OxLDL induces endothelial dysfunction and death via TRAF3IP2: inhibition by HDL3 and AMPK activators.
ABSTRACT: Oxidized low-density lipoprotein (oxLDL) induces endothelial cell death through the activation of NF-?B and AP-1 pathways. TRAF3IP2 is a redox-sensitive cytoplasmic adapter protein and an upstream regulator of IKK/NF-?B 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.
Project description:Hyperglycemia-induced endothelial dysfunction is characterized by enhanced inflammatory cytokine and adhesion molecule expression, and endothelial-monocyte adhesion. The adapter molecule CIKS (connection to IKK and SAPK/JNK; also known as Act1 or TRAF3IP2) is an upstream regulator of NF-?B and AP-1, and plays a role in inflammation and injury. Here we show that high glucose (HG; 25mM vs. 5mM d-glucose)-induced endothelial-monocyte adhesion and inhibition of endothelial cell (EC) migration were both reversed by CIKS knockdown. In EC, HG induced CIKS mRNA and protein expression via DPI-inhibitable Nox4-dependent ROS generation. Further, HG induced CIKS transcription and enhanced CIKS promoter-dependent reporter gene activation via Nox4, ROS, AP-1 and C/EBP. Coimmunoprecipitation and immunoblotting revealed CIKS/IKK?/JNK physical association under basal conditions that was enhanced by HG treatment. Importantly, CIKS knockdown inhibited HG-induced (i) IKK? and JNK phosphorylation, (ii) p65 and c-Jun nuclear translocation, and (iii) NF-?B- and AP-1-dependent proinflammatory cytokine, chemokine, and adhesion molecule expression. Similar to HG, the deleterious metabolic products of chronic hyperglycemia, AGE-HSA, AOPPs-HSA and oxLDL, also induced CIKS-dependent endothelial dysfunction. Notably, aortas from streptozotocin-induced and the autoimmune type 1 diabetic NOD and Akita mice showed enhanced DPI-inhibitable ROS generation and CIKS expression. Since CIKS mediates high glucose-induced NF-?B and AP-1-dependent inflammatory signaling and endothelial dysfunction, targeting CIKS may delay progression of vascular diseases during diabetes mellitus and atherosclerosis.
Project description:BACKGROUND:Plasmalogens are either phosphatidylcholine (PC P) or phosphatidylethanolamine (PE P) glycerophospholipids containing a vinyl ether moiety in sn-1-position and an esterified fatty acid in sn-2 position. Multiple functions have been proposed, including reservoir of precursors for inflammatory mediators, modulation of membrane fluidity, and anti-oxidative properties. They could therefore play a role under conditions of metabolic stress. Especially enzymatically modified LDL (eLDL) and oxidatively modified LDL (oxLDL) represent modifications of LDL that are taken up by macrophages in atherosclerotic plaques. The aim of this study was to analyze plasmalogen related effects of eLDL and oxLDL in human monocyte derived macrophages, as well as the effects of HDL3 mediated deloading. METHODS:Elutriated monocytes from nine healthy donors were differentiated in vitro for four days. Macrophages were then loaded with native LDL, eLDL and oxLDL for 24h and subsequently deloaded with HDL3 for another 24h. Lipidomic and transcriptomic profiles were obtained. RESULTS:Loading of macrophages with eLDL and oxLDL led to a transient but strong elevation of lysophosphatidylcholine (LPC) most likely through direct uptake. Only eLDL induced increased levels of total PC, presumably through an induction of PC synthesis. On the other hand treatment with oxLDL led to a significant increase in PC P. Analysis of individual lipid species showed lipoprotein and saturation specific effects for LPC, PC P and PE P species. Membrane fluidity was decreased by the large amount of FC contained in the lipoproteins, as indicated by a lower PC to FC ratio after lipoprotein loading. In contrast the observed changes in the saturated to mono-unsaturated fatty acid (SFA to MUFA) and saturated to poly-unsaturated fatty acid (SFA to PUFA) ratios in PE P could represent a cellular reaction to counteract this effect by producing more fluid membranes. Transcriptomic analysis showed considerable differences between eLDL and oxLDL treated macrophages. As a common feature of both lipoproteins we detected a strong downregulation of pathways for endogenous lipid synthesis as well as for exogenous lipid uptake. Deloading with HDL3 had only minor effects on total lipid class as well as on individual lipid species levels, most of the time not reaching significance. Interestingly treatment with HDL3 had no effect on membrane fluidity under these conditions, although incubation with HDL3 was partially able to counteract the oxLDL induced transcriptomic effects. To investigate the functional effect of lipoprotein treatment on macrophage polarization we performed surface marker flow cytometry. Under our experimental conditions oxLDL was able to partially shift the surface marker pattern towards a pro-inflammatory M1-like phenotype. This is consistent with the consumption of arachidonic acid containing PE P species in oxLDL treated cells, presumably for the synthesis of inflammatory mediators. SUMMARY:Our findings provide novel data on the lipoprotein induced, lipidomic and transcriptomic changes in macrophages. This can help us better understand the development of metabolic, inflammatory diseases as well as improve our background knowledge on lipid biomarkers in serum.
