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Oxidized phospholipids accumulate in atherosclerotic lesions, on lipoproteins, in other states of chronic inflammation, on apoptotic cells, necrotic cells and cells exposed to oxidative stress. These lipids regulate the transcription of over 1000 gene, regulating many endothelial functions, by activating several different cell surface receptors and multiple signaling pathways. These lipids also have important effects not involving transcription that regulate cell junctions and leukocyte binding. Thus these lipids are potent regulators of endothelial cell function with broad effects comparable in extent but differing from those of cytokines.
In the early 1990s, the first reports were published detailing the effect of phospholipid oxidation products on endothelial cell function. These studies grew out of the observation that minimally oxidized LDL (MM-LDL) could activate endothelial inflammatory functions.1 When MM-LDL was fractionated, oxidized phospholipids were found to be the major source of activity. Because there was very little oxidation of linoleic acid in MM-LDL, the studies with oxidized phospholipids focused on phospholipids containing oxidation products of arachidonic acid that were esterified into glycerol-phosphocholine. Studies showing effects of phospholipid oxidation products on specific endothelial inflammatory and pro-coagulant functions soon followed.2–4 Effects of ether containing phospholipid oxidation products were also reported.5 Though many oxidized phospholipids were shown to be present in MM-LDL and in 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) exposed to air oxidation, three oxidized PAPC (Ox-PAPC) products were extensively studied with respect to cell signaling.6 Two of these contained fragmentation products of arachidonic acid: a 5-carbon aldehyde (POVPC) and a 5-carbon acid (PGPC). The third, an especially active oxidized phospholipid, was shown to be a product of the addition of oxygen to arachidonic acid to form an epoxyisoprostane (PEIPC). The effects of other arachidonate and linoleate fragmentation products on endothelial cell inflammatory function have been more recently reported, including alpha beta unsaturated acids (e.g. Kodia PC)7 and aldehydes (e.g. HOOHA-PC).8 Thus, a large number of phospholipid oxidation products have been shown to effect endothelial inflammatory function.
Initially, bioactive phospholipid oxidation products were shown to be formed in lipoproteins oxidized in the test tubes with UV light and enzymes. It was then shown that cultured cells could cause the formation of active phospholipid oxidation products in LDL.5 Phospholipid oxidation products are increased in atherosclerotic lesions in rabbits, mice and humans. In addition, phospholipid oxidation products were shown to accumulate in cells exposed to oxidative stress, including necrotic cells, apoptotic cells and cells treated with IL-1 beta.9, 10 Within the last several years, phospholipid oxidation products have been demonstrated to accumulate in lipoproteins of individuals at risk for coronary events, especially in Lp (a) containing particles, where they may serve as prognostic indicators.11 These studies suggest the importance of phospholipid oxidation products in atherosclerosis. These oxidation products may also play a role in other chronic inflammatory diseases where they have been shown to accumulate.
This review will focus on signaling by phospholipids containing esterified oxidized fatty acids at the sn-2 position. We will also make reference to studies with ether containing phospholipid oxidation products and with lyso PC. We have chosen to focus this review on endothelial cell signaling because of the large amount of detailed work in this cell type. Separate reviews have and are being written to examine effects on other cells. In published studies, our group has demonstrated that Ox-PAPC regulates the transcription of over 1000 genes in endothelial cells.12 Of these, approximately 600 are upregulated and 400 are downregulated after a 4-hour treatment. The regulated genes include genes effecting inflammation, procoagulant activity, redox reaction, sterol metabolism, cell cycle, the unfolded protein response (UPR), and angiogenesis. More targeted in vivo analyses of aortas coated with Ox-PAPC have also demonstrated regulation of transcription of a number of the same genes.13 In addition to transcriptional regulation, several groups have demonstrated effects on signaling, as opposed to transcription, related to changes in cell shape and leukocyte binding to the endothelial cells.4, 14 Thus, the published studies demonstrate important effects of Ox-PAPC on all major aspects of endothelial cell function. The array and cell biology studies have highlighted several transcription factors important in Ox-PAPC action, such as STAT3, SREBP, Nrf2, erg-1, NFAT and transcription factors of the UPR system.15, 16 In this review, we will address the roles of these transcription factors as well as other signaling molecules in Ox-PAPC signaling. Since Ox-PAPC contains a mixture of oxidized phospholipids, studies with the individual phospholipids have also been reported and will be discussed in this review. Several other excellent reviews have covered other aspects of Ox-PAPC signaling. 15, 17
Oxidized phospholipids have been demonstrated to induce an inflammatory response in endothelial cells. This inflammatory response is characterized by an increase in the deposition of leukocyte binding molecules on the endothelial surface to bind monocytes, and by an increase in the production of chemotactic factors.
