Abstract 2387: Modulation Of P35/cyclin-dependent Kinase 5 Signaling Rectifies Defective Human Brain Endothelial Cell Migration Produced By In Vitro Simulation Of Ischemic Stroke

Uncontrolled activation of calcium-sensitive calpain pathways induces neuronal damage during ischemic stroke. Vascular remodelling plays a critical role in tissue recovery. The main molecular events underpinning angiogenesis in stroke are still imprecise. Recently, we highlighted a novel non-neuronal role of the cyclin-dependent kinase (Cdk)-5 operating through p35 activator, which modulates in vitro brain angiogenesis. Cdk5 activators p35 and p25 were recently shown to be involved in neuronal protection (p35) or death (p25) after brain ischemia. However, the impact of p35/Cdk5 on angiogenesis during hypoxic injury remains unclear. Here, using the calpain inhibitor MDL28170 and GFP-Cdk5 wild type (wt) or kinase mutant (D144N) transfectants in an in vitro model of stroke, we tested the hypothesis that activation of p35/Cdk5 pathway may positively modulate angiogenesis during hypoxia. Human brain microvascular endothelial cells (hCMEC/D3) were treated (24h) with MDL28170 (10μM) under conditions of hypoxia (1%O 2 ) or normoxia. Angiogenesis was investigated by wound-healing assay and capillary-tube formation on matrigel, using Cell-IQ® Imaging system (Chip-Man Technologies Ltd). Cell viability was evaluated by assay of MTT proliferation, propidium iodide nuclear inclusion (nPI) and heat shock protein 70 (Hsp70) level. p35, Cdk5 and activated pY(15)Cdk5 intracellular localization were analyzed by confocal microscopy. Protein levels were estimated by western blot. Inhibition of calpain activity increased angiogenesis (p