Project description:Hyperglycemia-induced production of endothelin (ET)-1 is a hallmark of endothelial dysfunction in diabetes. Although the detrimental vascular effects of increased ET-1 are well known, the molecular mechanisms regulating endothelial synthesis of ET-1 in the setting of diabetes remain largely unidentified. Here, we show that adapter molecule TRAF3 interacting protein 2 (TRAF3IP2) mediates high glucose-induced ET-1 production in endothelial cells and ET-1-mediated endothelial cell inflammation. Specifically, we found that high glucose upregulated TRAF3IP2 in human aortic endothelial cells, which subsequently led to activation of JNK and IKK?. shRNA-mediated silencing of TRAF3IP2, JNK1, or IKK? abrogated high-glucose-induced ET-converting enzyme 1 expression and ET-1 production. Likewise, overexpression of TRAF3IP2, in the absence of high glucose, led to activation of JNK and IKK? as well as increased ET-1 production. Furthermore, ET-1 transcriptionally upregulated TRAF3IP2, and this upregulation was prevented by pharmacological inhibition of ET-1 receptor B using BQ-788, or inhibition of NADPH oxidase-derived reactive oxygen species using gp91ds-tat and GKT137831. Notably, we found that knockdown of TRAF3IP2 abolished ET-1-induced proinflammatory and adhesion molecule (IL-1?, TNF-?, monocyte chemoattractant protein 1, ICAM-1, VCAM-1, and E-selectin) expression and monocyte adhesion to endothelial cells. Finally, we report that TRAF3IP2 is upregulated and colocalized with CD31, an endothelial marker, in the aorta of diabetic mice. Collectively, findings from the present study identify endothelial TRAF3IP2 as a potential new therapeutic target to suppress ET-1 production and associated vascular complications in diabetes. NEW & NOTEWORTHY This study provides the first evidence that the adapter molecule TRAF3 interacting protein 2 mediates high glucose-induced production of endothelin-1 by endothelial cells as well as endothelin-1-mediated endothelial cell inflammation. The findings presented herein suggest that TRAF3 interacting protein 2 may be an important therapeutic target in diabetic vasculopathy characterized by excess endothelin-1 production.
Project description:We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-?B and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18R?. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. These results demonstrate that the promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-xpressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo.
Project description:The IκBKinase (IKK) complex represents a central signaling nexus in the TNF-dependent activation of the pro-inflammatory NF-κB pathway. However, recent studies suggested that the distinct IKK subunits (IKK, IKK, and NEMO) might withhold additional NF-κB-independent functions in inflammation and cancer. Here, we generated mice lacking all three IKK subunits in liver parenchymal cells (LPC) (IKK//NEMOLPC-KO) and compared their phenotype with mice lacking both catalytic subunits (IKK/LPC-KO), allowing to functionally dissect putative I-κB-Kinase-independent functions of the regulatory subunit NEMO. We show that the additional deletion of NEMO rescues IKK/LPC-KO mice from lethal cholestasis and biliary ductopenia by triggering LPC apoptosis and inducing a strong compensatory proliferation of LPC including cholangiocytes. Beyond this beneficial effect, we show that increased hepatocyte cell-death and compensatory proliferation inhibit the activation of LPC-necroptosis but trigger spontaneous hepatocarcinogenesis in IKK//NEMOLPC-KO mice. Collectively, our data show that free NEMO molecules unbound to the catalytic IKK subunits control LPC programmed cell death pathways and proliferation, cholestasis and hepatocarcinogenesis independently of an IKK-related function. These findings support the idea of different functional levels at which NEMO controls inflammation and cancer in the liver.
Project description:TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates I?B kinase (IKK)/NF-?B, JNK/AP-1, and c/EBP? and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-?B, JNK/AP-1, c/EBP?, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.
Project description:BACKGROUND: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NF?B and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of a dominant negative AMPK?2 mutant in tumor necrosis factor-?-stimulated human endothelial cells resulted in increased NF?B activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPK?2(-/-) mice the interleukin (IL)-1? induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NF?B activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPK?2(-/-) mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of I?B and p65, indicating a link between AMPK and the I?B kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPK?2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPK?2(+/+) versus AMPK?2(-/-) mice. CONCLUSIONS: These results demonstrate that the IKK is a direct substrate of AMPK?2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NF?B activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.
Project description:Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKK?, a subunit of the I?B kinase (IKK) complex that is essential for the activation of canonical NF-?B signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.
Project description:UNLABELLED: RATIONALE:C-reactive protein (CRP) and lysophosphatidylcholine (LPC) are phosphorylcholine-(PC)-containing oxidized phospholipids (oxPLs) found in oxidized LDL (oxLDL), which trigger pro-atherogenic activities of macrophages during the process of atherosclerosis. It has been previously reported that CRP binds to the PC head group of oxLDL in a calcium-dependent manner. The aim of this study was to investigate the importance of binding between CRP and LPC to the pro-atherogenic activities of macrophages. OBJECTIVES AND FINDINGS:A chemiluminescent immunoassay and HPLC showed that human recombinant CRP formed a stable complex with LPC in the presence of calcium. The Kd value of the binding of the CRP-LPC complex to the receptors Fc?RIA or Fc?RIIA was 3-5 fold lower than that of CRP alone. The CRP-LPC complex triggered less potent generation of reactive oxygen species and less activation of the transcription factors AP-1 and NF-kB by human monocyte-derived macrophages in comparison to CRP or LPC alone. However, CRP did not affect activities driven by components of oxLDL lacking PC, such as upregulation of PPRE, ABCA1, CD36 and PPAR? and the enhancement of cholesterol efflux by human macrophages. The presence of CRP inhibited the association of Dil-labelled oxLDL to human macrophages. CONCLUSIONS:The formation of complexes between CRP and PC-containing oxPLs, such as LPC, suppresses the pro-atherogenic effects of CRP and LPC on macrophages. This effect may in part retard the progression of atherosclerosis.
Project description:Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5?,6? epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity.