PROJECT SUMMARY Novel therapeutic approaches to minimize neurovascular injury in stroke are needed. The cerebrovascular endothelium not only plays a critical role in blood brain barrier (BBB) disruption and exacerbation of neuronal injury but it also has a great therapeutic potential. However, the limited understanding of the endothelial specific molecular mechanisms involved in BBB disruption restricts progress towards developing novel therapeutic approaches specifically targeting the endothelium in stroke. Sphingosine-1-phosphate (S1P) is highly abundant in plasma, and is a potent modulator of endothelial function via its receptors (S1PR). We and others have shown that S1P via S1PR1 promotes endothelial barrier function in a Gi- phosphatidylinositol-3- kinase-(PI3K)-Akt dependent way in various organs. In sharp contrast, we found that S1PR2 expression is low under basal conditions but its upregulation upon injury induces endothelial permeability as well as endothelial inflammation via activation of G12/13-Rho-Rho kinase (ROCK)-Nuclear Factor-B (NFB) pathway. Therefore, upregulation of S1PR2 switches endothelial S1P signaling from anti-inflammatory to pro-inflammatory. In sepsis models, we have shown that endothelial S1PR2 plays a critical role in the induction of vascular permeability in various organs (e.g. lung, kidney) leading to perpetuation of systemic inflammation. However, in the cerebrovascular endothelium, the specific role of endothelial S1P signaling in BBB disruption upon ischemia and the impact on stroke outcomes are not understood. We found that S1PR2 is transcriptionally upregulated in cerebral microvessels in vivo after ischemia. In addition, global genetic or pharmacological inhibition of S1PR2 prevents the early breakdown of the BBB resulting in decreased neuronal injury. Our in vitro studies have revealed that S1PR2 regulates brain endothelial responses to ischemic injury (e.g. permeability), but not neuronal or glial responses. These findings have provided the basis for our central hypothesis that ischemia-induced transcriptional upregulation of S1PR2 in the endothelium exacerbates BBB dysfunction and brain injury and it can be targeted as novel vasoprotective therapy in stroke. Aim 1 will investigate the role of endothelial S1PR2 signaling in BBB dysfunction and its impact on brain injury, post- ischemic neuroinflammation and stroke outcome. Aim 2 will elucidate the molecular mechanisms governing the expression of S1PR2 in cerebral microvessels upon ischemia. Aim 3 will evaluate the therapeutic potential of targeting this pathway in stroke. Results of the proposed studies will define the novel role of endothelial S1P signaling in BBB dysfunction in stroke and its impact on brain injury. They will also help clarify the molecular mechanisms governing S1P signaling in the cerebrovascular endothelium upon ischemia. Overall, they are expected to have a positive translational impact for novel therapeutic interventions specifica...