Abstract WP254: Inhibition of Calcium/Calmodulin-Dependent Protein Kinase Kinase (CaMKK) Exacerbates Endothelial Cell Impairment After Cerebral Ischemia

CaMKK (α and β) is a major kinase activated by elevated intracellular calcium. We have previously demonstrated CaMKK is neuroprotective in stroke. Pharmacological inhibition of CaMKK exacerbated outcome while overexpression reduced brain injury in mice. Brain microvascular endothelial cells play an essential role in maintaining BBB integrity, disruption of which aggravates ischemic injury. Interestingly, CaMKK activates a key endothelial protectant Sirtuin1 (SIRT1), although this interaction has never been studied in stroke. We hypothesized that CaMKK protects brain endothelial cells (EC) through activation of SIRT1 after ischemia. Oxygen-glucose deprivation (OGD, 18 hours) with reoxygenation (24 hours) was performed in human brain microvascular endothelial cells (HBEC-5i). Cell viability was assessed using a CCK-8 assay. Middle cerebral artery occlusion (MCAO, 90 mins) was used to induce stroke in male mice (20 to 25 g). The CaMKK inhibitor STO-609, SIRT 1 inhibitor EX-527, or CaMKK β siRNA was added to EC before OGD. Data are presented as mean±SEM. STO-609 treatment reduced cell viability (%) after OGD (vehicle 71.45 ± 5.60 vs. STO-609 55.06 ± 5.02, n=4, p<0.05). Knockdown of CaMKK β using siRNA increased cell death following OGD. Mechanistically, inhibition of CaMKK by STO-609 led to significant down-regulation of phosphorated SIRT1 (Ser-27) after OGD. Levels of total SIRT1 were also reduced by STO-609, consistent with the notion that phosphorylation prevents the degradation of this protein. Changes in downstream targets (e.g., eNOS) of SIRT1 were accordingly observed with STO-609 treatment. Further, the effect of STO-609 on cell viability after OGD was absent when SIRT1 was concurrently inhibited by EX-527 (STO-609+EX-527 45.19 ± 2.09 vs. EX-527 44.42 ± 2.89, n=4, p>0.05). Finally, in a MCAO model, intracerebroventricular injection of STO-609 (pre-stroke) significantly worsened BBB integrity (Evans blue assay) after 24-hour reperfusion (MCAO+DMSO 17.83 ± 6.34 versus MCAO+STO-609 55.74 ± 8.88, n=5/6; P <0.05). In conclusion, we demonstrate that CaMKK inhibition reduces EC viability after ischemia via SIRT 1. Our data suggest CaMKK attenuates ischemic brain injury by protecting the microvascular system and preserving BBB integrity.