Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine Research Articles

Fatty acid epoxyisoprostane E2 stimulates an oxidative stress response in endothelial cells

Atherosclerosis is the main underlying cause of major cardiovascular diseases such as stroke and heart attack. Oxidized phospholipids such as oxidized 1-palmitoyl-2-arachidonoyl-sn-Glycero-3-phosphorylcholine (OxPAPC) accumulate in lesions of and promote atherosclerosis. OxPAPC activates endothelial cells, a critical early event of atherogenesis. Epoxyisoprostane E2 (EI) is an oxidized fatty acid contained at the sn-2 position of 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine (PEIPC), the most active component of OxPAPC in regulating inflammation. OxPAPC and its components including PEIPC activate endothelial cells to express an array of genes in different categories including oxidative stress response genes such as tumor suppressor gene OKL38 and Heme oxygenase-1 (HO-1). EI can be released by lipase from PEIPC. In this study, we examined the ability of EI to stimulate oxidative stress response in endothelial cells. EI released from OxPAPC and synthetic EI stimulated the expression of oxidative stress response gene OKL38 and antioxidant gene HO-1. Treatment of endothelial cells with EI increased the production of superoxide. NADPH oxidase inhibitor Apocynin and superoxide scavenger N-acetyl-cysteine (NAC) significantly attenuated EI-stimulated expression of OKL38 and HO-1. We further demonstrated that EI activated oxidative stress-sensitive transcription factor Nrf2. Silencing of Nrf2 with siRNA significantly reduced EI stimulated expression of OKL38 and HO-1. Thus, we demonstrated that EI induced oxidative stress in endothelial cells leading to increased expression of oxidative stress response gene OKL38 and HO-1 via Nrf2 signaling pathway relevant to atherosclerosis.

Oxidized phospholipids impair pulmonary antibacterial defenses: Evidence in mice exposed to cigarette smoke

Patients with COPD are associated with poor pulmonary anti-bacterial innate defenses, which increase the risk for frequent acute exacerbations caused by bacterial infection. Despite elevated numbers of phagocytes (macrophages and neutrophils), airways of patients with COPD show stable bacterial colonization. A defect in the phagocytic ability of alveolar macrophages (AMs) is one of the primary reasons for failure to clear the invading bacteria in airways of smokers and COPD patients and also in mice exposed to cigarette smoke (CS). Oxidative stress, as a result of CS exposure is implicated; however, the factors or mediators that inhibit phagocytic activity of AMs in lungs of smokers remain unclear. In the current study, we provide evidence that accumulation of oxidized phospholipids (Ox-PLs) mediate inhibition of phagocytic function of AMs in CS-exposed mice. Mice exposed to 6months of CS showed impaired bacterial phagocytosis and clearance by AMs and elevated levels of Ox-PLs in bronchoalveolar lavage fluid (BALF), compared to mice exposed to room air. Intratracheal instillation of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OX-PAPC) inhibited phagocytic activity of AMs and impaired pulmonary bacterial clearance in mice. In vitro studies demonstrated that exposure of J774 macrophages to OX-PAPC inhibited bacterial phagocytosis and clearance. However, pre-treatment of OX-PAPC with the monoclonal antibody EO6, which specifically binds to oxidized phospholipid but not native phospholipid, abolished OX-PAPC induced inhibition of bacterial phagocytosis and clearance. Incubation of BALF retrieved from CS-exposed mice impaired bacterial phagocytosis by J774 macrophages, which was abolished by pre-treatment of BALF with the EO6 antibody. In conclusion, our study shows that Ox-PLs generated following chronic CS exposure could play a crucial role in inhibiting phagocytic function of AMs and thus impair pulmonary anti-bacterial innate defenses in CS-exposed mice. Therapeutic approaches that augment pulmonary antioxidant defenses could be beneficial in reducing oxidative stress-driven impairment of phagocytosis by AMs in smokers and COPD patients.

