The overall objective of the proposed studies in this renewal application is to define the mechanisms by which the procoagulant thrombin, a serine protease released during clotting initiated by sepsis or vascular injury, regulates the expression of adhesion molecule, ICAM-1, in endothelial cells, and thereby endothelial adhesivity toward neutrophil (PMN). In the last grant cycle, we showed that PKC(, PI3 kinase, and the downstream target Akt mediate thrombin-induced IKK/NF-(B activation and ICAM-1 expression in endothelial cells. We now have evidence that thrombin-induced IKK/NF-(B activation and ICAM-1 expression are tightly regulated through the functional coupling of PKC( and PI3 kinase to the mammalian target of rapamycin (mTOR). Our data show that mTOR down-regulates thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-(B. In light of these findings, we hypothesize that thrombin activates mTOR complexes via PKC(- and PI3 kinase/Akt-dependent pathways and that activated mTOR complexes in turn down-regulate IKK/NF-(B activation and ICAM-1 expression in endothelial cells. We further postulate that loss of mTOR-mediated inhibition of thrombin- induced endothelial adhesivity leads to marked exacerbation in lung PMN sequestration and PMN-dependent lung vascular injury and tissue edema. We will address this hypothesis by completion of the following specific aims.
Aim 1 : Determine the role of mTOR complexes, mTOR-raptor and mTOR-rictor, in modulating thrombin-induced IKK/NF-(B activation, ICAM-1 expression, endothelial adhesivity, and PMN migration across endothelial barrier.
Aim 2 : Determine the role of PKC( in signaling thrombin-induced activation of mTOR complexes and in thereby modulating IKK/NF-(B activation, ICAM-1 expression, and PMN migration across endothelial barrier.
Aim 3 : Determine the role of PI3 kinase/Akt in mediating thrombin-induced activation of mTOR complexes, and in thereby modulating NF-(B activation, ICAM-1 expression, and PMN migration across endothelial barrier. Studies will utilize multidisciplinary approaches ranging from biochemical, cellular, and molecular biology to lung physiology to identify the critical inhibitory pathways that are integrated with the stimulatory pathways but in a temporally divergent fashion to tightly regulate ICAM-1 expression induced by thrombin. With the knowledge gained, we believe that it will be possible to block PMN-mediated lung vascular injury by promoting the specific inhibitory signaling events controlling ICAM-1 expression associated with intravascular coagulation. These studies may provide future directions for development of therapeutic strategies for inflammatory disease states such as Acute Respiratory Distress Syndrome (ARDS).
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