The Effects of Ozone-Induced Oxysterols on Thrombotic Risk

Abstract Almost a third of the US population is exposed to ozone (O3) levels that are unsafe according to US EPA standards. These levels are deemed unsafe primarily because O3 has been shown to cause inflammation and negatively impact the respiratory system. One mechanism by which O3 contributes to health complications is by forming oxysterols, a result of oxidizing cholesterol and cholesterol-like precursors. These oxysterols can cause endothelial cell stress and dysfunction, as well as contribute to the formation of atherogenic plaques in the vasculature. These effects of oxysterols have inspired the investigation of how oxysterols might also negatively impact the cardiovascular system. This interest is partially driven by the 30% increase in cardiovascular disease observed in the US over the last 30 years without an understanding of all the contributing mechanisms to this trend. A potential way O3-induced oxysterols could be impacting cardiovascular disease is by increasing risk of thrombosis. Oxysterols form protein adducts (particularly at lysine residues) that can disrupt the functions of other proteins, with an example being the oxysterol adduction of the liver X receptor (LXR). This also suggests that oxysterols in plasma could be forming protein adducts with coagulation factors. Interestingly, prothrombotic changes in plasma have been identified after O3 exposure via unbiased proteomics. This evidence supports our hypothesis that ozone exposure and oxysterol generation can increase risk of thrombosis. We will test this hypothesis through the following specific aims (SA): In SA1, to investigate the mechanisms by O3 oxysterols might contribute to thrombotic risk, we will investigate if O3-induced oxysterols induce prothrombotic endothelial activation. Culturing endothelial cells in vitro, we will expose them to various concentrations of O3-induced oxysterols and proinflammatory cytokines. Subsequently, we will perform RNA-seq to determine if any of the differentially expressed genes might exacerbate thrombotic risk. Additionally, we will assess endothelial barrier integrity using trans-endothelial electrical resistance (TEER). In SA2, we will use mass spectrometry and “click” cycloaddition chemistry to determine if any coagulation factors have adducted to any oxysterols in samples from O3-exposed participants. We will also determine how controlled O3-exposure affects in vitro thrombin generation (TG) and plasmin generation (PG) assays. We expect that we will observe more prothrombotic TG and/or PG parameters from the plasma of O3-exposed participants compared to controls. The specific regions of adduction on any coagulation factors will allow us to evaluate potential mechanistic consequences to protein function, and thrombotic risk by extension. Together, data from these experiments will inform how O3 exposure and O3- induced oxysterols might impact cardiovascular disease by identifying specific biomarkers and mechanisms to clearly link oxidant air pollutant exposure with thrombotic risk.