Abstract 889: Increase Cellular Senescence and Cerebral Infarct Size in Mice with Chronic Activation of Endothelial Protein Kinase Akt
Background: Protein kinase Akt is a multifunctional serine-threonine protein kinase implicated in diverse cellular functions including cell growth, proliferation, and survival. However, loss-of-function mutations of Akt in yeast and Drosophila lead to improved resistance to oxidative stress and prolongation of lifespan. Thus, the precise role of Akt in cellular senescence and survival remains unclear. We hypothesize that chronic activation of endothelial Akt increases endothelial cell senescence and apoptosis, and that this may contribute to worsening of endothelium-dependent cerebrovascular injury.
Methods and Results: Using the tet-off system for tissue-specific inducible gene expression, we have developed double transgenic mutant mice with chronic activation of endothelial Akt (ΔecAkt mice) by crossing endothelial-specific VE-cadherin-tTA transgenic mice with constitutively-active tet-o-myrAkt transgenic mice. Induction of endothelial mutant Akt expression and activity was performed by withdrawal of doxycycline at age of 8 weeks. Chronic activation of endothelial Akt was achieved by withholding doxycycline for 8 weeks. Control and ΔecAkt mice with and without doxycycline withdrawal were then subjected transient cerebral ischemia by intraluminal filamentous middle cerebral artery occlusion (MCAO) for 2 hrs, followed by 22 hrs of reperfusion. Chronic activation of endothelial AKT increased stroke size and brain edema by 38% and 50%, respectively, compared to control or ΔecAkt mice without doxycycline withdrawal (P<0.05 for both). This corresponded to worsening of neurologic deficit score from 2.0 to 2.8 (P<0.05). Endothelial cells from ΔecAkt mice with doxycycline withdrawal exhibited increased cellular senescence as determined by reduced trimethylation of Lys9 on histone H3, progeria-like dysmorphic nuclear shape, and decreased lamin-A associated proteins.
Conclusion: These findings indicate that chronic activation of Akt, as oppose to short-term transient activation, in the endothelium leads to endothelial cell senescence and loss of endothelium-dependent stroke protection. Thus, factors or conditions that chronically activates Akt in the endothelium may have deleterious effects on stroke outcomes.