Norbert Leitinger

Oxidized phospholipids induce expression of human heme oxygenase-1 involving activation of cAMP-responsive element-binding protein.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, protects against oxidative stress, and shows potent anti-inflammatory effects. Oxidized phospholipids, which are generated during inflammation and apoptosis, modulate the inflammatory response by inducing the expression of several genes including HO-1. Here we investigated the signaling pathways and transcriptional events involved in the induction of HO-1 gene expression by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in human umbilical vein endothelial cells. OxPAPC up-regulated HO-1 mRNA and protein in a time- and concentration-dependent manner, whereas pro-inflammatory agents like TNF-α and lipopolysaccharide did not significantly induce HO-1 expression in human umbilical vein endothelial cells. Signaling pathways involved in the OxPAPC-mediated HO-1 induction included protein kinases A and C, as well as the mitogen-activated protein kinases p38 and ERK. The cAMP-responsive element-binding protein (CREB) was phosphorylated via these pathways in response to OxPAPC treatment and expression of a dominant-negative mutant of CREB inhibited OxPAPC-induced activity of a human heme oxygenase-1 promoter-driven luciferase reporter construct. We identified a cAMP-responsive element and a Maf recognition element to be involved in the transcriptional activation of the HO-1 promoter by OxPAPC. In gel shift assays we observed binding of CREB to the cAMP-responsive element after OxPAPC treatment. Induction of HO-1 expression by lipid oxidation products via CREB may represent a feedback mechanism to limit inflammation and associated tissue damage.

Epoxycyclopentenone-Containing Oxidized Phospholipids Restore Endothelial Barrier Function via Cdc42 and Rac

After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.

Analysis of inflammatory gene induction by oxidized phospholipids in vivo by quantitative real-time RT-PCR in comparison with effects of LPS.

Oxidized phospholipids are thought to play a role in the development of atherosclerosis and other chronic inflammatory processes. In this study, we analyzed the expression of inflammatory genes induced by oxidized L-alpha-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholin (OxPAPC) in vitro and in vivo using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cultured human umbilical vein endothelial cells (HUVEC) and monocyte-like U937 cells were treated with OxPAPC or lipopolysaccharide (LPS) for 3 h. For in vivo studies, OxPAPC or LPS was injected intravenously into female C57Bl/6J mice and different tissues were isolated after 3 h. We found that both OxPAPC and LPS induced expression of early growth response factor 1 (EGR-1) and monocyte chemoattractant protein 1 (MCP-1) in HUVEC and of JE, the mouse homologue of MCP-1, in liver and heart. Interestingly, OxPAPC but not LPS increased expression of heme oxygenase 1 (HO-1) in U937 cells, HUVEC, aorta, heart, liver, and isolated blood cells. In contrast, E-selectin was selectively induced by LPS, but not by OxPAPC. Finally, OxPAPC-induced expression of HO-1 was blocked by a platelet-activating factor (PAF) receptor antagonist. We conclude that oxidized phospholipids are biologically active in vivo and exert a specific response inducing a pattern of genes that is different from that induced by LPS. In addition, we demonstrate that the quantitative real-time RT-PCR technology is a proper tool to investigate differential inflammatory gene induction in vivo.

Anti-inflammatory properties of lipid oxidation products.

Oxidative modification of lipids occurs during inflammatory processes and leads to the formation and accumulation of biologically active lipid oxidation products that induce specific cellular reactions. These reactions lead to a modulation of the inflammatory process and may determine the fate and outcome of the bodys reaction in acute inflammation during host defense. The processes by which oxidized lipids may play an important role include resolution of inflammation involving apoptosis, chronic inflammatory processes, and innate and adaptive immune responses. The classical view of lipid oxidation products is that they can induce and propagate chronic inflammatory reactions. However, evidence is accumulating that cells and tissues respond towards these oxidatively formed stress signals also by activation of anti-inflammatory processes. These include defense strategies such as (a) induction of signaling pathways leading to the upregulation of anti-inflammatory genes, (b) inhibition of signaling pathways coupled to the expression of proinflammatory genes, and (c) preventing the interaction of proinflammatory bacterial products with host cells. This contribution summarizes recent findings on the anti-inflammatory action of oxidized lipoproteins and lipid oxidation products. We discuss confirmed and suggested mechanisms as well as the (patho)physiological significance of these findings.

The influence of isoprostanes on ADP-induced platelet aggregation and cyclic AMP-generation in human platelets.

Isoprostanes are eicosanoids that are non-enzymatic products of free radical catalyzed peroxidation of arachidonyl containing phospholipids (1). They are subsequently released from the site of generation as esters of phospholipid (bound) or through the action of phospholipase(s) A2 in free form (2). One F2-isoprostane whose formation is highly favored is 8-iso-PGF2 alpha which has been shown to be a potent pulmonary and renal vasoconstrictor (3,4). Actions of 8-iso-PGF2 alpha were demonstrated to be mediated through a receptor related to but probably distinct from the thromboxane (TXA2)/endoperoxide (PGH2) receptor (5). Although 8-epi-PGF2 alpha is a potent agonist of TXA2/PGH2 receptors in vascular smooth muscle, interestingly it acts primarily as an antagonist of TXA2/PGH2 receptors on both human and rat platelets (6). There is also evidence for the generation of D- and E-ring isoprostanes (7) and their receptor-mediated action on smooth muscle cells (8) and platelets (9). Recent reports support the hypothesis that E2-isoprostane receptors are distinct from TXA2/PGH2 receptors, suggesting at least different subtypes, one of these specifically recognizing E2-isoprostanes (9). Isoprostanes have been suggested to be useful markers for oxidant injury. For example, F2-isoprostanes were significantly elevated in plasma of rats during reperfusion after hepatic ischemia (10) and in patients with hepatorenal syndrome (11). It has been suggested that the release of F2-isoprostanes from oxidized LDL in macrophages could be a contributory factor in the development of atherosclerosis and at sites of inflammation, locally elevated levels of isoprostanes could contribute to blood cell activation. In this study we investigate possible pro- or antiaggregatory properties of various F- and E-type isoprostanes on human platelets.

