Effects of silencing leukocyte-type 12/15-lipoxygenase using short interfering RNAs

The leukocyte-type 12/15-lipoxygenase (12/15-LO) has been implicated in the pathogenesis of atherosclerosis, hypertension, and diabetes. 12/15-LO and its products are associated with LDL oxidation, cellular growth, migration, adhesion, and inflammatory gene expression in monocytes/macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs). Our objective, therefore, was to develop novel expression vectors for short interfering RNAs (siRNAs) targeting 12/15-LO to evaluate its functional relevance in macrophages and VSMCs. We used a PCR-based approach to rapidly identify effective siRNA target sites on mouse 12/15-LO and initially tested their efficacy on a fusion construct of 12/15-LO cDNA and enhanced green fluorescent protein. We then cloned these U6 promoter+siRNA PCR products into plasmid vectors [short hairpin siRNAs (shRNAs)] to knockdown endogenous 12/15-LO expression in mouse macrophages and also rat and mouse VSMCs. Furthermore, the functional effects of shRNA-mediated 12/15-LO knockdown were noted by the reduced oxidant stress and chemokine [monocyte chemoattractant protein-1 (MCP-1)] expression in a differentiated mouse monocytic cell line as well as by the reduced cellular adhesion and fibronectin expression in VMSCs. Knocking down 12/15-LO expression also reduced the expression of inflammatory genes, MCP-1, vascular cell adhesion molecule-1, and interleukin-6 in VSMCs. Our results illustrate the functional relevance of 12/15-LO activation in macrophages and VSMCs and its relationship to oxidant stress and inflammation.     

TLR4-Mediated Expression of Mac-1 in Monocytes Plays a Pivotal Role in Monocyte Adhesion to Vascular Endothelium

Toll-like receptor 4 (TLR4) is known to mediate monocyte adhesion to endothelial cells, however, its role on the expression of monocyte adhesion molecules is unclear. In the present study, we investigated the role of TLR4 on the expression of monocyte adhesion molecules, and determined the functional role of TLR4-induced adhesion molecules on monocyte adhesion to endothelial cells. When THP-1 monocytes were stimulated with Kdo2-Lipid A (KLA), a specific TLR4 agonist, Mac-1 expression was markedly increased in association with an increased adhesion of monocytes to endothelial cells. These were attenuated by anti-Mac-1 antibody, suggesting a functional role of TLR4-induced Mac-1 on monocyte adhesion to endothelial cells. In monocytes treated with MK886, a 5-lipoxygenase (LO) inhibitor, both Mac-1 expression and monocyte adhesion to endothelial cells induced by KLA were markedly attenuated. Moreover, KLA increased the expression of mRNA and protein of 5-LO, suggesting a pivotal role of 5-LO on these processes. In in vivo studies, KLA increased monocyte adhesion to aortic endothelium of wild-type (WT) mice, which was attenuated in WT mice treated with anti-Mac-1 antibody as well as in TLR4-deficient mice. Taken together, TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis.     

12/15-Lipoxygenase activity mediates inflammatory monocyte/endothelial interactions and atherosclerosis in vivo