In human aortic EC (HAEC), Ox-PAPC has been demonstrated to promote surface deposition of the CS-1-containing variant of fibronectin (CS1), serving as a ligand for the α4β1 (VLA-4) integrin expressed on the surface of monocytes 4. The deposition of fibronectin is mediated through increasing cAMP levels, leading to the activation of R-Ras and inactivation of H-Ras. R-Ras was shown to activate PI3K, which then results in the activation of α5β1 integrin on the apical surface; this activated integrin binds the fibronectin 18. In fact, it has been shown that inactivation of VLA-4 using a synthetic peptide mimetic of CS1 (4), decreased leukocyte entry and fatty streak formation in mice fed an atherogenic diet (19, 20). Recent findings have also demonstrated a role for the prostaglandin receptor E2 (EP2), a G-protein coupled receptor (GPCR), in this pathway (Figure 1) 21. Agonists of this receptor increased activation of beta 1 integrin. Several groups have obtained evidence that P-Selectin protein expression is increased by oxidized phospholipid, resulting in enhanced release of P-Selectin onto the cell surface by histamine or Ox-LDL 13, 22. Targeted deletion of P-selectin slowed the progression of atherosclerosis in LDL receptor-null mice 23. There is also evidence for an additional pathway regulating monocyte adhesion involving lipoxygenase.24 Treatment of endothelial cells with MM-LDL was shown to cause the release of arachidonic acid; furthermore, monocytes binding induced by MM-LDL and POVPC was reduced by inhibition of the lipoxygenase pathway 25. Furthermore, 12-S-Hete mimicked the effect on monocyte binding and there was evidence for a role of CS-1 fibronectin. The sequence of events in this pathway is not yet determined. Interestingly, the mechanism of leukocyte binding induced by oxidized phospholipids in human EC is different from that of lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), or interleukin 1 (IL-1) which induce both monocyte and neutrophil binding. Ox-PAPC does not upregulate expression of ICAM-1, VCAM-1 and E-selectin and induces only monocyte binding. These properties of Ox-PAPC may indeed explain the selective accumulation of monocytes, rather than neutrophils, in early atherosclerotic lesions.
Array data and other studies indicate that Ox-PAPC increases the mRNA levels of a number of chemokines including MCP-1, MIP-1α, MIP-1β, CXCL3, IL-6, IL-8, and GROα 12. These cytokines play important roles in mediating enhanced monocyte/EC adhesion as well as monocyte migration. One interesting feature of Ox-PAPC signaling is the prolonged transcriptional regulation of the chemokines, such as IL-8 and MCP-1, by Ox-PAPC in comparison to traditional cytokines. Ox-PAPC-induced IL-8 transcription, for example, is sustained for at least 18 hours following treatment, with its peak mRNA induction observed at approximately 6 hours following Ox-PAPC treatment.26 This is in comparison to TNF-induced IL-8 transcription, which reaches its peak at 1 hour following treatment and returns to baseline levels at 4 hours. There are multiple signaling pathways controlling this prolonged transcriptional activity (Figure 2). The pathways most extensively studied have been in human aortic endothelial cells (HAEC). Furthermore, most of the pathways discussed below are also activated by PEIPC, the most active pro-inflammatory phospholipid in Ox-PAPC. The rapid activation of IL-8 transcription was demonstrated to be activated by a c-Src/JAK2/STAT3 pathway 27, which is turned on within seconds following Ox-PAPC treatment and returns to baseline levels within 4–6 hours. This pathway directly regulates IL-8 transcription through binding to a novel gamma-interferon activation sequence (GAS) element in the IL-8 promoter. The sustained regulation of IL-8 transcription was found to be mediated by cholesterol depletion and the activation of the uncoupled eNOS/SREBP pathway 28, 29, which is sustained for up to 16 hours after Ox-PAPC treatment in HAEC. Increased binding of SREBP to an SRE element in the IL-8 promoter was also demonstrated. The exact steps causing cholesterol depletion have not yet been determined. However, there is clear involvement of eNOS in SREBP activation. Ox-PAPC treatment was demonstrated to stimulate the activity of eNOS but also promote eNOS uncoupling and the generation of reactive oxygen species (ROS). L-NAME, an inhibitor of eNOS activity, was able to partially inhibit SREBP activation and the expression of known SREBP target genes, such as the LDL receptor (28), by Ox-PAPC. Furthermore, recent findings have also demonstrated a role for VEGFR2 in these pathways 30. In these studies, siRNA against VEGFR2 decreased the transcription of IL-8 and LDL receptor in response to Ox-PAPC, as well as decreasing the activation of SREBP (30). Furthermore, Ox-PAPC-induced VEGFR2 activation was c-Src dependent 30, suggesting potential cross talk between the c-Src/JAK2/STAT3 pathway and the uncoupled eNOS/SREBP pathway. While a role for VEGFR2 in Ox-PAPC induced eNOS activation and uncoupling was not reported, other studies have reported interaction between VEGFR2 and eNOS 31.
