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

Materials

human medium OxLDL (#770202), nLDL (#770200), and HDL3 (#770300) were purchased from KALEN Biomedical, LLC. (Montgomery Village, MD). Metformin hydrochloride (#2864; Tocris Bioscience), recombinant human adiponectin (1065-AP), anti-LOX-1 antibodies (#AF1798; used in immunoblotting and for blocking OxLDL/LOX-1 interaction), and normal goat IgG (#AB-108-C) were purchased from R & D Systems (Minneapolis, MN). Compound C (a cell-permeable pyrazolopyrimidine compound that acts as a potent, selective, reversible, and ATP-competitive inhibitor of AMPK), DPI (#300260; 10 µM in DMSO for 30 min) and the solvent control DMSO were purchased from EMD Biosciences (Gibbstown, NJ). Tiron (#D7389) was purchased from Sigma-Aldrich. AICAR (catalog # EI-330) was from Biomol (Plymouth Meeting, PA). 16:0 Lyso PC (16:0-LPC; 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine; #855675) and 18:0 Lyso PC (18:0-LPC; 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine; #855775) were supplied by Avanti Polar Lipids, Inc. (Alabaster, AL). Minimally modified LDL (mmLDL) was generously provided by Yury I. Miller of the University of California at San Diego, and is characterized as previously described [23, 24]. All tissue culture supplies, Vybrant Cell Adhesion Assay kit (V-13181), TRIzol and Alexa Fluor® 488 F(ab')₂ fragment of goat anti-rabbit IgG (H+L; #A11070) were purchased from Invitrogen (Chicago, IL). Anti-TRAF3IP2 (#IMG-563), and peroxidase-conjugated affinity purified goat anti-rabbit IgG (#111-035-046), and goat anti-mouse IgG were purchased from Imgenex (San Diego, CA) and Jackson Immuno Research (Baltimore, PA) respectively. Antibodies against JNK1 (#sc-1648), phospho-IKKα/β (Ser¹⁸⁰/Ser¹⁸¹; sc-23470-R), phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵; sc-6254), ICAM-1 (6.5B5; #sc-18853), and VCAM-1 (H-276; #sc-8304) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). AMPK and phospho-AMPK (#9957), Akt (#9272), pAkt (Thr308, #9275), phospho-p65 (Ser536; #3031), p65 (#3034), (),α-tubulin (# 2144), and Lamin A/C (# 2032) antibodies were from Cell Signaling Technology, Inc. (Beverly, MA). Affinity-purified rabbit polyclonal antibodies that detect the active (cleaved) p17/19 form of caspase-3, but not the p35 precursor form (AB3623) were purchased from Millipore (Billerica, MA, USA).Quantitect cDNA synthesis kit (# 205310) and EndoFree® Plasmid Maxi kit (#12362) were from Qiagen (Valencia, CA). Cell Death Detection ELISA^PLUS kit was from Roche Applied Science. SuperSignal West Pico Chemiluminescent substrate (#34080) and NE-PER Nuclear Extraction kit were purchased from Thermo Fisher Scientific Inc. (Houston, TX).

Cell Culture

Clonetics® human aortic endothelial cells (EC, #CC-2535; Lonza) were previously described [14], and cultured at 37°C in endothelial basal medium-2 (EBM-2, #CC-3156) supplemented with EGM-2 SingleQuots. THP-1 cells (human acute monocytic leukemia cell line) and the mouse monocytic cell line RAW264.7 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA). THP-1 cells were cultured as previously described in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum and 0.05 mM 2-mercaptoethanol [14]. RAW264.7 cells were cultured in DMEM+10% FCS, and used below passage number 10. EC were used between passages 4 to 8. At 60%–70% confluency the media was changed to EBM-2 (without supplements), and incubated with indicated agonists and antagonists.

