322 - Neonatal hyperoxia decreases delta-like 4 and claudin 5 expressions in brain endothelial cells and Dll4 regulates endothelial integrity through the Dll4-Notch-Cldn5 pathway pathway

Poster Number: 322
Publication Number: 322.3312

Background: Neonatal hyperoxia (HOX) exposure induces cognitive impairment in both humans and mice. Cognitive functions depend in part upon blood-brain barrier (BBB) integrity. One of the BBB components is cerebrovascular endothelial cells (EC). Brain EC expresses high levels of delta-like 4 (Dll4) and tight junction protein claudin 5 (Cldn5). Dll4 is a Notch ligand in EC and a key regulator of cerebrovascular development and Cldn5 regulates BBB permeability. We previously demonstrated that insufficiency of Dll4 leads to deviant angiogenesis, increases endothelial permeability and reduces Notch activation by decreasing cellular levels of NOTCH Intracellular domain (NICD) in brain EC. Further, decreased Cldn5 expression impairs BBB integrity.

Objective: : Demonstrate that neonatal HOX increases BBB permeability concurrently with decreased Dll4 and Cldn5 expression in mouse brain EC, and Dll4 regulates BBB through the Dll4-Notch-Cldn5 pathway.

Design/Methods: Albumin (a marker of BBB permeability), Dll4, NICD and Cldn5 levels were compared in mouse brain and EC exposed to 85% HOX vs. room air between postnatal days (P)1-14 at P14. Dll4 and Cldn5 protein in the cortex was determined by immunofluorescence. Additionally, cldn5 levels were determined in human brain microvascular EC (HBMEC) with Dll4 levels knock-down by 50% and in HBMEC exposed to hyperoxia. The density of Notch at Cldn5 promoter in HBMEC was determined by chromatin immunoprecipitation (ChIP) assays. Finally, Cldn5 promoter activities with or without NICD were measured by in vitro promoter activity assays.

Results: Neonatal HOX significantly increased albumin levels in brain lysates and decreased Dll4, NICD and Cldn5 expression in mouse brain EC with females being more affected. Neonatal HOX also significantly decreased Dll4 and Cldn5 abundancy in the mouse brain cortex (Fig. 1). The Cldn5 promoter contains a highly conserved binding site for the DNA-binding protein RBPJ that acts with Dll4-induced NICD to regulate target genes. Consistent with this, Dll4 repression in hyperoxia was associated with decreased NICD and Cldn5 levels in HBMEC. ChIP assays revealed that Notch bound to the Cldn5 promoter. Additionally, Cldn5 promoter activity were enhanced with the addition of NICD (Fig.2).

Conclusion(s): HOX impairs BBB integrity and suppresses brain endothelial Dll4 and Cldn5 expression both in vivo and in vitro. Dll4 regulates endothelial integrity through the Dll4-Notch-Cldn5 pathway. We speculate that decreased Dll4 and Cldn5 expression may impair BBB integrity and contribute to long-term cognitive impairment in individuals exposed to neonatal hyperoxia.