In addition, ER stress and activation of the unfolded protein response (UPR) pathway have also recently been shown to regulate IL-8 expression in HAEC 32. siRNA-mediated knockdown of transcription factors ATF4 and XBP1, key effectors of the UPR, downregulated both the basal and the Ox-PAPC simulated expression of IL-8 in HAEC. In these studies, a role for the UPR in both the basal and the Ox-PAPC induced expression of IL-6, CXCL3 and MCP-1 in HAEC was also shown.
Several other molecules and receptors have been implicated in the regulation of IL-8 and MCP-1/JE induced by Ox-PAPC. Ox-PAPC, POVPC and PEIPC were demonstrated to activate PPAR alpha and PPAR gamma.33 Using murine aortic EC obtained from PPARα-null mice, a role for PPARα has been demonstrated in the induction of MCP-1 by Ox-PAPC. The role of PPARα in the regulation of chemokine expression by Ox-PAPC in human EC, however, remains controversial. In HeLa cells, a role for TLR4 in activation of IL-8 transcription has also been demonstrated.34 However, effects in HAEC were inconsistent. Interestingly, TLR4 was shown to regulate cell shape changes and MIP-2 synthesis in response to MM-LDL in macrophages.35 These studies suggest differential regulation of chemokine expression by MM-LDL and oxidized lipids across different cell types.
Interestingly, the signaling events activated in EC by Ox-PAPC that regulate chemokine expression are different from those induced by mediators of acute inflammation such as LPS, TNFα and IL-1. The latter agonists induce expression of inflammatory genes rapidly and acutely (with transcriptional activation returning to baseline by 4 hours), and largely act through the activation of the transcription factor, nuclear factor-kappa beta (NFκB). In contrast, Ox-PAPC induces transcriptional regulation of chemokines in a prolonged manner, and through the activation of a different set of transcription factors: STAT3, SREBP, PPAR alpha and UPR. These properties may in part explain the selective chronic inflammatory picture, characterized by monocyte accumulation, observed in atherosclerotic lesions.
Interestingly treatment of cells with Ox-PAPC or its constituent lipids inhibits the effect of LPS, TLR2 ligand and TLR9 ligand on inflammation both in vitro and in vivo.15 The most active Ox-PAPC lipids regulating this inhibition have been identified as alpha beta unsaturated lipids, such as KoDiA PC.7 Several mechanisms have been demonstrated to contribute to this inhibition. Ox-PAPC can form complexes in vitro with CD14 and LBP, thus blocking the binding of LPS.36 In addition, Ox-PAPC effects formation of the TLR4 complex with CD14 by altering caveolar membranes.7 This latter effect appears to be due to activation of neutral sphingomyelinase, which has been demonstrated to alter caveolar function.37 While Ox-PAPC inhibits LPS effects on inflammation, it is actually synergistic with effects of fine diesel exhaust particles found as air-pollutants38 and with TNF alpha.37 The mechanisms regulating these synergisms are not yet known.
Effects of MM-LDL and Ox-PAPC on the synthesis of proteins regulating pro-coagulant activity have been reported in both capillary and aortic endothelial cells. Regulation of this activity can be mediated by changes in molecules that initiate coagulation (tissue factor, TF) or those that regulate the breakdown of small thrombi (plasminogen activator, TPA; plasminogen activator inhibitor PAI; thrombomodulin TM). MM-LDL was shown to regulate all of these activities to promote a procoagulant state.2, 28, 39–42 The signal transduction pathway regulating the induction of TF mRNA levels by Ox-PAPC in HUVEC was shown to be mediated through both a PKC/Erk/EGR-1 pathway and a Ca++/calcineurin/NFAT pathway.39 Both transcription factors, EGR-1 and NFAT, act together in regulating the TF promoter. In a separate study, VEGFR2 was demonstrated to regulate ERK activation and TF expression.30 Thus, VEGFR2 appears to be an important receptor in the regulation of inflammation and coagulation. The mechanism mediating the reduction in TM mRNA levels by Ox-PAPC has also been examined. This inhibition was shown to be due decreased activation of transcription factors RXR, SP1 and RARbeta, known regulators of TM transcription. These studies demonstrate differential regulation of TF and TM transcription by Ox-PAPC, mediated through activation of distinct and separate signaling pathways. In addition, Ox-PAPC was also show to reduce the activity of tissue factor pathway inhibitor (TFPI), a protease inhibitor that inhibits the initial reactions of blood coagulation 42. In these studies, it was concluded that TFPI activity was inhibited through direct interaction of oxidized lipids with the C-terminal basic region of TFPI. In summary, the pro-coagulant activity of oxidized phospholipids is mediated through the activation of several molecular mechanisms.