We have shown that the 12/15-lipoxygenase (12/15-LO) product 12S-hydroxyeicosatetraenoic acid increases monocyte adhesion to human endothelial cells (EC) in vitro. Recent studies have implicated 12/15-LO in mediating atherosclerosis in mice. We generated transgenic mice on a C57BL/6J (B6) background that modestly overexpressed the murine 12/15-LO gene (designated LOTG). LOTG mice had 2.5-fold elevations in levels of 12S-hydroxyeicosatetraenoic acid and a 2-fold increase in expression of 12/15-LO protein in vivo. These mice developed spontaneous aortic fatty streak lesions on a chow diet. Thus, we examined effects of 12/15-LO expression on early events leading to atherosclerosis in these mice. We found that, under basal unstimulated conditions, LOTG EC bound more monocytes than B6 control EC (18 +/- 2 versus 7 +/- 1 monocytes/field, respectively; p < 0.0001). Inhibition of 12/15-LO activity in LOTG EC using a 12/15-LO ribozyme completely blocked monocyte adhesion in LOTG mice. Thus, 12/15-LO activity is required for monocyte/EC adhesion in the vessel wall. Expression of ICAM-1 in aortic endothelia of LOTG mice was increased severalfold. VCAM-1 expression was not changed. In a series of blocking studies, antibodies to alpha(4) and beta(2) integrins in WEHI monocytes blocked monocyte adhesion to both LOTG and B6 control EC. Inhibition of ICAM-1, VCAM-1, and connecting segment-1 fibronectin in EC significantly reduced adhesion of WEHI monocytes to LOTG EC. In summary, these data indicate that EC from LOTG mice are "pre-activated" to bind monocytes. Monocyte adhesion in LOTG mice is mediated through beta(2) integrin and ICAM-1 interactions as well as through VLA-4 and connecting segment-1 fibronectin/VCAM-1 interactions. Thus, 12/15-LO mediates monocyte/EC interactions in the vessel wall in atherogenesis at least in part through molecular regulation of expression of endothelial adhesion molecules.     

15-Lipoxygenase-mediated modification of high-density lipoproteins impairs SR-BI- and ABCA1-dependent cholesterol efflux from macrophages

Elevated plasma levels of high-density lipoprotein cholesterol (HDL-C) are atheroprotective and HDL-dependent reverse cholesterol transport has been related to this effect. HDL particles may, however, undergo modifications that affect their biological activities. Lipoxygenases (LOs) belong to a family of lipid peroxidizing enzymes; among them, reticulocyte-type 15-lipoxygenase (15-LO-1) appears to play a pathophysiological role in atherosclerosis, as its expression is increased in atherosclerotic plaques and it has been shown to oxidize low-density lipoproteins to an atherogenic form. In this work we investigated the impact of in vitro 15-lipoxygenase-catalyzed modification of HDL3 on their ability to act as cholesterol acceptor and found that 15-LO-modified HDL3 were less effective in mediating cholesterol efflux from lipid-laden J774 cells. A reduced binding of 15-LO-modified HDL3 to scavenger receptor class B, type I (SR-BI), due to HDL apoproteins cross-linking, explained, at least in part, the observed reduction of cholesterol efflux. In addition, ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux was also reduced, as a consequence of pre-beta-particles loss after HDL3 modification. These results suggest that 15-lipoxygenase might induce structural alterations of HDL3 particles that impair their capability of triggering reverse cholesterol transport.     

Role of 12/15-lipoxygenase in the expression of MCP-1 in mouse macrophages

Monocyte chemoattractant protein (MCP)-1 plays a key role in atherosclerosis and inflammation associated with visceral adiposity by inducing mononuclear cell migration. Evidence shows that mouse peritoneal macrophages (MPM) express a 12-lipoxygenase (12/15-LO) that has been clearly linked to accelerated atherosclerosis in mouse models and increased monocyte endothelial interactions in both rodent and human cells. However, the role of 12/15-LO products in regulating MCP-1 expression in macrophages has not been clarified. In this study, we tested the role of 12/15-LO products using MPM and the mouse macrophage cell line, J774A.1 cells. We found that 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] increased MCP-1 mRNA and protein expression in J774A.1 cells and MPM. In contrast, 12(R)-HETE, a lipid not derived from 12/15-LO, did not affect MCP-1 expression. 15(S)-HETE also increased MCP-1 mRNA expression, but the effect was less compared with 12(S)-HETE. MCP-1 mRNA expression was upregulated in a macrophage cell line stably overexpressing 12/15-LO (Plox-86 cells) and in MPM isolated from a 12/15-LO transgenic mouse. In addition, the expression of MCP-1 was downregulated in MPM isolated from 12/15-LO knockout mice. 12(S)-HETE-induced MCP-1 mRNA expression was attenuated by specific inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein kinase (p38). 12(S)-HETE also directly activated NADPH oxidase activity. Two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium chloride, blocked 12(S)-HETE-induced MCP-1 mRNA. Apocynin attenuated 12(S)-HETE-induced MCP-1 protein secretion. These data show that 12(S)-HETE increases MCP-1 expression by inducing PKC, p38, and NADPH oxidase activity. These results suggest a potentially important mechanism linking 12/15-LO activation to MCP-1 expression that induces inflammatory cell infiltration.     

Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B₄ (LTB₄), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB₄ production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB₄ production and activity. We found that treatment with ox-LDL increased the production of LTB₄ and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB₄ receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB₄ receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB₄ production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.     

High density lipoproteins (HDL) interrupt the sphingosine kinase signaling pathway. A possible mechanism for protection against atherosclerosis by HDL.

The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-alpha (TNF), resulting in the expression of various adhesion proteins. We have recently reported that sphingosine 1-phosphate, generated by sphingosine kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated sphingosine kinase activity in endothelial cells resulting in a decrease in sphingosine 1-phosphate production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-kappaB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.     

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did.We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.     

Endothelial surface N-glycans mediate monocyte adhesion and are targets for anti-inflammatory effects of peroxisome proliferator-activated receptor γ ligands

Endothelial-monocyte interactions are regulated by adhesion molecules and key in the development of vascular inflammatory disease. Peroxisome proliferator-activated receptor (PPAR) γ activation in endothelial cells is recognized to mediate anti-inflammatory effects that inhibit monocyte rolling and adhesion. Herein, evidence is provided for a novel mechanism for the anti-inflammatory effects of PPARγ ligand action that involves inhibition of proinflammatory cytokine-dependent up-regulation of endothelial N-glycans. TNFα treatment of human umbilical vein endothelial cells increased surface expression of high mannose/hybrid N-glycans. A role for these sugars in mediating THP-1 or primary human monocyte rolling and adhesion was indicated by competition studies in which addition of α-methylmannose, but not α-methylglucose, inhibited monocyte rolling and adhesion during flow, but not under static conditions. This result supports the notion that adhesion molecules provide scaffolds for sugar epitopes to mediate adhesion with cognate receptors. A panel of structurally distinct PPARγ agonists all decreased TNFα-dependent expression of endothelial high mannose/hybrid N-glycans. Using rosiglitazone as a model PPARγ agonist, which decreased TNFα-induced high mannose N-glycan expression, we demonstrate a role for these carbohydrate residues in THP-1 rolling and adhesion that is independent of endothelial surface adhesion molecule expression (ICAM-1 and E-selectin). Data from N-glycan processing gene arrays identified α-mannosidases (MAN1A2 and MAN1C1) as targets for down-regulation by TNFα, which was reversed by rosiglitazone, a result consistent with altered high mannose/hybrid N-glycan epitopes. Taken together we propose a novel anti-inflammatory mechanism of endothelial PPARγ activation that involves targeting protein post-translational modification of adhesion molecules, specifically N-glycosylation.     

Induction of scavenger receptor class B type I is critical for simvastatin enhancement of high-density lipoprotein-induced anti-inflammatory actions in endothelial cells

