Homocysteine Induces Phosphorylation of Y731 on VE-cadherin by Activating a Metabotropic-to-Ionotropic Glutamate Receptor Cascade

Elevated plasma homocysteine (Hcy) induces multiple pathologic effects in vascular endothelium, but the mechanisms are poorly understood. Endothelial cell-cell (EC-EC) junctions are critical for maintaining the integrity of the endothelium. The adherens junction protein VE-cadherin is important in maintaining and regulating EC-EC junctions. Phosphorylation of VE-cadherin is a major signaling mechanism controlling EC-EC adherence. In neuronal tissue, Hcy activates both ionotropic (NMDA r ) and metabotropic (mGluR 5 ) glutamate receptors, which we have found to be expressed on cerebral endothelial cells. We tested the hypotheses that 1) Hcy induces phosphorylation of Y731 on VE-cadherin by activating mGluR 5  and NMDA r  and 2) this phosphorylation is dependent on activation of PKC. Using cerebral microvascular endothelial cells (bEnd.3), we found that Hcy increases phosphorylation (western blot and ELISA) of Y731 on VE-cadherin in a dose- and time-dependent manner (p<0.05). This response is attenuated with NMDA r  and mGluR 5 specific antagonists, memantine (100 μM) and MPEP (10 μM) respectively (p<0.05). Treating bEnd.3 with the NMDA r  and mGluR 5  specific agonists, NMDA (25 μM) and CHPG (25 μM), also increases Y731 phosphorylation on VE-cadherin (p<0.05). Activating the mGluR 5 receptor with CHPG and simultaneously inhibiting the NMDA r  receptor with memantine ameliorates the response of CHPG alone, while activating NMDA r  and inhibiting mGluR 5 does not (p<0.05). PKC inhibition with bisindolylmaleimide-I (1 μM) reduced Hcy, NMDA, and CHPG induced Y731 phosphorylation on VE-cadherin (p<0.05). All of the p-values expressed here represent n=10–32 where experiments were repeated 2–4 times. Our data show that Hcy induces phosphorylation of Y731 on VE-cadherin by activating mGluR 5 receptors, which in turn drives an NMDA r -dependent response. PKC activation is required in this signaling cascade. These data demonstrate a fundamentally new mechanism for Hcy-mediated vascular dysfunction and present a new set of targets for testing therapeutic strategies in hyperhomocysteinemia.