Abstract 5216: Transient modulation of the blood-brain barrier with time dependent adenosine receptor agonism
Authors: Vezina Amelie , McCord Matthew McCord , Gilbert Mark R. , Jackson Sadhana 
Published: July 2017

Background:

The blood-brain barrier (BBB), a dynamic, highly regulated and poorly permeable blockade, is the key obstacle to effective drug delivery to the central nervous system (CNS). Its main function is to protect the brain from toxic agents such as chemotherapy. The BBB regulates drug entry by the presence of tight junctions, adherens junctions and multidrug resistance proteins. Agonism of adenosine A2A receptor (A2AR) by Regadenoson, a FDA approved A2AR agonist, in non-tumor bearing rodents has been shown to transiently disrupt BBB permeability. Here, we studied the time dependent impact of Regadenoson on brain endothelial cells to transiently decrease tight and adherens junctions, while impairing ABC transporter function, thus increasing BBB permeability.

Methods:

Mouse brain endothelial cells (bEnd.3), were treated with 10μM Regadenoson on a time-course of 0-24 hours. Claudin-5, Occludin, ZO-1, VE-Cadherin, and PECAM-1 expression studies were assessed by flow cytometry, immunofluorescence and western blotting of subcellular fractions at varied time points. Additionally, tight and adherens junctions’ gene expression was assessed by qRT-PCR using SYBR green. Expression and function of ABCB1, ABCG2 was reported by western blotting of whole cell lysates, qRT-PCR and flow cytometry measurement of cellular uptake of MRP substrates. Transendothelial electrical resistance (TEER) was monitored by cellular impedance of endothelial cell monolayers with and without Regadenoson to further describe permeability changes.

Results:

Membrane expression of Claudin-5, Occludin, and ZO-1 was decreased approximately 30 minutes after Regadenoson treatment. Claudin-5 and Occludin expression returned to basal levels after 8 hours whereas, ZO-1 expression returned to baseline expression by 4 hours after treatment. Total protein expression was unchanged with Regadenoson treatment, but the cytoplasmic expression of tight and adherens junctions’ proteins were increased; suggesting that changes in protein localization is the cause of transient disruption. Eight hours after treatment, protein concentration and tRNA expression of ABCB1 and ABCG2 was decreased by 50%. TEER measurements demonstrated a 40% reduction in cell impedance after Regadenoson.

Conclusion:

Our results demonstrate that A2AR agonism causes transient changes in junctional and ABC transporter expression and functionality. The short duration of Regadenoson effects on BBB function may have important implications for its clinical use to enhance drug delivery of chemotherapy and other therapies for CNS diseases.

Citation Format: Amelie Vezina, Matthew McCord McCord, Mark R. Gilbert, Sadhana Jackson. Transient modulation of the blood-brain barrier with time dependent adenosine receptor agonism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5216. doi:10.1158/1538-7445.AM2017-5216