Ox-PAPC activation of PMET system increases expression of heme oxygenase-1 in human aortic endothelial cell.

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) has been demonstrated to accumulate in atherosclerotic lesions and regulates expression of more than 1,000 genes in human aortic endothelial cell (HAEC). Among the most highly induced is heme oxygenase-1 (HO-1), a cell-protective antioxidant enzyme, which is sensitively induced by oxidative stress. To identify the pathway by which Ox-PAPC induces HO-1, we focused on the plasma membrane electron transport (PMET) complex, which contains ecto-NADH oxidase 1 (eNOX1) and NADPH:quinone oxidoreductase 1 (NQO1) and affects cellular redox status by regulating levels of NAD(P)H. We demonstrated that Ox-PAPC and its active components stimulated electron transfer through the PMET complex in HAECs from inside to outside [as determined by extracellular 2-(4-iodophenyl)-3-(44-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) reduction] and from outside to inside of the cell (as determined by intracellular NBT reduction). Chemical inhibitors of PMET system and siRNAs to PMET components (NQO1 and eNOX1) significantly decreased HO-1 induction by Ox-PAPC. We present evidence that Ox-PAPC activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in HAEC plays an important role in the induction of HO-1 and PMET inhibitors blocked Nrf2 activation by Ox-PAPC. We hypothesized that PMET activation by Ox-PAPC causes intracellular NAD(P)H depletion, which leads to the increased oxidative stress and HO-1 induction. Supporting this hypothesis, cotreatment of cells with exogenous NAD(P)H and Ox-PAPC significantly decreased oxidative stress and HO-1 induction by Ox-PAPC. Taken together, we demonstrated that the PMET system in HAEC plays an important role in the regulation of cellular redox status and HO-1 expression by Ox-PAPC.

Role of the Jak/STAT pathway in the regulation of interleukin-8 transcription by oxidized phospholipids in vitro and in atherosclerosis in vivo.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipid, 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine, induce endothelial cells (EC) to synthesize chemotactic factors, such as interleukin 8 (IL-8). Previously, we demonstrated a role for c-Src kinase activation in Ox-PAPC-induced IL-8 transcription. In this study, we have examined the mechanism regulating IL-8 transcription by Ox-PAPC downstream of c-Src. Our findings demonstrate an important role for JAK2 in the regulation of IL-8 transcription by Ox-PAPC. Treatment of human aortic EC with Ox-PAPC and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine induced a rapid yet sustained activation of JAK2; activation of JAK2 by Ox-PAPC was dependent on c-Src kinase activity. Furthermore, pretreatment with selective JAK2 inhibitors significantly reduced Ox-PAPC-induced IL-8 transcription. In previous studies, we also demonstrated activation of STAT3 by Ox-PAPC. Here we provide evidence that STAT3 activation by Ox-PAPC is dependent on JAK2 activation and that STAT3 activation regulates IL-8 transcription by Ox-PAPC in human EC. Transfection with small interfering RNA against STAT3 significantly reduced Ox-PAPC-induced IL-8 transcription. Using chromatin immunoprecipitation assays, we demonstrated binding of activated STAT3 to the sequence flanking the consensus gamma-interferon activation sequence (GAS) in the IL-8 promoter; site-directed mutagenesis of GAS inhibited IL-8 transcription by Ox-PAPC. Finally, these studies demonstrate a role for STAT3 activation in atherosclerosis in vivo. We found increased staining for activated STAT3 in the inflammatory regions of human atherosclerotic lesions and reduced fatty streak formation in EC-specific STAT3 knock-out mice on the atherogenic diet. Taken together, these data demonstrate an important role for the JAK2/STAT3 pathway in Ox-PAPC-induced IL-8 transcription in vitro and in atherosclerosis in vivo.

High-density lipoprotein modulates oxidized phospholipid signaling in human endothelial cells from proinflammatory to anti-inflammatory.

OBJECTIVE: Oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine (Ox-PAPC) and its component phospholipid, 1-palmitoyl-2-(5,6 epoxyisoprostanoyl)-sn-glycero-3-phosphocholine (PEIPC), which are present in atherosclerotic lesions, activate endothelial cells to induce a complex inflammatory and pro-oxidant response. Previously, we demonstrated induction of genes regulating chemotaxis, sterol biosynthesis, the unfolded protein response, and redox homeostasis by Ox-PAPC in human aortic endothelial cells (HAECs). Activation of the c-Src kinase/signal transducer and activator of transcription 3 and the endothelial nitric oxide synthase/sterol regulatory element binding protein (SREBP) pathways were shown to regulate several of these inflammatory effects of Ox-PAPC in HAECs. The goal of the current studies was to determine the role of high-density lipoprotein (HDL) in regulating Ox-PAPC signaling in HAECs. METHODS AND RESULTS: Using quantitative real-time polymerase chain reaction, Western analysis, and functional studies, we demonstrated that pretreatment of HAECs with HDL reduced the induction of inflammatory, sterol biosynthetic, and unfolded protein response genes by Ox-PAPC and PEIPC; Ox-PAPC-induced chemotactic activity and monocyte binding were also decreased. These effects were associated with HDL inhibition of Ox-PAPC-induced c-Src, signal transducer and activator of transcription 3, and SREBP activation, alterations in endothelial nitric oxide synthase phosphorylation (previously associated with the inflammatory action of Ox-PAPC), and a decrease in superoxide formation. Finally, we demonstrated that treatment with HDL did not inhibit Ox-PAPC and PEIPC-induced activation of redox pathways, which protect the cell from the effects of oxidative stress. CONCLUSIONS: Taken together, these studies demonstrated that HDL inhibits the pro-inflammatory effects of Ox-PAPC and PEIPC, while maintaining the antioxidant activities of these lipids.