There have been two studies reporting regulation of angiogenesis by phospholipid oxidation products. In the first report, based on hydrolysis by PAF-AH, the authors concluded that oxidized phospholipids in OxLDL for 4 hours inhibited the synthesis of basic FGF by HUVEC and thus were likely to inhibit angiogenesis.40 Their studies, like a number of others, demonstrated that a PAF receptor antagonist could block this effect. However, PAF itself did not have the same effects as OxPAPC. In a more recent report, Bochkov et al. demonstrated both in vivo and in vitro that Ox-PAPC treatment of endothelial cells for 2 days stimulated angiogenesis by increasing level of IL-8, VEGF and COX-2.39 Two specific Ox-PAPC components, POVPC and PGPC, were demonstrated to have this activity when tested at 50ug/ml. They also reported that ERK-1 and NFAT, previously shown to be activated by Ox-PAPC, might play a role in propagating the angiogenic response.
Many of the studies discussed above have involved transcriptional regulation. However, other important non-transcriptional effects of oxidized phospholipids have also been described. A large amount of work demonstrates that oxidized phospholipids activate junction formation in pulmonary artery endothelial cells.43 These studies have demonstrated that, both in vitro and in vivo, Ox-PAPC and specifically the epoxycylopentenone-containing phospholipids, increase transendothelial resistance.14 They have shown that the increase in junction formation is due to an increase in Rac activation mediated by activation of PKA, PKC and tyrosine kinases. Furthermore, GTP exchange factors TIAM1 and beta PIX have been identified as the regulators of Rac activation.44 Downstream of Rac, as seen in other systems, Ox-PAPC treatment induces the phosphorylation FAK and Paxillin, which are involved in focal adhesion formation. These studies suggest that oxidized phospholipids protect barrier function in lung endothelial cells.
Several studies have demonstrated that treatment of endothelial cells with Ox-PAPC induces oxidative stress. Rouhanizadeh et al. demonstrated that glutathione was depleted in response to Ox-PAPC treatment in bovine aortic endothelial cells and that NADPH oxidase was the source superoxide induction by Ox-PAPC, which led to glutathione depletion.45 Therond et al. observed a decrease in glutathione levels in response to short chain polar lipid derivatives in endothelial cells.46 Gharavi et al. demonstrated that superoxide produced by uncoupled e-NOS also is a source of ROS.28 Increased mitochondrial metabolism may also play a role as has been seen for fatty acid oxidation products 47. Whatever the source of ROS, Ox-PAPC clearly induces transcription of anti-oxidant enzymes including HO-1, glutathione synthase, thioredoxin reductase and glutathione reductase. A newly recognized anti-oxidant gene, OKL 38, is also induced by Ox-PAPC. Regulation of HO-1 transcription has been examined by two groups, who have identified two different pathways involved in this regulation: Kronke et al. identified a MAP kinase/CREB pathway of activation in HUVEC48, while Li et al. identified a pathway involving the activation of Nrf216 in HAEC. Induction of these antioxidant enzymes is likely to protect the cell from cell death, which is negligible in response to Ox-PAPC.
We are beginning to understand the multiple signaling mechanisms by which oxidized phospholipids interact with endothelial cells to activate some processes and inhibit others. Ox-PAPC and individual phospholipids from Ox-PAPC were shown to activate multiple cell signaling pathways and transcription factors. The fact that oxidized phospholipids are present at many sites of chronic inflammation and in blood suggests that they play a prominent role in the regulation of endothelial function. Because these lipids are more difficult to prepare and test, we are at an early stage in our understanding of lipid signaling, that is, in comparison to our understanding of cytokine signaling. However, similar to cytokine signaling, it is clear that multiple pathways are involved in lipid signaling that effect all of the major functions of endothelial cells.
Grant Support: PO1 HL30568 and RO1 HL 064731 (JB), AHA Western States Fellowship (NG)
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