Inflammatory mediators reduce surface PrP on human BMVEC resulting in decreased barrier integrity.

The cellular prion protein (PrP) is a surface adhesion molecule expressed at junctions of various cell types including brain microvascular endothelial cells (BMVEC) that are important components of the blood-brain barrier (BBB). PrP is involved in several physiological processes including regulation of epithelial cell barrier function and monocyte migration across BMVEC. BBB dysfunction and disruption are significant events in central nervous system (CNS) inflammatory processes including HIV neuropathogenesis. Tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) are two inflammatory factors that have been implicated in the processes that affect BBB integrity. To examine the effect of inflammation on PrP expression in BMVEC, we used these mediators and found that TNF-α and VEGF decrease surface PrP on primary human BMVEC. We also showed that these factors decrease total PrP protein as well as mRNA, indicating that they regulate expression of this protein by de novo synthesis. To determine the effect of PrP loss from the surface of BMVEC on barrier integrity, we used small hairpin RNAs to knockdown PrP. We found that the absence of PrP from BMVEC causes increased permeability as determined by a fluorescein isothiocyanate (FITC)-dextran permeability assay. This suggests that cell surface PrP is essential for endothelial monolayer integrity. To determine the mechanism by which PrP downregulation leads to increased permeability of an endothelial monolayer, we examined changes in expression and localization of tight junction proteins, occludin and claudin-5, and found that decreased PrP leads to decreased total and membrane-associated occludin and claudin-5. We propose that an additional mechanism by which inflammatory factors affect endothelial monolayer permeability is by decreasing cell-associated PrP. This increase in permeability may have subsequent consequences that lead to CNS damage.