Isolevuglandin peptide modification and proteasomal processing is responsible for autoimmune mediated hypertension

Vanderbilt University Medical Center, Nashville TN

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Abstract

PROJECT SUMMARY An estimated one-third of the world?s population suffers from hypertension. Over the last decade, it has become clear that immune activation contributes to hypertension. Reactive oxygen species (ROS) have been shown to be an important activator of this disease. Isolevuglandins (isoLG) are oxidation products of fatty acids that form as a result of ROS. These molecules adduct covalently to lysine residues of proteins. IsoLG adducted proteins are presented by antigen presenting dendritic cells (DCs) and result in CD8+ T-cell activation and resultant hypertension. This subset of T-cells is exclusively activated by peptides presented within MHC-I, implying a role of the proteasome in peptide processing. In preliminary studies I have found that isoLG-adducted peptides are markedly enriched in monocytes of patients with systemic lupus erythematosus (SLE). I propose to address the hypothesis that IsoLG modification of proteins is responsible for hypertension in SLE and that the presentation of IsoLG-modified peptides is proteasome dependent. In Aim 1, I will test the hypothesis that IsoLG modification of native proteins and their presentation on dendritic cells contribute to hypertension and vascular inflammation in a mouse model of SLE. This will be accomplished by utilizing the B6.SLE123 mouse model of SLE. IsoLG modified peptide presentation will be analyzed by flow cytometry at baseline, and after treatment with high salt. Blood pressure will be measured by carotid artery telemetry. Tissue inflammation will be analyzed by flow cytometry and histologic analysis. Co-treatment with an isoLG scavenger molecule 2-HOBA and hypertensive stimulus will then be performed. I predict that treatment of SLE mice with 2-HOBA will attenuate hypertension and tissue inflammation. In Aim 2, I will test the hypothesis that proteasomal processing of peptides mediates hypertension and isoLG-adduct presentation.. It is known that treatment of mouse dendritic cells with the oxidant tert-butyl hydroperoxide (TBHP) confers isoLG-mediated sensitivity to hypertensive stimulus in recipient mice. I have found that co-treatment of dendritic cells with TBHP and proteasome inhibitor (PI) attenuates surface isoLG presentation. I will co-treat wild-type mice with hypertensive stimulus and a PI. IsoLG surface protein presentation and tissue inflammation will be measured. I have also shown that proteasomal activity is increased in DC?s from hypertensive mice. This correlates with increased expression of the inflammatory immunoproteasomal subunit LMP7. We have generated an LMP7 conditional knockout mouse and will study the effects of LMP7 deletion on hypertension and isoLG-adduct presentation in DCs. !