Afadin controls p120‐catenin–ZO‐1 interactions leading to endothelial barrier enhancement by oxidized phospholipids

Afadin is a novel regulator of epithelial cell junctions assembly. However, its role in the formation of endothelial cell junctions and the regulation of vascular permeability remains obscure. We previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in the in vitro and in vivo models of lung endothelial barrier dysfunction and acute lung injury, which were mediated by Rac GTPase. This study examined a role of afadin in the OxPAPC-induced enhancement of interactions between adherens junctions and tight junctions as a novel mechanism of endothelial cell (EC) barrier preservation. OxPAPC induced Rap1-dependent afadin accumulation at the cell periphery and Rap1-dependent afadin interaction with adherens junction and tight junction proteins p120-catenin and ZO-1, respectively. Afadin knockdown using siRNA or ectopic expression of afadin mutant lacking Rap1 GTPase binding domain suppressed OxPAPC-induced EC barrier enhancement and abolished barrier protective effects of OxPAPC against thrombin-induced EC permeability. Afadin knockdown also abolished protective effects of OxPAPC against ventilator-induced lung injury in vivo. These results demonstrate for the first time a critical role of afadin in the regulation of vascular barrier function in vitro and in vivo via coordination of adherens junction-tight junction interactions.

Oxidized phospholipids reduce ventilator-induced vascular leak and inflammation in vivo

BackgroundMechanical ventilation at high tidal volume (HTV) may cause pulmonary capillary leakage and acute lung inflammation resulting in ventilator-induced lung injury. Besides blunting the Toll-like receptor-4-induced inflammatory cascade and lung dysfunction in a model of lipopolysaccharide-induced lung injury, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exerts direct barrier-protective effects on pulmonary endothelial cells in vitro via activation of the small GTPases Rac and Cdc42. To test the hypothesis that OxPAPC may attenuate lung inflammation and barrier disruption caused by pathologic lung distension, we used a rodent model of ventilator-induced lung injury and an in vitro model of pulmonary endothelial cells exposed to pathologic mechanochemical stimulation.MethodsRats received a single intravenous injection of OxPAPC (1.5 mg/kg) followed by mechanical ventilation at low tidal volume (LTV) (7 mL/kg) or HTV (20 mL/kg). Bronchoalveolar lavage was performed and lung tissue was stained for histological analysis. In vitro, the effects of OxPAPC on endothelial barrier dysfunction and GTPase activation were assessed in cells exposed to thrombin and pathologic (18%) cyclic stretch.ResultsHTV induced profound increases in bronchoalveolar lavage and tissue neutrophils and in lavage protein. Intravenous OxPAPC markedly attenuated HTV-induced protein and inflammatory cell accumulation in bronchoalveolar lavage fluid and lung tissue. In vitro, high-magnitude stretch enhanced thrombin-induced endothelial paracellular gap formation associated with Rho activation. These effects were dramatically attenuated by OxPAPC and were associated with OxPAPC-induced activation of Rac.ConclusionOxPAPC exhibits protective effects in these models of ventilator-induced lung injury.

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.

The Therapeutic Effect of Oxidized 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphorylcholine in Rodents With Acute Necrotizing Pancreatitis and Its Mechanism

This study aims to investigate the therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OXPAPC) in rodents with acute necrotizing pancreatitis (ANP) and its mechanism. The ANP model was induced by cerulein challenged by lipopolysaccharide in mice and taurocholic acid in Sprague-Dawley rats. Both ANP models were treated with OXPAPC. Twenty animals of each group were separated to investigate mortality. Detection included serum levels of amylase and lactate dehydrogenase, histological changes of pancreas, activity of myeloperoxidase in pancreas, mRNA expression of inflammatory factors, expression of signal transduction factor proteins, and binding activity of transcriptional factors. After treatment with OXPAPC, survival rate was improved in the rat model. In both models, OXPAPC significantly decreased serum amylase and lactate dehydrogenase levels. Histologically, OXPAPC reduced the severity of pancreatic injury. There was a significant decline of myeloperoxidase activity. The mRNA levels of intrapancreatic inflammatory factors were depressed. Activated p38, C-jun N-terminal kinase 1, and inhibitor of kappa-B kinase beta proteins were down-regulated. Electrophoretic mobility shift assay showed that the binding activity of nuclear factor-kappaB and activator protein 1 to DNA was inhibited. The OXPAPC decreased the severity of experimental ANP in rodents. The protective effect of OXPAPC was mediated, at least in part, through blocking the lipopolysaccharide signal pathway.