Adenoviral vectors and lentiviral shRNA particles

Ad.siNox2, Ad.siGFP, Ad.GFP, Ad.dnAMPK and Ad.kdAMPK were previously described [11, 12]. At 70–80% confluency, EC were infected at ambient temperature with adenovirus in PBS at the indicated multiplicity of infection (MOI). After 1 h, the adenovirus was replaced with culture medium. Assays were carried out 24 or 48 h later. At 50% confluency, EC were infected with lentiviral particles in complete medium for 48 hrs. Lentiviral particles expressing shRNA against human TRAF3IP2 IKKβ, p65, JNK1, c-Jun, and GFP were purchased from Santa Cruz Biotechnology Inc., and were previously described [14]. Lentiviral particles expressing LOX-1 shRNA (OLR1; TRCN0000060518) were from Sigma-Aldrich. To increase lentivirus infection efficiency, cells were co-treated with the cationic polymer Polybrene® (5 µg/ml in water; sc-134220). Neither shRNA nor Polybrene® affected cell viability. The siRNA and shRNA had no off-target effects, and at the indicated moi and duration of infection, did not affect EC adherence, shape and viability (trypan blue-dye exclusion; data not shown).

Endothelial-monocyte adhesion assay

Adhesion of monocytic cells to endothelial cells was analyzed by the Vybrant Cell Adhesion assay kit as previously described [14]. In brief, EC were plated in a 24-well plate. When at 70 to 80% confluency, the complete medium was replaced with EBM-2 containing OxLDL for 2 hours. THP-1 cells were labeled with 5 µM calcein AM in serum-free RPMI 1640 for 30 minutes at 37°C, washed twice with pre-warmed RPMI 1640, and resuspended in the same medium. The labeled cells (5×10⁴ cells) were then added to EC and incubated for 1 hr at 37° C. The non-adherent cells were removed by gently washing three times with ice-cold PBS. Finally 200 µl of PBS were added to each well. Endothelial-monocyte adhesion was quantitated by measuring fluorescence at excitation and emission wavelengths of 485 and 535nm, respectively, using BioTek Fluorimeter. Wells containing EC without THP-1 cells served as blanks.

RT-qPCR

Total RNA was isolated using the TRIzol method. One microgram of RNA was reverse transcribed using the Quantitect cDNA synthesis kit. TRAF3IP2, cytokine, chemokine and adhesion molecule expression was analyzed by RT-qPCR using TaqMan® probes and Eppendorf Realplex⁴ system [11, 12, 14]. Data were shown as fold change (2^−ΔΔCt).

Nuclear protein extraction

The nuclear and cytoplasmic fractions were separated using the NE-PER nuclear extraction kit according to the manufacturer’s instructions and has been previously described [14]. Protein levels were quantified using the Bradford method. Lamin B served as an invariant control.

Immunoblotting and ELISA

EC were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM Na₃VO₄, 1 mM PMSF, protease inhibitor cocktail, and 1% NP-40) for 30 min at 4°C and lysates were collected after centrifugation at 14,000g for 20 min at 4°C. Protein concentrations in clear cell lysates were determined using Bradford’s reagent and BSA as a standard. For western blots, equal amounts of total protein (lysates were boiled in 6x Laemmli buffer containing SDS) were separated by SDS-PAGE [11, 12, 14]. Separated proteins were transferred to PVDF membrane and blocked in PBST (phosphate-buffered saline + 0.2% Tween-20) containing 5% non-fat dry milk powder. The membrane was then incubated with primary antibody overnight at 4°C. The primary antibodies were diluted in PBST solution containing 2.5% nonfat dry milk powder. The membranes were washed in PBST (3×5 minutes) and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The washed blot was then developed using enhanced chemiluminiscence reagent and exposed to X-ray film. Images of the bands were scanned by reflectance scanning densitometry, and the intensity of the bands was quantified using Scion Image software.

Immunoprecipitation (IP) and immunoblotting (IB)

IP/IB experiments were performed as previously described [25, 26]. For IP, equal amounts of whole cell extracts were incubated overnight with anti-TRAF3IP2 antibodies attached to agarose beads at 4 °C under slow rotation. After washing 3 times in a buffer containing 50 mM Tris-Cl, 150 mM NaCl, 0.1% Nonidet P-40, the bound proteins were eluted from the beads by boiling in SDS sample buffer for subsequent SDS-PAGE and IB. The antibodies against LOX1 and AMPK are described above.

