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

Abstract

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<0.01), augmenting p35 protein levels (30%). Hypoxia induced cell stress, as evidenced by increased Hsp70 (0.8 ±0.02 vs 0.5±0.01, ratio on GAPDH optical density) and nPIs, and hampered cell migration and tube formation (p<0.01). This was associated with reduced p35 (56%, ratio on GAPDH optical density) contents. Blocking Cdk5 activity with D144N transfectant reduced migration (p<0.05). Cdk5 (wt) overexpression enhanced migration, which was further augmented in hypoxia (p<0.05). Hypoxia impaired reorganization of the cytoskeleton and reduced p35/actin co-localization, without affecting the distribution of Cdk5 or pY(15)Cdk5 in association with actin filaments and focal tips. MDL28170 significantly reversed the effects of hypoxia on cell migration and p35 localization, suggesting a role of p35 in cytoskeleton stability. MDL28170 further increased cell migration in Cdk5(wt) or D144N either in hypoxia or normoxia, and Cdk5 protein levels in hypoxia (0.2 ±0.01 vs 0.18±0.01, ratio on GAPDH optical density), implying that increased p35 may be protective during stroke. In conclusion, our data suggest that p35/Cdk5 signaling supports angiogenesis in stroke. p35 may be a signalling intermediate, involved in cytoskeleton organization and cell dynamics during angiogenesis.