Changes in plasma lipoprotein profiles, especially low levels of high-density lipoprotein (HDL), are a common biomarker for several inflammatory and immune diseases, including atherosclerosis and rheumatoid arthritis. We examined the effect of simvastatin on HDL-induced anti-inflammatory actions. HDL and sphingosine 1-phosphate (S1P), a bioactive lipid component of the lipoprotein, inhibited TNF alpha-induced expression of VCAM-1, which was associated with NO synthase (NOS) activation, in human umbilical venous endothelial cells. The HDL- but not S1P-induced anti-inflammatory actions were enhanced by a prior treatment of the cells with simvastatin in a manner sensitive to mevalonic acid. Simvastatin stimulated the expression of scavenger receptor class B type I (SR-BI) and endothelial NOS. As for S1P receptors, however, the statin inhibited the expression of S1P(3) receptor mRNA but caused no detectable change in S1P(1) receptor expression. The reconstituted HDL, a stimulator of SR-BI, mimicked HDL actions in a simvastatin-sensitive manner. The HDL- and reconstituted HDL-induced actions were blocked by small interfering RNA specific to SR-BI regardless of simvastatin treatment. The statin-induced expression of SR-BI was attenuated by constitutively active RhoA and small interfering RNA specific to peroxisome proliferator-activated receptor-alpha. Administration of simvastatin in vivo stimulated endothelial SR-BI expression, which was accompanied by the inhibition of the ex vivo monocyte adhesion in aortas from TNF alpha-injected mice. In conclusion, simvastatin induces endothelial SR-BI expression through a RhoA- and peroxisome proliferator-activated receptor-alpha-dependent mechanism, thereby enhancing the HDL-induced activation of NOS and the inhibition of adhesion molecule expression.     

A Novel Anti-Inflammatory Effect for High Density Lipoprotein

High density lipoprotein has anti-inflammatory effects in addition to mediating reverse cholesterol transport. While many of the chronic anti-inflammatory effects of high density lipoprotein (HDL) are attributed to changes in cell adhesion molecules, little is known about acute signal transduction events elicited by HDL in endothelial cells. We now show that high density lipoprotein decreases endothelial cell exocytosis, the first step in leukocyte trafficking. ApoA-I, a major apolipoprotein of HDL, mediates inhibition of endothelial cell exocytosis by interacting with endothelial scavenger receptor-BI which triggers an intracellular protective signaling cascade involving protein kinase C (PKC). Other apolipoproteins within the HDL particle have only modest effects upon endothelial exocytosis. Using a human primary culture of endothelial cells and murine apo-AI knockout mice, we show that apo-AI prevents endothelial cell exocytosis which limits leukocyte recruitment. These data suggest that high density lipoprotein may inhibit diseases associated with vascular inflammation in part by blocking endothelial exocytosis.     

Apelin–APJ induces ICAM-1, VCAM-1 and MCP-1 expression via NF-κB/JNK signal pathway in human umbilical vein endothelial cells

Apelin receptor (APJ) deficiency has been reported to be preventive against atherosclerosis. However, the mechanism of this effect remains unknown. In this study, quantitative real-time RT-PCR, Western blotting and ELISA analyses revealed a significant increase in the expression of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) treated with apelin. Inhibitors of cellular signal transduction molecules were used to demonstrate involvement of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in apelin–APJ-induced activation of adhesion molecules and chemokines. Inhibition of APJ expression by RNA interference abrogated apelin-induced expression of adhesion molecules and chemokines and apelin-stimulated cellular signal transduction in HUVECs. The apelin–APJ system in endothelial cells is involved in the expression of adhesion molecules and chemokines, which are important for the initiation of endothelial inflammation-related atherosclerosis. Therefore, apelin–APJ and the cell signaling pathways activated by this system in endothelial cells may represent targets for therapy of atherosclerosis.     

The role of the chemokines MCP-1, GRO-alpha, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-α, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-α and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.     

On the role of vitamin C and other antioxidants in atherogenesis and vascular dysfunction

