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In a previous study, we identified the regulated in development and DNA damage response 2 (REDD2) gene as a highly expressed gene in human atherosclerotic lesions in comparison to normal artery, as well as in cultured human macrophages, and showed its implication in oxidized low-density lipoprotein (LDL)-induced macrophage death sensitivity. In this article, we attempt to identify the mechanism by which REDD2 induces such a phenomenon. Transient transfection of U-937 monocytic cells with a pCI.CMV.REDD2 expression vector increased by approximately twofold the mRNA levels of REDD2 in comparison to control cells transfected with pCI.CMV.GFP. Reactive oxygen species (ROS) production was significantly induced in REDD2-transfected cells compared with control cells (157+/-48 and 100+/-8 arbitrary units/mg cell protein, respectively; p<0.05). Moreover, a significant increase in parameters known to reflect the oxidative modifications of LDL was observed. Among enzymes involved in ROS production or degradation, we found a specific reduction in thioredoxin-1 (Trx-1) mRNA ( approximately 52+/-7% decrease, p<0.01 vs control cells) and protein ( approximately 60+/-4% decrease, p<0.001 vs control cells) levels in cells overexpressing REDD2 in comparison to control cells. In contrast, transfection of U-937 cells with siRNA against REDD2 decreased the mRNA levels of REDD2 by approximately 60% and increased Trx-1 mRNA and protein levels. Moreover, we observed no or a moderate increase in Bax (proapoptotic) and a significant decrease in Bcl2 (antiapoptotic) gene expression in cells that overexpress REDD2 compared to control cells. In addition, we showed that Trx-1 mRNA and protein levels were increased at low H(2)O(2) doses and decreased at higher doses. Interestingly, macrophages isolated from human atherosclerotic lesions differentially express REDD2 and Trx-1. Indeed, in certain patients, levels of REDD2 mRNA were low and those of Trx-1 mRNA were high. In contrast, in other patients, levels of REDD2 were high and levels of Trx-1 mRNA were low.

Macrophage-derived reactive oxygen species contribute to the initiation and development of atherosclerosis. The cellular balance between oxidative and reductive states depends on the endogenous antioxidant capacity, with the thioredoxin-1 (Trx-1) system playing a major role. Peroxisome proliferator-activated receptor-alpha (PPARalpha) is expressed by human macrophages and exhibits anti-inflammatory properties. Here we show that the selective PPARalpha activator GW647 significantly increased the Trx-1 mRNA and protein expression in human macrophages as determined by quantitative polymerase chain reaction and Western immunoblotting. Consistently, the Trx-1 activity was significantly increased by PPARalpha activation. By contrast, PPARalpha activation led to the down-regulation of vitamin D(3) up-regulated protein 1 (VDUP-1), the physiological inhibitor of Trx-1. Analysis of the Trx-1 and VDUP-1 promoters with gene reporter assays, mutational analysis, gel shift assays and chromatin immunoprecipitation analyses revealed the presence of a functional response element specific for PPARalpha in the Trx-1 promoter and the presence of a functional activator protein 1 (AP-1) site in the VDUP-1 promoter. The interference of PPARalpha/retinoid X receptor alpha with the AP-1 transcription factor elements c-Jun/c-Fos resulted in the inhibition of AP-1 binding and down-regulation of the VDUP-1 gene expression. Finally, PPARalpha activation reduced the lidocaine-induced caspase-3 activity and apoptosis, which might be due to the VDUP-1-mediated regulation of the Bax/Bcl-2 ratio. Together these data indicate that stimulation of PPARalpha in human macrophages might reduce arterial inflammation through differential regulation of the Trx-1 and VDUP-1 gene expression.

