Hypoxia induces purinergic receptor signaling to disrupt endothelial barrier function

Abstract

Blood-brain-barrier permeability is regulated by endothelial junctional proteins and is vital in limiting access to and from the blood to the CNS. When stressed, several cells, including endothelial cells, can release nucleotides like ATP and ADP that signal through purinergic receptors on these cells to disrupt BBB permeability. While this process is primarily protective, unrestricted, uncontrolled barrier disruption during injury or inflammation can lead to serious neurological consequences. Purinergic receptors are broadly classified into two families: the P1 adenosine and P2 nucleotide receptors. The P2 receptors are further sub-classified into the P2XR ion channels and the P2YR GPCRs. While ATP mainly activates P2XRs, P2YRs have a broader range of ligand selectivity. The P2Y1R, essential for platelet function, is reportedly ubiquitous in its expression. Prior studies using gene knockout and specific antagonists have shown that these approaches have neuroprotective effects following occlusive stroke. Here we investigated the expression of P2Y1R in primary cultured brain endothelial cells and its relation to the maintenance of BBB function. Results show that following in vitro hypoxia and reoxygenation, P2Y1R expression is upregulated in both control and diabetic cells. At the same time, endothelial junctional markers, ZO-1 and VE-cadherin, were downregulated, and endothelial permeability increased. siRNA knockdown of P2Y1R and MRS 2500 effectively blocked this response. Thus, we show that P2Y1R signaling in endothelial cells leads to the downregulation of endothelial barrier function.

Keywords: blood-brain barrier; diabetes; endothelial cells; hypoxia; purinergic receptor.

FIGURE 1

Upregulation of endothelial P2Y1 receptor post-H/RO decreases junctional protein expression. Representative Western blots showing the expression of VE-cadherin, ZO-1, P2Y1 receptor and actin in (A), control and (C), diabetic endothelial cells. (B,D) Bar graphs for the same presented fold change in total protein and as mean ± SE. n = 5 for each, * indicates p < 0.05 vs. normoxic control, # indicates p < 0.05 vs. untreated normoxic diabetic (U in bar chart). (E) Representative Western blots after surface biotinylation in control and diabetic cells showing P2Y1R expression in surface (S) and intracellular (I) fractions with or without H/RO (24 h). (F) Mean data showing the surface expression of the protein alone expressed as % total protein.

FIGURE 2

P2Y1 receptor signaling decreases endothelial junctional protein expression. Representative Western blots from control cell lysates showing the expression of VE-cadherin, ZO-1, P2Y1 receptor and actin in (A), with or without H/RO and P2Y1R siRNA and (B), bar graph for the same showing fold change in total protein. Mean ± SE. n = 5 for each, * indicates p < 0.05 vs. normoxic scrambled or no-treatment controls, # indicates p < 0.05 vs. H/RO (24 h) treatment. (C) Representative Western blots from control cell lysates showing the expression of P2Y1R after addition of ADP to the extracellular media at specific concentrations. (D) Mean data. n = 5 for each, * indicates p < 0.05 vs. untreated controls. (E) Representative Western blots from control cell lysates showing the expression of proteins in control cells with or without H/RO and Xestospongin C (Xes C) or bisindolylmaleimide II (BIM II) treatment. (F) Bar graphs of mean protein expression post-H/RO presented as fold change. n = 5 for each, * indicates p < 0.05 vs. normoxic controls, # indicates p < 0.05 vs. untreated H/RO.

FIGURE 3

P2Y1R antagonist, MRS 2500, rescues endothelial function after H/RO. Representative Western blots showing the expression of VE-cadherin, ZO-1, P2Y1 receptor and actin in (A), control and diabetic endothelial cells with or without H/RO and MRS 2500 treatment. Bar graphs for protein expression in control, (B), and diabetic cells, (C), presented as fold change in total protein and mean ± SE. n = 5 for each, * indicates p < 0.05 vs. untreated control, # indicates p < 0.05 vs. respective H/RO (24 h) treatment. (D). Bar graph showing fold change in endothelial transwell permeability in normoxic, after H/RO (24 h) alone and H/RO (24 h) +MRS 2500 treated cells. n = 6 for each. * indicates p < 0.05 vs. normoxic control, # indicates p < 0.05 vs. normoxic diabetic and δ indicates p < 0.05 vs. respective H/RO (24 h).