Detection of superoxide

Superoxide (O₂⁻) generation was quantified as previously described [26] using the lucigenin-enhanced chemiluminescence assay. In brief, EC were rinsed twice in PBS and resuspended in Krebs/HEPES buffer (in mM: 115 NaCl, 20 HEPES, 1.17 K₂HPO₄, 1.17 MgSO₄, 4.3 KCl, 1.3 CaCl₂, 25 NaHCO₃, and 11.7 glucose pH 7.4) that contained no NADPH. After incubation at 37 °C for 30 min, the tubes were placed in a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany). Dark-adapted lucigenin (5 µM) and either OxLDL alone or OxLDL combined with DPI or Tiron were added. Luminescence was measured for 10 s with a delay of 5 s. After background luminescence was subtracted, results were expressed as relative light units (RLU) per second per 5 × 10⁴ cells. Studies were also performed using EC infected with Ad.siNox2 (moi 100 for 48 h) prior to the assay. Ad.siGFP served as a control.

Cell death detection

Following 48 h incubation in ECM plus 0.5% bovine serum albumin, EC were treated with the indicated concentration of OxLDL for 24 h. Cell death was analyzed by a photometric enzyme immunoassay (cell death detection ELISA^PLUS kit, catalog No. 11920685001, Roche Applied Science) [14]. The assay is based on the quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones. This allows the specific determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. After removal of the unbound antibodies, the amount of peroxidase retained in the immunocomplex was determined photometrically using ABTS (2,2 - azinobis-(3 ethylbenzothiazoline-6-sulfonic acid)) as the substrate. Cell viability was also assessed by a microplate-based MTT cell viability assay, and viability was expressed as percentage of MTT absorbance at 562/650 nm in the absence of OxLDL. In addition, cell death was analyzed by DNA laddering using the Apoptotic DNA Ladder Kit (11 835 246 001; Roche Applied Science) and by determining the levels of cleaved caspase-3.

Animals

The investigation conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health, and all protocols were approved by the Institutional Animal Care and Use Committees at University of Texas Health Sciences Center in San Antonio, TX and Tulane University in New Orleans, LA. All animals were housed in a temperature- (22°C) and light-controlled (12 h light and 12 h dark) environment with ad libitum access to water and food. TRAF3IP2-null mice on a C57Bl/6 background are previously described [27]. These mice exhibit no basal phenotypic abnormalities. Breeding, litter size and the sex ratio, growth, body and heart weights were comparable between TRAF3IP2-null and wild type C57Bl/6 control mice [27]. All studies were carried out on male mice 3 months of age.

Monocyte adhesion to endothelium ex vivo

The assay describing ex vivo adhesion of monocytes to endothelium was previously reported [28].Thoracic aortas were isolated from WT and TRAF3IP2-null mice, opened longitudinally, cut into pieces, layered and pinned onto agarose gels, washed, treated with OxLDL (50 µg/ml for 3 h), and then incubated with RAW264.7 mouse monocytes loaded with calcein AM [14]. After 15 min the vessels were washed in DMEM+0.5% BSA to remove unbound cells, and the cells firmly adhered to the vessels were photographed. The number of adhered monocytes was counted in four fields using fluorescent microscopy, and results from 6 animals summarized.

Collection of thoracic aorta and contraction studies

Thoracic aortas were collected, placed in gassed (95% O₂+5%CO₂) Krebs-Hensleit solution composed of 119 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO₄, 1.8 mmol/L CaCl₂, 1.2 mmol/L KH₂PO₄, 24.9 mmol/L NaHCO₃ and 11.1 mmol/L glucose, and then cut into 3 mm length rings. Aortic rings were attached to holders and then placed in an organ bath filled with the buffer solution. Following 1 h equilibration, aortic rings were incubated with nLDL or OxLDL (100 µg/ml) for 1 h. Tissues were washed three times, and vasorelaxant responses were measured as previously described [29]. Rings were contracted to submaximal tone with 1 µM phenylephrine (PE) and subsequently relaxed with cumulative concentrations of the endothelium-dependent vasodilator acetylcholine (ACh). The percentage was calculated by considering contractions obtained immediately prior to the addition of ACh as 100%.

