Etiology of microvascular changes in Gram-positive sepsis: mechanisms and therape

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DESCRIPTION (provided by applicant): Methicillin-resistant Staphylococcus aureus (MRSA) infection has become a serious health problem both in the community and in hospitals. ~20,000 people die annually in the U.S. because of MRSA, the rate is greater than those die from AIDS. The goal of the present proposal is to characterize novel mechanistic aspects of microvascular hyper-permeability during MRSA pneumonia/sepsis and offer novel and efficient therapeutic options that will reduce morbidity and mortality. Our general hypothesis is that stimulation of arginine vasopressin V1a receptors (V1aR) will mitigate MRSA sepsis-induced vascular hyper-permeability and reverse the hypotension, whereas the stimulation of the arginine vasopressin V2 receptors (V2R) will augment the vascular hyper- permeability and will not affect the hypotension. We propose to elucidate the downstream mechanisms, focusing on the interaction between V1aR, V2R, reactive nitrogen species (RNS), and potent angiogenic growth factors such as angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF). Aim 1: In an ovine MRSA sepsis model, establish mechanisms of severe vascular hyper-permeability, focusing on opposing effects of V1aR and V2R on release of potent permeability factor Ang-2 and microvascular hyper- permeability. Hypotheses: 1) V1aR activation reduces vascular hyper-permeability by inhibiting Ang-2 release; 2) V2R stimulation increases vascular hyper-permeability by promoting Ang-2 release; 3) a selective V1aR agonist is superior to a mixed V1aR/V2R agonist arginine vasopressin (AVP) for preventing cardiovascular collapse. Aim 2: In an ovine MRSA sepsis model, determine that novel and selective V1aR agonist will mitigate severe vascular leakage by preventing RNS formation through inhibiting excessive nitric oxide production. The down- stream mechanisms of how RNS cause vascular leakage will be also determined. Hypotheses: 1) The vascular hyper-permeability caused by RNS is mediated by Ang-2 release and VEGF expression; 3) RNS cause hypo- reactivity of V1aR: 3) V1aR agonist reduces RNS formation by inhibiting excessive nitric oxide production. Aim 3: In human cell culture studies, determine the molecular mechanisms of MRSA-induced severe vascular hyper-permeability with especial reference to downstream mechanisms of how V2R activation causes, and of how V1aR agonist inhibits, vascular hyper-permeability in MRSA sepsis. Hypotheses: 1) V2R stimulation pro- motes Ang-2 release in a MAPK-dependent pathway; 2) The V1aR agonist suppresses V2R and thus prevents Ang-2 release; 3) V1aR agonist modulates interaction of Ang-2 with Tie-2 receptors, most probably by sup- pressing the Tie-2 activation; 4) VEGF promotes Ang-2 release by its direct action or via V2R stimulation; 5) V2R activation in MRSA sepsis is mediated by RNS; 6) V1aR is oxidized/nitrated by RNS. To achieve the aims, the study will utilize a novel ovine model of MRSA sepsis and sophisticated cell culture techniques. We are particularly optimistic about the possibility that the proposed research will lead to translational clinical studies for agonists of the selective V1aR and peroxynitrite modulators.