Oxidative stress has been implicated as an important etiologic factor in atherosclerosis and vascular dysfunction. Antioxidants may inhibit atherogenesis and improve vascular function by two different mechanisms. First, lipid-soluble antioxidants present in low-density lipoprotein (LDL), including alpha-tocopherol, and water-soluble antioxidants present in the extracellular fluid of the arterial wall, including ascorbic acid (vitamin C), inhibit LDL oxidation through an LDL-specific antioxidant action. Second, antioxidants present in the cells of the vascular wall decrease cellular production and release of reactive oxygen species (ROS), inhibit endothelial activation (i.e., expression of adhesion molecules and monocyte chemoattractants), and improve the biologic activity of endothelium-derived nitric oxide (EDNO) through a cell- or tissue-specific antioxidant action. alpha-Tocopherol and a number of thiol antioxidants have been shown to decrease adhesion molecule expression and monocyte-endothelial interactions. Vitamin C has been demonstrated to potentiate EDNO activity and normalize vascular function in patients with coronary artery disease and associated risk factors, including hypercholesterolemia, hyperhomocysteinemia, hypertension, diabetes, and smoking.     

Alpha and beta subunits of the LFA-1 membrane molecule are involved in human monocyte-endothelial cell adhesion

Human monocyte adhesion to vascular endothelium is an important transitional event in mononuclear phagocyte development. The molecular mechanism involved in monocyte adhesion to endothelial cells was studied using purified human monocytes and a panel of monoclonal antibodies (MAb). The purified human monocytes were phenotypically characterized and expressed relatively low levels of HLA class II antigens. The monocytes were labeled with Indium-111 to provide high specific activity and a sensitive measure of adhesion. Using this radionuclide adhesion assay, monocytes demonstrated consistent and reproducible adhesion to a confluent monolayer of human umbilical vein-derived endothelial cells. To identify the cell surface molecules involved in human monocyte-endothelial cell adhesion, 15 MAb to 11 monocyte surface structures were used to attempt to inhibit adhesion. MAb recognizing 10 monocyte cell surface molecules did not inhibit adhesion. In contrast, MAb recognizing the alpha and beta subunits of LFA-1 (lymphocyte function-associated) significantly inhibited monocyte adhesion to endothelial cells. Monocyte adhesion was comparably inhibited by F(ab')2 and intact MAb. Significant inhibition was observed at 5 micrograms/ml of anti-LFA-1 MAb. These results indicate that the alpha and beta subunits of the LFA-1 membrane molecule are involved in human monocyte-endothelial cell adhesions.     

OxLDL induces endothelial dysfunction and death via TRAF3IP2: Inhibition by HDL3 and AMPK activators

Oxidized low-density lipoprotein (oxLDL) induces endothelial cell death through the activation of NF-κB and AP-1 pathways. TRAF3IP2 is a redox-sensitive cytoplasmic adapter protein and an upstream regulator of IKK/NF-κB and JNK/AP-1. Here we show that oxLDL-induced death in human primary coronary artery endothelial cells (ECs) was markedly attenuated by the knockdown of TRAF3IP2 or the lectin-like oxLDL receptor 1 (LOX-1). Further, oxLDL induced Nox2/superoxide-dependent TRAF3IP2 expression, IKK/p65 and JNK/c-Jun activation, and LOX-1 upregulation, suggesting a reinforcing mechanism. Similarly, the lysolipids present in oxLDL (16:0-LPC and 18:0-LPC) and minimally modified LDL also upregulated TRAF3IP2 expression. Notably, whereas native HDL3 reversed oxLDL-induced TRAF3IP2 expression and cell death, 15-lipoxygenase-modified HDL3 potentiated its proapoptotic effects. The activators of the AMPK/Akt pathway, adiponectin, AICAR, and metformin, attenuated superoxide generation, TRAF3IP2 expression, and oxLDL/TRAF3IP2-mediated EC death. Further, both HDL3 and adiponectin reversed oxLDL/TRAF3IP2-dependent monocyte adhesion to endothelial cells in vitro. Importantly, TRAF3IP2 gene deletion and the AMPK activators reversed oxLDL-induced impaired vasorelaxation ex vivo. These results indicate that oxLDL-induced endothelial cell death and dysfunction are mediated via TRAF3IP2 and that native HDL3 and the AMPK activators inhibit this response. Targeting TRAF3IP2 could potentially inhibit progression of atherosclerotic vascular diseases.