Abstract 16456: Homocysteine Increases Cerebral EC Barrier Permeability Through the NMDA Receptor in vitro and in vivo
Hyperhomocysteinemia (HHcy) is a risk factor for vascular and neurologic diseases. HHcy increases blood-brain barrier (BBB) permeability, but the mechanisms are unknown. Claudin-5 (CLDN5) is a tight junction protein critical to BBB integrity though its role in Hcy-mediated BBB permeability is unexplored. Memantine, an antagonist for the N-methyl-D-aspartic acid (NMDA) receptor, protects against glutamate-mediated BBB permeability. Hcy can activate NMDA receptors expressed on neurons; therefore, we tested the hypothesis that Hcy decreases CLDN5 expression and increases BBB permeability through NMDA receptors expressed on endothelial cells. Expression of NMDA receptors on brain microvascular endothelial cells was confirmed by immunocytochemistry, western-blotting and flow-cytometry. All data are expressed in relative units as mean±SEM, all experiments n=3–10. In Vitro: Endothelial cells (bEnd.3) were grown to confluence in 24 well plates or transwell inserts (0.4μm pores) and culture media was conditioned with saline, Hcy (20 μM), or Hcy (20 μM) + memantine (100 μM) every 12 hrs for 3 days. Hcy reduced CLDN5 expression compared to control (3.59±0.54 vs. 7.17±0.68; p<0.05), which was restored with memantine treatment (6.98±0.89; p<0.05). Hcy treatment led to a significant increase in endothelial monolayer permeability (dye flux through transwell), which was restored by memantine (p<0.05). In Vivo: Brain homogenates from HHcy mice (CBS +/−) also had a reduction in CLDN5 expression compared to wild-type littermates (0.3±0.04 vs. 0.14±0.006; p<0.05), which was attenuated by supplementing the drinking water of CBS (+/−) mice with memantine for 2 wks (0.20±0.01; p<0.05). Increased BBB permeability observed in CBS +/− mice (0.13±0.01 vs. 0.51±0.09) was reversed with memantine treatment (0.2±0.01; p<0.05). We conclude that Hcy decreases endothelial cell CLDN5 expression and increases BBB permeability by a NMDA-receptor dependent mechanism. Modulating NMDA receptor activation by Hcy in the cerebral microvasculature may provide a novel therapeutic approach to the vascular and neurodegenerative impact of HHcy.
- © 2010 by American Heart Association, Inc.