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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) A 2 in free form [2]. One F 2 -isoprostane whose formation is highly favored is 8-iso-PGF 2α which has been shown to be a potent pulmonary and renal vasoconstrictor [3, 4]. Actions of 8-iso-PGF 2α were demonstrated to be mediated through a receptor related to but probably distinct from the thromboxane (TXA 2 ) / endoperoxide (PGH 2 ) receptor [5]. Although 8-epi-PGF 2α is a potent agonist of TXA 2 /PGH 2 receptors in vascular smooth muscle, interestingly it acts primarily as an antagonist of TXA 2 /PGH 2 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 E 2 -isoprostane receptors are distinct from TXA 2 /PGH 2 receptors, suggesting at least different subtypes, one of these specifically recognizing E 2 -isoprostanes [9]. Isoprostanes have been suggested to be useful markers for oxidant injury. For example, F 2 -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 F 2 -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.

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.

Our group has previously demonstrated that oxidized phospholipids derived from mildly oxidized low density lipoprotein (MM-LDL) or oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) increase monocyte-endothelial interactions. There are indications that the effects of these phospholipids are receptor mediated. Oxidized phospholipids have previously been shown to activate cells via the platelet activating factor (PAF) receptor, therefore, we pretreated human aortic endothelial cells (HAECs) with a PAF-receptor antagonist (WEB 2086) prior to treatment with oxPAPC. WEB 2086 inhibited monocyte binding to endothelial cells induced by oxPAPC (200 μg/ml) at concentrations from 1 nM to 10 μM, but had no effect on the induction of monocyte binding induced by lipopolysaccharide (LPS). We were able to isolate and identify several active oxidized phospholipids by combined normal phase high performance liquid chromatography (HPLC) and electrospray mass spectrometry (LC/MS). The induction of monocyte binding to HAECs by two of these partially purified phospholipids was totally abolished by the pretreatment of HAECs with WEB2086 (100 nM). PAF itself, however, when tested at concentrations from 10 nM to 10 μM had no effect on monocyte binding. These results suggest that several of the oxidized phospholipids present in MM-LDL and oxPAPC induce monocyte binding through a receptor which is perhaps distinct from the PAF-receptor, but can be blocked by the PAF-receptor antagonist WEB 2086.

Low-density lipoproteins (LDL)-apheresis is a well established treatment of severe hypercholesterolemia resulting in fast clinical improvement and angiographically proven regression after 6 months of therapy. The underlying mechanisms, beside lipoprotein removal, are still under debate. Recently, oxidized LDL were shown to be of key importance in foam cell formation and atherosclerotic lesion development. We examined the influence of dextran-sulfate LDL-apheresis on the susceptibility of LDL to oxidation in 6 patients (5 males, 1 female, age: 41-60 years) suffering from severe heterozygous hypercholesterolemia or combined hyperlipidemia. LDL-apheresis influenced the oxidizability of LDL by a significant (P < 0.01) prolongation of the median of lag time (min) for LDL samples (before treatment 75, range: 31-176 versus after treatment 129.5, range 45-286). A significant (P < 0.01) difference could be also observed in the amount of conjugated dienes as expressed by the maximum rate in absorbance (before treatment 15.39, range: 5.29-21.22 versus after treatment 20.20, range 12.88-72.33). Thiobarbituric acid reactive substances (TBARS) formation was significantly decreased in LDL obtained after apheresis treatment as compared to pretreatment LDL. Electrophoretic mobility (EM) of LDL obtained before and after LDL-apheresis revealed a significant increase (P < 0.05) from a mean of 8.8 +/- 0.5 to a mean of 10.5 +/- 0.5 mm. The titers of plasma autoantibodies against oxLDL (oLAb) which varied considerably interindividually, were not influenced by LDL-apheresis treatment. Levels of F2-isoprostanes, as measured by plasma levels of 8-iso-prostaglandin-F2 alpha (8-iso-PGF2 alpha), reflecting oxidative stress, did not change, either. In summary, our findings provide evidence that even one single dextran sulfate LDL-apheresis treatment decreases LDL-oxidizability, which is an additional beneficial effect to that of lipid lowering.

ADP-ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with 32P-NAD; then the nucleolar proteins were analyzed by 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE-cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP-ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP-ribosylation.

Oxidized phospholipids (OxPL) derived from oxidized lipoproteins or from membranes of apoptotic cells accumulate in damaged tissue. We hypothesized that OxPL generated during apoptosis may protect from inflammation through inducing a tolerogenic response. Our data shows that i.v. injection of C57BL/6 (B6) mice with OxPAPC (oxidation product of the membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-3-glycero-phosphoryl-choline) results in a reduced proportion of the CD11b-Hi CD11c+ dendritic cells (DC) in the spleen with much lower expression of MHC II and co-stimulatory molecules CD80 and CD86 as compared to saline injected controls. Furthermore, the number of CD4+Foxp3+ regulatory T cells (Tregs) was significantly higher in the spleens of mice treated with OxPAPC. Mechanistically, both the splenic CD11c+ DC purified from OxPAPC-treated mice and the OxPAPC-treated bone marrow-derived DC induced a more potent in vitro differentiation of naïve T cells to inducible Tregs as compared to the untreated controls. In a multiple low dose streptozotocin (MLD-STZ)-induced model of T1D, the B6 mice pre-treated with OxPAPC had a higher proportion of Tregs and were protected against MLD-STZ induced hyperglycemia as compared to saline injected controls. The data suggests that OxPAPC has therapeutic potential for treatment of T1D and other autoimmune diseases by inducing a DC-mediated increase of Treg differentiation.

Acute inflammation and vascular leak are cardinal features of acute lung injury and the acute respiratory distress syndrome. Nonspecific tissue inflammation and injury in response to infectious and noninfectious insults lead to oxidative stress and the generation of lipid oxidation products, which, in turn, may inhibit the acute inflammatory response to bacterial components. In this study, we used animal and endothelial cell culture models of lipopolysaccharide-induced lung injury to test the hypothesis that oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine may attenuate the acute lung inflammatory response to bacterial wall lipopolysaccharide (LPS) and enhance lung vascular barrier recovery. Rats received aerosolized LPS (5 mg/kg) or sterile water with concurrent intravenous oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine (0.5-6 mg/kg) or saline. At 18 hours, bronchoalveolar lavage was performed and the lungs were removed for histological analysis. Measurements of transendothelial electrical resistance and histochemical analysis of endothelial monolayer disruption were used as an in vitro model of LPS-induced lung barrier dysfunction. Intratracheal instillation of LPS induced lung injury with profound increases in bronchoalveolar lavage and tissue neutrophils, protein content, and production of inflammatory cytokines IL-6 and IL-1b. Intravenous injection of oxidized phospholipids markedly attenuated LPS-induced tissue inflammation, barrier disruption, and IL-6 and IL-1b production over a range of doses. In vitro, oxidized phospholipids attenuated LPS-induced endothelial permeability and reversed LPS-induced cytoskeletal remodeling and disruption of monolayer integrity. These studies demonstrate in vivo and in vitro protective effects of oxidized phospholipids on LPS-induced lung dysfunction.

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.