Project Summary/Abstract Numerous studies show the mechanisms by which sickle cell disease (SCD) induces vasculopathy and increases vasocongestion are complex and multifactorial. In this revised, renewal application, we hypothesize that SCD induces vasculopathy as one of the first steps in the mechanism by which SCD increases vaso- occlusion. Our studies show that SCD induces a destructive cycle that is initiated by MPO and propagated by high mobility group box-1 (HMGB1), and one other inflammatory component that alters pulmonary physiology, impairs vascular function, and increases vasocongestion. Previously, we reported L-acetyl-lysyltyrosylcysteine amide (KYC) inhibits MPO, improves vascular function and reduces liver injury induced by excessive vasocongestion in SCD mice. New studies suggest that KYC not only reduces sickle RBC (sRBC) congestion but also increases the number of round sRBC in the lungs of SCD mice. Mechanistic studies reveal that KYC isn't just an inhibitor of MPO toxic oxidant production, but rather, is a unique tripeptide substrate that exploits MPO peroxidase activity to be converted into a novel anti-inflammatory agent that inactivates HMGB1 and activates the cellular pathways that are responsible for antioxidant defense enzyme expression in the lung. As KYC inhibits multiple inflammatory components in our hypothesized destructive cycle, and even activates a component that mediates antioxidant gene expression, new studies using mechanistic inhibitors are required for determine which components increase vasculopathy and vasocongestion in SCD. While a systems pharmaceutical agent may be useful for treating multifactorial diseases, they cannot be used to identify causal mechanisms directly. In this revised application, we hypothesize that SCD induces a destructive cycle, mediated by at least three major components. Our working hypothesis is SCD induces a destructive cycle that is composed of MPO, HMGB1 and a novel, dysregulate gene and together induce vasculopathy and increase vasocongestion. By treating sickle mice, sickle MPO knockout (ko) mice, chimeric sickle Tamoxifen- inducible HMGB1 ko mice and another chimeric sickle ko mice with highly selective mechanistic inhibitors we will be able to determine if and the extent to which MPO, HMGB1 and the third gene product, alone and/or in combination induces vasculopathy and increases vasocongestion. To assess vasculopathy, we will quantify differences in pulmonary artery relaxation, pulmonary permeability, sRBC vasocongestion, and susceptibility of each mouse strain to sRBC vasocongestion induced by hypoxia-reoxygenation injury (HRI) in Townes homozygote sickle Hb (SS) wt SS Mpo ko mice, and chimeric SS novel gene ko mice. Our long-term goals are to confirm the identities of each component and develop novel therapies aimed at improving vascular function and reducing sRBC vasocongestion.