OKL38 is an oxidative stress response gene stimulated by oxidized phospholipids

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) is present in oxidative modified LDL and accumulates in lesions of many chronic inflammatory diseases, such as atherosclerosis. In a microarray study, OxPAPC has been demonstrated to modulate the expression of >700 genes in human aortic endothelial cells. We found that the levels of mRNA for OKL38 [also named Bone marrow Derived Growth Factor (BDGI)], a tumor growth inhibitor, were strongly increased by OxPAPC. Here, we report that OKL38 is regulated by an oxidative signal induced by OxPAPC and its component lipid 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine. The stimulation of OKL38 by OxPAPC depends on superoxide production, because the NADPH oxidase (Nox) inhibitor apocynin and the superoxide scavenger N-acetyl cysteine block this stimulation. Oxidative stress by tert-butylhydroquinone treatment also induced the expression of OKL38. The stimulation of OKL38 expression by OxPAPC is mediated via transcription factor nuclear factor E2-related factor (Nrf2), a common factor involved in the regulation of oxidative stress-stimulated genes. Activation of Nrf2 induces the expression of OKL38, whereas small interfering RNA knockdown of Nrf2 blocks the stimulation of OKL38 by OxPAPC. Our results suggest that OKL38 is regulated via the Nox/Nrf2 pathway in response to oxidative stress stimuli.

Tiam1 and βPIX mediate Rac‐dependent endothelial barrier protective response to oxidized phospholipids

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibits potent barrier protective effects on pulmonary endothelium, which are mediated by small GTPases Rac and Cdc42. However, upstream mechanisms of OxPAPC-induced small GTPase activation are not known. We studied involvement of Rac/Cdc42-specific guanine nucleotide exchange factors (GEFs) Tiam1 and betaPIX in OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective responses in the human pulmonary endothelial cells (EC). OxPAPC induced membrane translocation of Tiam1, betaPIX, Cdc42, and Rac, but did not affect intracellular distribution of Rho and Rho-specific GEF p115-RhoGEF. Protein depletion of Tiam1 and betaPIX using siRNA approach abolished OxPAPC-induced activation of Rac and its effector PAK1. EC transfection with Tiam1-, betaPIX-, or PAK1-specific siRNA dramatically attenuated OxPAPC-induced barrier enhancement, peripheral actin cytoskeletal enhancement, and translocation of actin-binding proteins cortactin and Arp3. These results show for the first time that Tiam1 and betaPIX mediate OxPAPC-induced Rac activation, cytoskeletal remodeling, and barrier protective response in pulmonary endothelium.

Oxidized Phospholipids Inhibit Phagocytosis and Impair Outcome in Gram-Negative Sepsis In Vivo

Oxidized phospholipids that are generated during inflammation exert anti-inflammatory properties and prevent death during murine endotoxemia. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) inhibits the interaction of LPS with LPS-binding protein and CD14. In this study, we determined the functional properties of OxPAPC and potential interference with CD14 during abdominal sepsis caused by Escherichia coli. Administration of OxPAPC rendered mice highly susceptible to E. coli peritonitis, as indicated by an accelerated mortality and enhanced bacterial outgrowth and dissemination. CD14(-/-) mice also displayed increased mortality and bacterial outgrowth and OxPAPC did not further impair host defense in these animals. The mechanisms by which OxPAPC and CD14 deficiency impaired the immune response differed: whereas CD14(-/-) mice demonstrated a strongly reduced recruitment of phagocytes to the site of the infection, OxPAPC did not influence the influx of inflammatory cells but strongly diminished the phagocytosing capacity of neutrophils and macrophages by a CD14-independent mechanism. Furthermore, OxPAPC potently inhibited uptake of fluorospheres as well as receptor-mediated endocytosis and fluid-phase pinocytosis. These data suggest that oxidized phospholipids such as produced during inflammatory reactions may contribute to mortality during Gram-negative sepsis in vivo via impairment of the phagocytic properties of professional phagocytes.