Oxidized Cholesteryl Linoleates Stimulate Endothelial Cells to Bind Monocytes via the Extracellular Signal–Regulated Kinase 1/2 Pathway

Oxidation products of cholesteryl esters have been shown to be present in oxidized low density lipoprotein and in atherosclerotic lesions. Monocyte adhesion to the endothelium is an initiating crucial event in atherogenesis. Here, we show that in vitro oxidized cholesteryl linoleate (oxCL) stimulated human umbilical vein endothelial cells (HUVECs) to bind human peripheral blood mononuclear cells as well as monocyte-like U937 cells but not peripheral blood neutrophils or neutrophil-like HL-60 cells. Among the oxidation products contained in oxCLs, 9-oxononanoyl cholesterol (9-ONC) and cholesteryl linoleate hydroperoxides stimulated U937 cell adhesion. OxCL-induced U937 cell adhesion was inhibited by an antibody against the connecting segment-1 region of fibronectin. Neither oxCL nor 9-ONC induced activation of the classical nuclear factor-&kgr;B pathway. In contrast, stimulation of HUVECs with oxCL resulted in phosphorylation of the extracellular signal–regulated kinase 1/2. Moreover, U937 cell adhesion induced by 9-ONC and oxCL was blocked by a mitogen-activated protein kinase/extracellular signal–regulated kinase inhibitor and a protein kinase C inhibitor. Taken together, oxCLs stimulate HUVECs to specifically bind monocytes, involving endothelial connecting segment-1 and the activation of a protein kinase C– and mitogen-activated protein kinase–dependent pathway. Thus, oxidized cholesteryl esters may play an important role as novel mediators in the initiation and progression of atherosclerosis.

20 MOLECULAR MECHANISMS OF LUNG ENDOTHELIAL BARRIER REGULATION BY OXIDIZED PHOSPHOLIPIDS

Increased levels of oxidized phospholipids generated by oxidation of shed cell membranes released into local circulation have been recently detected in acute lung injury and cardiac ischemia. This study characterized signaling pathways and barrier regulation induced in human pulmonary artery endothelial cells (EC) by a bioactive component of cell membrane-derived oxidized phospholipids, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). OxPAPC induced time-dependent activation of protein kinase C (PKC), protein kinase A (PKA), Raf/MEK1,2/Erk-1,2 MAP kinase cascade, JNK MAP-kinase, protein tyrosine phosphorylation, and small GTPases Rac and Cdcd42 without noticeable activation of Rho. These effects were specific to oxidized phospholipids, as non-oxidized species (PAPC, PLPC, DMPC) were without effect. OxPAPC caused sustained concentration-dependent increases in EC transendothelial resistance (TER) and alleviated increased permeability elicited by thrombin. This barrier-protective effect was linked to oxygenated, epoxyisoprostane-containing phospholipids, as confirmed by mass-spectrometric analysis of OxPAPC bioactive components. Pharmacological inhibitors of OxPAPC-induced signaling and depletion of small GTPases using siRNA approach showed direct involvement of PKC, PKA, Rac and Cdc42, but not MAP-kinase cascades and Rho GTPase in EC barrier-protective response. In addition, inhibition of PKC and PKA attenuated OxPAPC-induced activation of Rac, suggesting that PKC and PKA are upstream of Rac in OxPAPC-activated signaling cascade. These results demonstrate for the first time barrier-protective properties of cell membrane-derived oxidized phospholipids generated in acute lung or cardiac injury and suggest PKC-, PKA-, Cdc42- and Rac-dependent cytoskeletal mechanisms of the vascular barrier restoration.

Thromboxane and Prostacyclin Production of Trophoblast Cells After Very Low-Density Lipoprotein Incubation

Objective: To test the effects of native VLDL (nVLDL) and oxidized VLDL (oxVLDL) on thromboxane and prostacyclin release from syncytiotrophoblasts in an in vitro model.Study Design: After isolation and Percoll centrifugation, three series of syncytiotrophoblast cell cultures were prepared for determination of thromboxane B2 (T×B2) and 6-keto prostaglandin (PG)lα, the stable metabolites of thromboxane and prostacyclin: a standard cell culture, a cell culture incubated with nVLDL, and a cell culture incubated with ox VLDL. Samples were taken at 2 and 24 h of incubation, and the changes in concentration were compared among cultures.Results: Production of T×B2 decreased in the standard cell culture (P = 0.0001), but increased after incubation with nVLDL (P = 0.01) or ox VLDL. Release of 6-keto PG1α remained largely unchanged in the standard cell culture, but increased in nVLDL (P = 0.02) and oxVLDL incubated cells. Comparisons among cell cultures showed that the production of T×B2 was significantly higher in ...