Impact of Wnt/β‐catenin signaling on ethanol‐induced changes in brain endothelial cell permeability

Chronic exposure to ethanol is associated with enhanced leakiness in the brain microvessel endothelial cells that form the blood–brain barrier (BBB). As previous studies suggested Wnt/β‐catenin signaling could improve the BBB phenotype of brain endothelial cells, we examined the extent to which Wnt signaling is altered following ethanol exposure, using both a cell culture model of the BBB and mice exposed to ethanol, and the ability of Wnt activation to reverse the permeability effects of ethanol. The human brain endothelial cells, hCMEC/D3, were exposed to ethanol (17–200 mM) for various periods of time (0–96 hr) and Wnt signaling, as well as expression of downstream genes influencing BBB integrity in the cell monolayers were monitored. Determination of Wnt signaling in both brain homogenates and brain microvessels from mice exposed to ethanol was also performed. The effects of ethanol on the permeability of the hCMEC/D3 monolayers were examined using both small molecular weight (sodium fluorescein) and large molecular weight (IRdye 800CW PEG) fluorescent markers. Exposure of hCMEC/D3 to ethanol (50 mM) caused a down‐regulation of Wnt/β‐catenin signaling, a reduction of tight junction protein expression and up‐regulation of plasmalemma vesicle associated protein (PLVAP). A similar reduction in Wnt/β‐catenin activity in both cortical brain homogenates and isolated cortical cerebral microvessels were observed in mice. Other areas such as cerebellum and striatum displayed as much as 3–6 fold increases in Dkk‐1, an endogenous Wnt inhibitor. Ethanol exposure caused significant changes in both sodium fluorescein and IRdye 800CW PEG permeability (2‐fold compared to control). The ethanol‐induced increases in permeability were attenuated by treatment with known Wnt activators (i.e. LiCl or Wnt3a). Additional screens of CNS active agents with possible Wnt activity indicated fluoxetine could also prevent the permeability effects of ethanol. These studies suggest that ethanol‐induced changes in brain microvessel permeability can be reversed through activation of Wnt signaling. Wnt/β‐catenin is important for blood‐brain barrier (BBB) development and maintenance. In the current studies we report that Wnt/β‐catenin signaling is downregulated in the BBB following prolonged ethanol exposure. Activation of Wnt/β‐catenin signaling reversed the detrimental effects of ethanol on brain endothelial cell function. Examination of various CNS drugs for Wnt activation suggested tha