Statistical analysis

Comparisons between controls and various treatments were performed by ANOVA with post hoc Dunnett's t-tests. All assays were performed at least three times, and the error bars in the figures indicate the S.E. Though a representative immunoblot is shown in the main figures, changes in protein/phosphorylation levels from three independent experiments were semi-quantified by densitometry, and were shown as ratios and fold changes from untreated or respective controls at the bottom of the panels whenever the results are less clear.

OxLDL induces cell death in primary human EC via TRAF3IP2

Previously, we demonstrated that TRAF3IP2, an upstream regulator of NF-κB and AP-1 pathways, plays a critical role in AOPPs-induced cardiomyocyte injury [12]. Here we have investigated its role in OxLDL- induced EC death. Consistent with earlier reports [30, 31], incubation of EC with OxLDL induced cell death in a concentration dependent manner, with a peak effect observed at 50 µg/ml (Supplementary Fig. S1-A). These results were confirmed by the detection of increased levels of the active (cleaved) form of caspase-3 (Supplementary Fig. S1-A, inset) and by an apoptotic genomic DNA fragmentation assay (Supplementary Fig. S1-B). Furthermore, knockdown of LOX-1, the cell surface receptor mainly responsible for OxLDL signaling in EC [4, 5], or blocking the interaction between OxLDL and LOX-1 with antibodies, each markedly attenuated OxLDL-induced EC death (Fig. 1A). An increase in the levels of the cleaved fragment of caspase-3 (Fig. 1B) supported these results. Notably, knockdown of TRAF3IP2 blunted OxLDL-induced EC death (Fig. 1C, cleaved caspase-3 levels are shown in Fig 1D). Thus OxLDL induces EC death via a LOX-1/TRAF3IP2 pathway.

OxLDL induces endothelial cell death via TRAF3IP2-mediated IKK/NF-κB and JNK/AP-1 activation

TRAF3IP2 is an upstream regulator of NF-κB and AP-1, and both are reported to contribute to cell death in a stimulus- and cell type-specific manner. We and others have previously reported that TRAF3IP2 physically interacts with IKK and JNK, and mediates activation of both NF-κB and AP-1. Here, we investigated whether OxLDL-induced EC death is mediated by IKK/NF-κB and JNK/AP-1. Indeed, OxLDL induced activation of both IKK (Fig. 2A) and JNK (Fig. 2B) in EC in part via TRAF3IP2, and while knockdown of IKKβ attenuated OxLDL-induced p65 activation (Fig. 2C), knockdown of JNK1 inhibited c-Jun phosphorylation (Fig. 2D). Furthermore, knockdown of TRAF3IP2 attenuated activation of both p65 and c-Jun, as evidenced by the reduced levels of nuclear p-p65 and p-c-Jun levels (Fig. 1E). Importantly, knockdown of p65 and JNK1 each inhibited OxLDL-induced caspase-3 activation (Fig. 1F, inset) and EC death (Fig. 1F). These results indicate that OxLDL induces EC death in part via TRAF3IP2-dependent IKK/NF-κB and JNK/AP-1 activation.

To date, TRAF3IP2 has been shown to play a critical role in IL-17 signaling. TRAF3IP2 physically associates with IL-17R via a SEFIR-SEFIR interaction, and activates both IKK and JNK. Since OxLDL induces TRAF3IP2 expression via LOX-1, and activates IKK/NF-κB and JNK/AP-1 in part via TRAF3IP2, we next investigated whether OxLDL-induced NF-κB and AP-1 activation is mediated via LOX-1/TRAF3IP2 physical interaction. Amino acid analysis of LOX-1 coding region revealed neither a SEFIR nor a TIR domain (data not shown), suggesting that TRAF3IP2 may not bind LOX-1. Further, immunoblotting of TRAF3IP2 immunoprecipitates with anti-LOX-1 antibodies revealed no significant binding between TRAF3IP2 and LOX-1 either at basal conditions or following OxLDL treatment (Fig. 2G, left hand panel). However, confirming our earlier published report, TRAF3IP2 was found associated with IKKβ, and OxLDL enhanced their binding (Fig. 2G, right hand panel). These results indicate that OxLDL-mediated TRAF3IP2-dependent NF-κB and AP-1 activations are not mediated by LOX-1/TRAF3IP2 physical association.

