Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function

ABSTRACT: Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-2-phosphatidylcholine (PAPC), referred to as OxPAPC, accumulate in atherosclerotic lesions and regulate over 1000 genes in human aortic endothelial cells (HAEC). We previously demonstrated that OxPAPC covalently binds to a number of proteins in HAEC. The goal of these studies was to gain insight into the binding mechanism and determine if binding regulates activity. In whole cells N-acetylcysteine inhibited gene regulation by OxPAPC, and blocking cell cysteines with N-ethylmaleimide strongly inhibited OxPAPC binding. Using MS, we demonstrate that most of the binding of OxPAPC to cysteine is mediated by PEIPC. We also show that OxPAPC binds to a model protein, H-Ras, at cysteines previously shown to regulate activity in response to 15dPGJ2. This binding was observed with recombinant protein and in cells overexpressing H-Ras. 15dPGJ2 and OxPAPC increased H-Ras activity at comparable concentrations. Using microarray analysis, we demonstrate a considerable overlap of gene regulation by OxPAPC and 15dPGJ2 in HAEC, suggesting that some effects attributed to 15dPGJ2 may also be regulated by OxPAPC since OxPAPC lipids and 15dPGJ2 both accumulate in inflammatory sites. Overall, we provide evidence for the importance of OxPAPC-cysteine interactions in regulating HAEC function. Control, OxPAPC, and 15dPGJ2-treated HAECs Samples. 'Control' is media (1%FBS, 99%M199)-only treated HAECs. 15dPGJ2 and OxPAPC are treated with those respective compounds in media.

ORGANISM(S): Homo sapiens

SUBMITTER: James Springstead

PROVIDER: E-GEOD-35709 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Regulatory T cells (Tregs) are protective in atherosclerosis but reduced during disease progression due to cell death and loss of stability. However, the mechanisms of Treg dysfunction remain unknown. Oxidized phospholipids (oxPLs) are abundant in atherosclerosis and can activate innate immune cells, but little is known regarding their impact on T cells. Given Treg loss during atherosclerosis progression and oxPL levels in the plaque microenvironment, we investigated whether oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), an oxPL associated with atherosclerotic plaques, alters Treg differentiation and function. Methods: CD4+ T cells were polarized to Treg, Th1, and Th17 cells with or without oxPAPC and assessed by flow cytometry. Gene expression in oxPAPC-treated Tregs was analyzed by bulk RNA sequencing. Functional studies of oxPAPC-induced Tregs were performed by co-culturing Tregs with CTV-labeled cells in vitro, and by adoptively transferring Tregs to hyperlipidemic Ldlr-/- mice to measure atherosclerosis progression. Results: Compared to controls, oxPAPC-treated Tregs were less viable, but surviving cells expressed higher levels of the Th1-associated markers T-bet, CXCR3, and IFN-. Th1 and Th17 skewing cultures were unaltered by oxPAPC. IFN- is linked to Treg instability, thus Treg polarization experiments were repeated using Ifngr1-/- CD4+ T cells. IFNR1 deficiency did not improve cell viability in oxPAPC-treated Tregs, however, T-bet and IFN- expression was not increased in surviving cells suggesting a role for IFN-signaling. OxPAPC-treated Tregs were less suppressive in vitro, and adoptive transfer studies in hyperlipidemic Ldlr-/- mice showed that oxPAPC-induced Tregs possessed altered tissue homing and were insufficient to inhibit atherosclerosis progression. Conclusions: OxPAPC elicits Treg-specific changes altering Treg differentiation and inducing a Th1-like phenotype in surviving cells partially through IFN- signaling. This is biologically relevant as oxPAPC-treated Tregs do not reduce atherosclerosis progression in Ldlr-/- mice. This study supports the role for oxPLs in negatively impacting Treg differentiation and atheroprotective function.

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Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function

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