ELUCIDATING PRECONDITIONING-INDUCED, AKT-MEDIATED SIGNALING MECHANISMS OPERATIVE IN CEREBRAL ENDOTHELIUM THAT PROTECT THE MICROVASCULATURE FROM STROKE. Somalee Banerjee1, Yolanda Rangel2, Angela Freie2, Jeffrey M. Gidday2. Department of Biology1 and Department of Neurosurgery2, Washington University, St. Louis, MO.
Hypoxic preconditioning (HP) enhances cellular tolerance to ischemic injury in a variety of organs; however the mechanisms underlying this protective effect have yet to be fully elucidated. Protein Kinase B (Akt) is an intracellular kinase that regulates, by phosphorylation, the activity of a number of different proteins that have a role in proliferation, apoptosis, angiogenesis, and cell survival. It is also known that Akt is phosphorylated under hypoxic conditions. Since endothelial cells are the first to be affected in the pathway to ischemic injury, maintaining their integrity is the body’s primary line of defense to limit tissue injury. Therefore, a study of the mechanisms that give brain microvascular endothelial cells their unusual resistance to injury could lead to the identification of novel pathways that could also reduce injury in neurons following stroke. Thus, the focuses of my project was to characterize the temporal expression of Akt and phosphorylated Akt (pAkt) in the cerebral microcirculation and identify some of its downstream effectors mediating vascular protection in an adult mouse model of stroke. I hypothesized that HP would increase pAkt levels in the microvessel endothelial cells, and that this would lead to the phosphorylation of one or more downstream molecular targets of Akt that would then account for the vascular protection. One possible pro-survival effector I chose to examine was endothelial nitric oxide synthase (eNOS), which, when activated by phosphorylation, produces nitric oxide which, in turn, exhibits anti-apoptotic and other protective effects within endothelial cells and at the blood-endothelial interface.
Groups of adult male Swiss Webster/ND4 mice (8-10 weeks of age) were exposed to a HP stimulus (4 hours at 8% oxygen) after which they were recovered under normoxic conditions prior to being sacrificed either immediately after preconditioning, or at 2, 6, 24, or 48 hours later. Cerebral microvessel-rich preparations from four individual mice were pooled together and used for immunoblotting analysis. Results from this set of experiments demonstrated that Akt showed a marked increase immediately after HP and stayed elevated above the naïve levels at all other time-points. Expression levels of pAkt also showed a maximum upregulation immediately after HP and returned to naïve levels by 6 h after HP.
In order to facilitate the identification of downstream pAkt phosphorylation targets, the PI3-kinase inhibitor LY294002 (LY), which inhibits the phosphorylation of Akt, was administered to separate groups of mice 45 minutes prior to HP. Expression levels of eNOS and phosphorylated eNOS (p-eNOS) were measured to examine the necessity of pAkt in activating and maintaining eNOS protein levels. Protein profiles in cerebral microvessel fractions from mice treated with LY and untreated control mice were examined at identical post-HP timepoints by immunoblotting. Results from this set of experiments showed that LY markedly decreased the expression of both Akt and pAkt normally observed to be elevated immediately after HP. Also, there was a marked decrease in the expression levels of both eNOS and p-eNOS immediately following hypoxic preconditioning in mice treated with LY as compared to those without the inhibitor, suggesting that pAkt phosphorylates and maintains p-eNOS levels in microvessel endothelial cells in response to HP.
Collectively, these results are the first to demonstrate the temporal profile of cerebral microvascular-specific expression of Akt and pAkt following hypoxic preconditioning. That HP did not increase p-eNOS levels was unexpected; this result will be investigated further. Moreover, I will continue to leverage this p-Akt inhibitor-based experimental design to identify other phosphorylated proteins regulated by pAkt that are integral to HP-induced protection of the endothelial cells that comprise the cerebral microcirculation.