OxLDL induces proinflammatory gene expression and monocyte adhesion to EC via TRAF3IP2

OxLDL induces NF-κB and AP-1 activation, through which it regulates both LOX1 and other proinflammatory genes [4, 5]. We next investigated the role of TRAF3IP2 in these pathways. Addition of OxLDL markedly elevated both LOX-1 mRNA and protein expression in the EC, and this response was attenuated by TRAF3IP2 knockdown (Fig. 3A). Further, OxLDL induced the expression of IL-18 and other proinflammatory genes important in the induction of EC dysfunction and death, and these responses were similarly attenuated by TRAF3IP2 knockdown (Fig. 3B and Table 1).

Since OxLDL induced ICAM-1 and VCAM-1 expression in TRAF3IP2-dependent manner (Table 1), and monocyte adhesion to EC is a critical initial step in atherogenesis, we next investigated whether OxLDL-induced endothelial-monocyte adhesion in vitro and ex vivo is TRAF3IP2-dependent. EC transduced with lentiviral TRAF3IP2 shRNA were incubated with OxLDL, and then layered with calcein AM-loaded THP-1 monocytes. While a significant increase in monocyte-endothelial cell adhesion was detected in the control GFP shRNA treated cells, THP-1 cell adhesion was markedly reduced following TRAF3IP2 knockdown (Fig. 3C). Importantly, in supporting ex vivo studies, segments of thoracic aorta from both wild type and TRAF3IP2-null mice were preincubated with OxLDL and then layered with calcein AM-loaded RAW264.7 cells. There was a marked increase in RAW264.7 adherence to WT vessels as compared to vessels isolated from TRAF3IP2-null mice (Fig. 3D). However, very few monocytes were found adhered to the endothelium of vessels isolated from either WT or TRAF3IP2-null mice treated with nLDL. These results indicate that TRAF3IP2 plays a critical role in several of the OxLDL-induced inflammatory changes that underlie the initiation and progression of atherosclerosis.

OxLDL induces TRAF3IP2 expression via Nox2-dependent superoxide generation

Having determined that TRAF3IP2 mediates OxLDL induction of its receptor LOX-1 (Fig. 2), as well as other proinflammatory genes (Fig. 2B and Table 1), we next investigated whether TRAF3IP2 itself is regulated by OxLDL. Addition of OxLDL significantly increased the expression of TRAF3IP2 mRNA and protein in the EC, a response that was inhibited by either LOX-1 knockdown or the addition of LOX-1 blocking antibodies (Fig. 4A). Furthermore, OxLDL enhanced the generation of superoxide in EC, and this enhancement could be partially inhibited by the superoxide scavenger Tiron, by the NADPH oxidase inhibitor DPI, or by the knockdown of Nox2 (Fig. 4B). Similarly, Tiron, DPI and Nox2 knockdown each inhibited TRAF3IP2 induction (Fig. 4C). Thus OxLDL-induces TRAF3IP2 expression in part via Nox2/superoxide-dependent signaling and suggests a positive reinforcing mechanism in the Ox-LDL proinflammatory pathway.

Lysophosphatidylcholine (LPC) constitutes up to 40% of the total lipid content of OxLDL and is formed during the oxidation of LDL. Since LPCs induce LOX-1 [32] and exert proinflammatory effects [33, 34], we next investigated whether 16:0-LPC (Palmitoyl-LPC) and 18:0-LPC (Stearoyl-LPC) induce TRAF3IP2 expression. In addition, we also investigated whether minimally modified LDL (mmLDL), which exerts both proinflammatory and pro-atherogeneic effects [35, 36], also induce TRAF3IP2 expression in EC. Our results indicate that pretreatment with 16:0-LPC and 18:0-LPC each induced TRAF3IP2 expression in EC (Fig. 4D). Similarly, mmLDL upregulated TRAF3IP2 expression (Fig. 4D). However, OxLDL appears to be a more effective inducer of TRAF3IP2 in EC (Fig. 4D). These results indicate that OxLDL and its lipid constituents, as well as mmLDL, can all upregulate TRAF3IP2 expression in EC, and strongly suggest that targeting TRAF3IP2 may blunt endothelial cell dysfunction and possibly death.