Oxidized Phospholipids Reduce Vascular Leak and Inflammation in Rat Model of Acute Lung Injury

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 may inhibit the acute inflammatory response to bacterial components. To test the hypothesis that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) may attenuate the acute lung inflammatory response to lipopolysaccharide (LPS) and enhance lung vascular barrier recovery, we used in vivo and in vitro models of LPS-induced lung injury. Rats received intratracheal aerosolized LPS (5 mg/kg) or sterile water with concurrent intravenous injection of OxPAPC (0.5-6.0 mg/kg) or saline alone. Nonoxidized PAPC was used as a control. At 18 h, bronchoalveolar lavage was performed and the lungs were removed for histologic analysis. Measurements of endothelial transmonolayer electrical resistance and immunofluorescent analysis of monolayer integrity were used in an in vitro model of LPS-induced lung vascular barrier dysfunction. In vivo, aerosolized intratracheal LPS induced lung injury with profound increases in bronchoalveolar lavage neutrophils, protein content, and the inflammatory cytokines interleukin 6 and interleukin 1beta, as well as tissue neutrophils. OxPAPC, but not nonoxidized PAPC, markedly attenuated the LPS-induced tissue inflammation, barrier disruption, and cytokine production over a range of doses. In vitro, oxidized phospholipids attenuated LPS-induced endothelial barrier disruption 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.

Identification of prostaglandin E2 receptor subtype 2 as a receptor activated by OxPAPC

Oxidized 1-palmitoyl-2-arachidonoyl-sn-Glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by the activation of endothelial β1 integrin. Our previous studies suggest this function of OxPAPC is mediated via a Gs-protein coupled receptor (GPCR). To identify the GPCR for OxPAPC, we screened a number of candidate GPCRs for their interaction with OxPAPC using a reporter gene assay. We identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes and macrophages. OxPAPC was able to compete with PGE2 for binding to EP2. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC50 of 108.6nM. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of β1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of TNF-α and IL-10 in monocyte-derived cells. Additionally, EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the IL-10 response to PEIPC in THP-1 cells. These results suggest that EP2 functions as an OxPAPC receptor in both endothelial cells and monocytes/macrophages. This project was funded by USPHS grants HL30568 and HL 064731

Identification of Prostaglandin E2 Receptor Subtype 2 As a Receptor Activated by OxPAPC

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by activation of endothelial beta1 integrin and subsequent deposition of fibronectin on the apical surface. Our previous studies suggest this function of OxPAPC is mediated via a Gs protein-coupled receptor (GPCR). PEIPC (1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine) is the most active lipid in OxPAPC that activates this pathway. We screened a number of candidate GPCRs for their interaction with OxPAPC and PEIPC, using a reporter gene assay; we identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes, and macrophages. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC50 of 108.6 nmol/L. OxPAPC and PEIPC were also able to compete with PGE2 for binding to EP2 in a ligand-binding assay. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of beta1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of tumor necrosis factor-alpha and interleukin-10 in monocyte-derived cells. EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the interleukin-10 response to PEIPC in monocytic THP-1 cells. These results suggest that EP2 functions as a receptor for OxPAPC and PEIPC, either as the phospholipid ester or the released fatty acid, in both endothelial cells and macrophages.

The therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine in mice with acute necrotizing pancreatitis

To investigate the therapeutic effect of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OXPAPC) in animal model of acute necrotizing pancreatitis (ANP) and its possible mechanism. Forty-eight C57BL/6J mice were randomly divided into two groups, ANP group and ANP treated with OXPAPC group. ANP model was induced by seven times administration of cerulein (50 microg/kg) challenged by lipopolysaccharide (LPS) (5 mg/kg) intraperitoneal injection. Treatment with OXPAPC was started 5 mins before LPS injection. At the 9th hour, 12nd hour and 24th hour after the first injection of cerulein, serum levels of amylase and lactate dehydrogenase (LDH) were measured. Severity of pancreatitis was evaluated by histological scoring system. The activity of myeloperoxidase (MPO) in pancreas was determined by zymohistochemistry. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA expressions in pancreas were studied by semi-quantitative RT-PCR. The activation of nuclear factor kappaB (NF-kappaB) and c-Jun-N-terminal kinase 1 (JNK1) were investigated by the methods of western blot. Treatment with OXPAPC significantly decreased serum amylase and LDH levels at the 9th hr and 12nd hour. Histologically, OXPAPC reduced the severity of pancreatic injury including inflammatory cell infiltration and necrosis at 12nd hour and 24th hour. There was a significant decrease of MPO activity in OXPAPC group as compared to ANP group. Intrapancreatic TNF-alpha, IL-1beta, ICAM-1 and E-selectin mRNA levels were reduced in OXPAPC-treated group. Western blot showed down-regulation of NF-KappaB p65 and JNK1 expression were found in OXPAPC-treated mice. OXPAPC decreased the severity of experimental acute pancreatitis in mice and inhibited the intrapancreatic inflammatory mediators expression in vivo. The protective effect of OXPAPC was mediated, at least in part, through blocking endotoxin signal pathway.

