunofluorescence revealed that these effects were predominantly non-cell-autonomous, with endothelial cells showing minimal transcriptional alterations despite severe barrier disruption. The second component of this work addressed the contribution of pericytes to BBB maintenance. We demonstrate that PDGFB expression in the adult vasculature is required to sustain pericyte coverage and preserve BBB integrity. Developmental loss of PDGFB caused vascular enlargement, disrupted arteriovenous zonation, and microvascular calcification, whereas deletion in adulthood led to progressive pericyte loss and age-dependent BBB leakage without overt vascular rarefaction or enlargement. These findings highlight the distinct consequences of developmental versus adult pericyte deficiency. The final part of the thesis investigated ANGPT2, which was consistently upregulated in Cldn5- and pericyte-deficient models, as well as in hereditary haemorrhagic telangiectasia, glioblastoma, and stroke. Contrary to its classical destabilizing role, dual loss of pericytes and ANGPT2 further impaired BBB stability, revealing a protective function for ANGPT2 in the adult brain. A global Angpt2 exon 4 knockout model exhibited region-specific vascular malformations, tracer leakage, and accumulation of reactive mural cells, fibroblasts, astrocytes, and microglia, with BBB defects also present in unaffected regions. Together, these studies delineate distinct molecular pathways of BBB disruption – from tight-junction loss to altered pericyte signalling and ANGPT2 dysregulation – and provide a comprehensive framework for understanding how endothelial, mural, and glial interactions preserve cerebrovascular stability in health and disease.