Project Summary: Particulate matter (PM) air pollution is a common promoter for the development of thrombosis and cardiovascular disease. Specifically, exposure and deposition of PM in the alveolar capillary region (ACR) of the lung promotes thrombosis through platelet activation, impaired fibrinolysis, and increased coagulation factor production; however, the molecular mechanisms producing these adverse effects following PM exposure remain unclear. Our goal is to identify the vascular endothelium's role in promoting these thrombotic effects and elucidate the molecular mechanisms that result in a pro-thrombotic endothelium. To do this we have developed an organotypic in vitro model of the ACR in which we expose alveolar epithelial cells within the model to PM and determine the trans-epithelial (TE) effect in the underlying endothelial cells within the model. Our preliminary data suggests this TE exposure induces endothelial redox dysfunction, decreases the expression of anti-coagulant and fibrinolytic genes, and increases the expression of procoagulant genes. Our central hypothesis is TE exposure-induced redox dysfunction activates a pro-thrombotic endothelium by alternatively regulating the NF-κB and MAPK pathways. To test this hypothesis, our first aim is to define the relationship between the induced endothelial redox dysfunction and the activation of a pro-thrombotic endothelium. To define this relationship, we will compare the kinetics of redox dysfunction with the temporal expression of pro-thrombotic targets identified in our preliminary data and with the kinetics of functional thrombotic endpoints. Antioxidant intervention studies will then be performed to determine whether TE exposure-induced redox dysfunction drives the activation of a pro- thrombotic endothelium. Our second aim is to determine how the NF-κB and MAPK signaling pathways activate a thrombotic endothelium following a TE exposure. To determine these pathways' role we will evaluate the activation of the NF-κB, c-Jun N-terminal kinase (JNK), and p38 signaling pathways and will assess promoter binding at the activated pro-thrombotic targets of the downstream transcription factors of these pathways, p65, c-Fos/c-Jun, and ATF-2. Antioxidant intervention studies will then be performed to determine if TE-induced redox dysfunction mediates the activation of a pro-thrombotic endothelium by alternatively regulating these cellular pathways. The molecular mechanisms identified upon completion of these aims will help elucidate the vascular endothelium's role in promoting PMI-thrombosis. With these results, preventative and therapeutic strategies against PMI-thrombosis and related cardiovascular diseases can be developed. Completion of these aims will advance my training as a molecular toxicologist and support my development into a successful principal investigator.