Oxidized Phospholipids Increase Interleukin 8 (IL-8) Synthesis by Activation of the c-src/Signal Transducers and Activators of Transcription (STAT)3 Pathway

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) and its component phospholipids 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine induce endothelial cells to synthesize chemotactic factors, such as interleukin 8 (IL-8). We have shown recently that Ox-PAPC-mediated induction of IL-8 transcription is independent of NF-kappaB activation, a major transcription factor utilized by cytokines and lipopolysaccharide for the induction of IL-8 transcription. In this study, we provide evidence for the role of c-src in Ox-PAPC and, specifically, PEIPC-mediated IL-8 induction. Ox-PAPC and its component phospholipids induced a rapid and transient phosphorylation of c-src Tyr418, a hallmark of c-src activation, in human aortic endothelial cells (HAEC). Ox-PAPC-mediated IL-8 protein synthesis in HAEC was inhibited by Src family kinase inhibitors, PP1 and PP2, but not by an inactive analog, PP3. Transient expression of plasmids containing C-terminal Src kinase or kinase-deficient dominant-negative c-src resulted in a 72 and 50% reduction in Ox-PAPC-induced IL-8 promoter activation in human microvascular endothelial cells, respectively. In contrast, overexpression of v-src kinase resulted in a 4-fold increase in IL-8 promoter activation, without inducing NF-kappaB promoter activation. Furthermore, treatment of HAEC with Ox-PAPC and its component PEIPC induced the activation of STAT3 by phosphorylating Tyr705, a feature of STAT3 activation. STAT3 is a known downstream effector of c-src. Ox-PAPC-induced activation of STAT3 resulted in the translocation of STAT3 from the cytoplasm of HAEC into their nuclear compartment. Transient expression of a dominant-negative STAT3beta construct in HMEC strongly inhibited IL-8 induction by Ox-PAPC. Taken together, these data demonstrate the role of the c-src kinase/STAT3 pathway in Ox-PAPC-mediated IL-8 expression in endothelial cells.

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-alpha 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.

Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins

The bioactive component of mildly oxidized low-density lipoproteins, oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphorylcholine (OxPAPC), activates tissue factor expression and monocyte adhesion to endothelial cells (EC) from systemic circulation, but blocks expression of inflammatory adhesion molecules (VCAM, E-selectin) and neutrophil adhesion associated with EC acute inflammatory response to bacterial lypopolysacharide (LPS). Due to constant exposure to oxygen free radicals, lipids in the injured lung are especially prone to oxidative modification and increased OxPAPC generation. In this study, we focused on OxPAPC-mediated intracellular signaling mechanisms that lead to physiological responses in pulmonary endothelial cells. Our results demonstrate that OxPAPC treatment activated in a time-dependent fashion protein kinase C (PKC), protein kinase A (PKA), Raf/MEK1,2/Erk-1,2 MAP kinase cascade, JNK MAP kinase and transient protein tyrosine phosphorylation in human pulmonary artery endothelial cells (HPAEC), whereas nonoxidized PAPC was without effect. Pharmacological inhibition of PKC and tyrosine kinases blocked activation of Erk-1,2 kinase cascade upstream of Raf. OxPAPC did not affect myosin light chain (MLC) phosphorylation, but increased phosphorylation of cofillin, a molecular regulator of actin polymerization. Finally, OxPAPC induced p60Src-dependent tyrosine phosphorylation of focal adhesion proteins paxillin and FAK. Our results suggest a critical involvement of PKC and tyrosine phosphorylation in OxPAPC-induced activation of Erk-1,2 MAP kinase cascade associated with regulation of specific gene expression, and demonstrate rapid phosphorylation of cytoskeletal proteins, which indicates OxPAPC-induced EC remodeling.

Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca++/NFAT

Activation of endothelial cells by lipid oxidation products is a key event in the initiation and progression of the atherosclerotic lesion. Minimally modified low-density lipoprotein (MM-LDL) induces the expression of certain inflammatory molecules such as tissue factor (TF) in endothelial cells. This study examined intracellular signaling pathways leading to TF up-regulation by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a biologically active component of MM-LDL. OxPAPC induced TF activity and protein expression in human umbilical vein endothelial cells (HUVECs). However, OxPAPC neither induced phosphorylation or degradation of I kappa B alpha nor DNA binding of nuclear factor-kappa B (NF-kappa B). Furthermore, OxPAPC-induced TF expression was not inhibited by overexpression of I kappa B alpha. These results strongly indicate that OxPAPC-induced TF expression is independent of the classical NF-kappa B pathway. However, OxPAPC stimulated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and expression of early growth response factor 1 (EGR-1). Inhibitors of mitogen-activated kinase/ERK (MEK) or protein kinase C (PKC) blocked elevation of both EGR-1 and TF. Furthermore, overexpression of NAB2, a corepressor of EGR-1, inhibited effects of OxPAPC. In addition, OxPAPC induced rapid and reversible elevation of free cytosolic Ca(++) levels and nuclear factor of activated T cells (NFAT)/DNA binding. Induction of TF expression by OxPAPC was partially inhibited by cyclosporin A, known to block calcineurin, a Ca(++)-dependent phosphatase upstream of NFAT. Treatment of OxPAPC with phospholipase A(2) destroyed its biologic activity and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine was identified as one biologically active component of OxPAPC that induces TF expression. Together, the results demonstrate that OxPAPC induces TF expression in HUVECs through activation of PKC/ERK/EGR-1 and Ca(++)/calcineurin/NFAT pathways rather than by NF-kappa B-mediated transcription. Thus, oxidized phospholipids may contribute to inflammation by activating pathways alternative to the classical NF-kappa B pathway.

Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.

Interleukin-10 blocks atherosclerotic events in vitro and in vivo.

Atherosclerosis can be viewed in part as an inflammatory disease process and may therefore be susceptible to manipulation of the immune state. Interleukin 10 (IL-10) is an inhibitory cytokine produced by activated lymphocytes and monocytes. These studies present evidence that IL-10 can inhibit minimally oxidized LDL (MM-LDL)-induced monocyte-endothelium interaction as well as inhibit atherosclerotic lesion formation in mice fed an atherosclerotic diet. Pretreatment of human aortic endothelial cells (HAECs) for 18, but not 4, hours with recombinant IL-10 caused a significant decrease in MM-LDL-induced monocyte binding. IL-10 was found to be maximally effective at 10 ng/mL. Transfection of HAECs with adenovirus expressing viral bcrf-1 IL-10 (Ad-vIL-10) in a sense but not antisense orientation completely inhibited the ability of MM-LDL to induce monocyte binding. Similar results were obtained with IL-10 or Ad-vIL-10 in HAECs stimulated with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). We have previously shown increases in cAMP associated with MM-LDL activation of endothelial cells. The MM-LDL-induced increase in cAMP levels was not inhibited by preincubation with IL-10. In vivo studies demonstrated that mice with a murine IL-10 transgene under the control of the human IL-2 promoter have decreased lesions versus controls on an atherogenic diet (5433+/-4008 mm(2) versus 13 574+/-4212 mm(2); P<0.05), whereas IL-10 null mice have increased lesions (33 250+/-9117 mm(2); P<0.0001) compared with either controls or IL-10 transgenic mice. These studies suggest an important role for IL-10 in the atherosclerotic disease process.

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-kappaB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.