Project Summary/Abstract Control of lung permeability by oxidized phospholipids Oxidative stress during pneumonia or septic lung injury generates a spectrum of bioactive oxidized lipids and proteins. While terminal products of lipid peroxidation exhibit deleterious effects, other groups, such as cyclopenthenone-containing products of phosphatidyl choline oxidation (OxPAPC) exhibited anti-inflammatory and vasoprotective properties in experimental models of inflammation. We have previously described potent effects of OxPAPC in preventing the lung injury and vascular barrier dysfunction. Products of phospholipid oxidation are unstable and may convert to barrier disruptive fragmented phospholipids. To overcome this problem, we developed and successfully tested a new class of synthetic phosholipase-resistant OxPAPC analogs based on structure-function analysis of barrier protective OxPAPC products. Testing synthetic OxPL compounds generated on our lab revealed that D-ring prostanoids not only showed barrier-protective and anti- inflammatory effects, but also effectively attenuated inflammation-associated apoptosis/necroptosis of lung vascular endothelium. In pilot studies, phospholipase resistant phospholipid containing D-ring prostanoid (D2PC) showed strong protective effects in the in vitro and in vivo models of Staphylococcus aureus (SA)- induced ALI. Pilot analysis of anti-inflammatory mechanisms triggered by D2PC revealed increased expression of KLF2 and KLF4, the two transcription factors regulating anti-inflammatory defense in vascular endothelium. Our pilot studies also suggest a link between D2PC-induced KLF2/4 upregulation and inhibition of inflammation-induced expression of thioredoxin interacting protein (TXNIP) involved in activation of NLRP3 inflammasome. In addition, D2PC upregulated guanine nucleotide exchange factor Epac1. Besides the documented role of Epac1-Rap1 GTPase signaling in the maintenance of lung EC barrier, our pilot studies show that D2PC suppressed SA-induced EC apoptosis/necroptosis via Epac1-dependent inhibition of death receptor 5 (DR5) within the caveolin enriched microdomains (CEM). The overall hypothesis of this study is that synthetic phospholipid D2PC mitigates SA-induced ALI and promotes lung recovery by protecting lung barrier function, attenuating inflammation and endothelial apoptosis via mechanisms that involve: a) KLF2/4- dependent downregulation of TXNIP and inactivation of NLRP3 inflammasome; and b) Epac1-mediated inhibition of DR5-dependent apoptotic/necroptotic signaling caused by SA. Aim-1 will investigate protective effects of synthetic D2PC in cell and animal models of SA?induced injury and evaluate CEM-associated receptors mediating D2PC effects. Aim-2 will study the anti-inflammatory mechanism of D2PC via KLF2/4- dependent suppression of TXNIP/NLRP3 cascade. Aim-3 will investigate anti-apoptotic/necroptotic properties of D2PC via Epac1-mediated inhibition of DR5 signaling. Characterization of a novel synthetic lipid compound and identification of molecular mechanisms governing its anti-inflammatory and cytoprotective properties may open a new avenue for treatment of ARDS and sepsis by mitigating lung injury and promoting lung repair.