HDL3, but not modified HDL3, reverses OxLDL-induced endothelial cell death and dysfunction

While OxLDL promotes oxidative stress, inflammation and injury, native HDL exerts potent anti-oxidative, anti-inflammatory and pro-survival effects [15]. Based on their composition, HDLs differ in their anti-atherogenic potential. While HDL2 is large and lipid-rich, HDL3 is small and protein-rich, and is considered to be more effective in accepting cellular cholesterol and promoting its efflux [37, 38]. To investigate the role of HDL on the OxLDL/LOX-1/TRAF3IP2 pathway, we confined our studies to HDL3. HDL3 can undergo oxidative modification under chronic oxidative and inflammatory conditions, and lose its anti-atherogenic properties, and become proinflammatory [16]. For example, 15-lipoxygenase (15LO), an enzyme present at undetectable levels in normal vessels, is expressed at high levels in atherosclerotic vessels, and can oxidatively modify HDL3 (15LO-HDL3) [17–19]. Therefore, we investigated whether native HDL3 and 15LO-HDL3 differentially affect OxLDL-induced TRAF3IP2 expression. Native HDL3 markedly attenuated OxLDL-induced TRAF3IP2 expression, whereas in contrast, 15LO-HDL3 enhanced its expression (Fig. 5A; results from three independent experiments are summarized on the right). Further, native HDL3 inhibited OxLDL-induced EC death, whereas 15LO-HDL3 potentiated its pro-apoptotic effects (Fig. 5B; cleaved caspase-3 levels confirmed these results, Fig. 5B, inset). Similarly, native HDL3 inhibited OxLDL-induced ICAM-1 and VCAM-1 expression (Fig. 5C; left and right panels respectively) and endothelial-monocyte adhesion (Fig. 5D), whereas the effects of 15LO-HDL3 on the OxLDL-induced changes were additive. Importantly, incubation with 15LO-HDL3 alone induced TRAF3IP2 expression, and LOX-1 knockdown attenuated this effect (Fig. 5E). These results indicate that while native HDL3 reverses OxLDL-induced TRAF3IP2-dependent endothelial dysfunction and death, the effects of 15LO-HDL3 are reinforcing.

Adiponectin reverses OxLDL-induced TRAF3IP2 expression and endothelial cell death

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects [39]. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory and proapoptotic cytokines. Adiponectin exerts anti-inflammatory and anti-apoptotic effects via AMPK phosphorylation and Akt activation [21]. Therefore we examined whether adiponectin blocks OxLDL-induced TRAF3IP2 expression and endothelial cell death. Initially, we determined whether adiponectin activates Akt in EC via AMPK. Our results show adiponectin induced AMPK phosphorylation (Fig. 6A), a response that was markedly attenuated by the forced expression of mutant AMPK by adenoviral transduction (Fig. 6A), or by Compound C, a pharmacological AMPK inhibitor (Supplementary Fig. S2-A). Further, forced expression of mutant AMPK inhibited adiponectin-induced Akt activation (Fig. 6B). Importantly, in addition to attenuating OxLDL-induced TRAF3IP2 expression (Fig. 6C), adiponectin reversed OxLDL-induced Akt suppression (Fig. 6D). Adiponectin also blunted OxLDL-induced EC death, an effect reversed by mutant AMPK (Fig. 6E). The AMPK inhibitor Compound C similarly reversed the pro-survival effects of adiponectin on OxLDL-induced EC death (Supplementary Fig. S2-B), as did mutant Akt (Supplementary Fig. S3). These results indicate that adiponectin blocks OxLDL-induced EC death via AMPK-dependent Akt activation.

AICAR and metformin reverse OxLDL-induced TRAF3IP2 expression and endothelial cell death

AICAR, a pharmacological activator of AMPK, exerts anti-inflammatory and anti-apoptotic effects [21]. Metformin, an anti-diabetic insulin-sensitizing drug, also has an anti-inflammatory effect. AMPK activation is considered as one of the mechanisms by which metformin exerts its protective effects [22]. Since adiponectin reversed OxLDL-induced TRAF3IP2 expression and EC death, we hypothesized that AICAR and metformin would antagonize the pro-apoptotic effects of OxLDL via AMPK activation and TRAF3IP2 suppression. Similar to adiponectin, both AICAR and metformin induced AMPK phosphorylation (Supplementary Fig. S4-A), and AMPK-dependent Akt activation (Supplementary Fig. S4-B and C). Moreover, both reversed OxLDL-induced suppression of Akt activation (Supplementary Fig. S4-D). Importantly, AICAR and metformin inhibited OxLDL-induced TRAF3IP2 expression in a dose-dependent manner (Fig. 7A and 7B) and reversed its pro-apoptotic effects (Fig. 7C). These results indicate that the AMPK activators AICAR and metformin block the pro-apoptotic effects of OxLDL via AMPK-dependent Akt activation.

So far we have demonstrated that HDL3 (Fig. 5A), adiponectin (Fig. 6C), AICAR (Fig. Fig. 7A) and metformin (Fig. 7B) all attenuate OxLDL-induced TRAF3IP2 expression in EC. Since TRAF3IP2 is a redox-sensitive adapter molecule, and AMPK activation is known to block oxidative stress, we next investigated whether HDL3, adiponectin, AICAR and metformin block superoxide generation in EC via AMPK. Therefore, EC transduced or not with a dominant negative mutant form of AMPK (dnAMPK) were treated with HDL3, adiponectin, AICAR or metformin prior to OxLDL addition. Superoxide (O₂⁻) production was quantified using the lucigenin-enhanced chemiluminescence assay. Confirming the results in Fig. 4B, OxLDL induced a robust increase in superoxide, and this effect was markedly attenuated by HDL3, adiponectin, AICAR or metformin pretreatment (Fig. 7D). However, forced expression of dnAMPK partially reversed this inhibitory effect (Fig. 7D). The superoxide scavenger Tiron served as a positive control, and significantly attenuated OxLDL-induced superoxide generation. Together, these results indicate that reduction in oxidative stress by the AMPK activators HDL3, adiponectin, AICAR or metformin might have contributed to TRAF3IP2 inhibition. We further investigated whether a physical association between AMPK and TRAF3IP2 might have contributed to attenuated downstream signaling. To this end, TRAF3IP2 was immunoprecipitated from EC treated or not with OxLDL, and then blotted with anti-AMPK antibodies. Using this technique, no binding between AMPK and TRAF3IP2 was evident, either at basal conditions or after OxLDL treatment (Fig. 7D). These results suggest that the AMPK activators may attenuate TRAF3IP2 expression by inhibiting ROS generation.

OxLDL impairs acetylcholine (ACh)-mediated vasorelaxation in part via TRAF3IP2, and this response is reversed by the AMPK activators

We have demonstrated that TRAF3IP2 plays a role in OxLDL-induced endothelial dysfunction (Fig. 2) and death (Fig. 1C) in vitro. We next investigated whether TRAF3IP2 contributes to OxLDL-induced impaired vasorelaxation ex-vivo. Our results show that while OxLDL impairs ACh-mediated, endothelium-dependent vasorelaxation ex-vivo, TRAF3IP2 gene deletion reverses this effect (Fig. 8A). Further, all three AMPK activators, adiponectin, AICAR and metformin reversed OxLDL-induced impaired vasorelaxation (Fig. 8B). These results indicate that TRAF3IP2 mediates OxLDL-induced impaired vasorelaxation ex vivo, and that the AMPK activators antagonize OxLDL–induced responses.