MMP-12, a macrophage-specific matrix metalloproteinase with large substrate specificity, has been reported to be highly expressed in mice, rabbits and human atherosclerotic lesions. Increased MMP-12 from inflammatory macrophages is associated with several degenerative diseases such as atherosclerosis. In this manuscript, we show that IL-1beta, a proinflammatory cytokine found in atherosclerotic plaques, increases both mRNA and protein levels of MMP-12 in human monocyte-derived macrophages (HMDM). Since peroxisome proliferator-activated receptors (PPARs), such as PPARalpha and PPARgamma, are expressed in macrophages and because PPAR activation exerts an anti-inflammatory effect on vascular cells, we have investigated the effect of PPARalpha and gamma isoforms on MMP-12 regulation in HMDM. Our results show that MMP-12 expression (mRNA and protein) is down regulated in IL-1beta-treated macrophages only in the presence of a specific PPARalpha agonist, GW647, in a dose-dependent manner. In contrast, this inhibitory effect was abolished in IL-1beta-stimulated peritoneal macrophages isolated from PPARalpha(-/-) mice and treated with the PPARalpha agonist, GW647. Moreover, reporter gene transfection experiments using different MMP-12 promoter constructs showed a reduction of the promoter activities by approximately 50% in IL-1beta-stimulated PPARalpha-pre-treated cells. However, MMP-12 promoter analysis did not reveal the presence of a PPRE response element. The IL-1beta effect is known to be mediated through the AP-1 binding site. Mutation of the AP-1 site, located at -81 in the MMP-12 promoter region relative to the transcription start site, followed by transfection analysis, gel shift and ChIP experiments revealed that the inhibitory effect was the consequence of the protein-protein interaction between GW 647-activated PPARalpha and c-Fos or c-Jun transcription factors, leading to inhibition of their binding to the AP-1 motif. These studies suggest that PPARalpha agonists may be used therapeutically, not only for lipid disorders, but also to prevent inflammation and atheromatous plaque rupture, where their ability to inhibit MMP-12 expression in HMDM may be beneficial.

Objective: In this article, we studied the effect of acetyl-11-keto-beta-boswellic acid (AKbetaBA), a natural inhibitor of the proinflammatory transcription factor NF-kappaB on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE-/-) mice., Methods and Results: Atherosclerotic lesions were induced by weekly LPS injection in apoE-/- mice. LPS alone increased atherosclerotic lesion size by approximately 100%, and treatment with AKbetaBA significantly reduced it by approximately 50%. Moreover, the activity of NF-kappaB was also reduced in the atherosclerotic plaques of LPS-injected apoE-/- mice treated with AKbetaBA. As a consequence, AKbetaBA treatment led to a significant downregulation of several NF-kappaB-dependent genes such as MCP-1, MCP-3, IL-1alpha, MIP-2, VEGF, and TF. By contrast, AKbetaBA did not affect the plasma concentrations of triglycerides, total cholesterol, antioxidized LDL antibodies, and various subsets of lymphocyte-derived cytokines. Moreover, AKbetaBA potently inhibited the IkappaB kinase (IKK) activity immunoprecipitated from LPS-stimulated mouse macrophages and mononuclear cells leading to decreased phosphorylation of IkappaB alpha and inhibition of p65/NF-kappaB activation. Comparable AKbetaBA-mediated inhibition was also observed in LPS-stimulated human macrophages., Conclusions: The inhibition of NF-kappaB activity by plant resins from species of the Boswellia family might represent an alternative for classical medicine treatments for chronic inflammatory diseases such as atherosclerosis.

Lipid accumulation alters macrophage biology and contributes to lipid retention within the vessel wall. In this study, we investigated the role of adipophilin on triglyceride accumulation and lipid-droplet formation in THP-1-derived macrophages (THP-1 macrophages). In the presence of acetylated low-density lipoprotein, macrophages infected with an adenovirus expressing human adipophilin showed a 31% increase in triglyceride content and a greater number of lipid droplets compared with control cells. Incubation of macrophages with very low-density lipoprotein (VLDL) dramatically increased cellular triglyceride content similarly in control and adipophilin-overexpressing cells. By itself, VLDL increased adipophilin expression, which explains the lack of effect of adipophilin overexpression on cellular triglyceride content in macrophages loaded with VLDL. The lipid-droplet content of macrophages was increased by overexpression of adipophilin and/or loading with VLDL. In contrast, inhibition of adipophilin expression using siRNA prevented lipid-droplet formation and significantly reduced intracellular triglyceride content. Using inhibitors of beta-oxidation and acyl-coenzyme A synthetase, results were obtained which suggest that adipophilin elevates cellular lipids by inhibition of beta-oxidation and stimulation of long-chain fatty acid incorporation into triglycerides. Adipophilin expression in THP-1 macrophages altered the cellular content of different lipids and enhanced the size of lipid droplets, consistent with a role for adipophilin in human foam cell formation.

Aims: We investigated the effect of plasma levels of human tissue inhibitor of metalloproteinase (hTIMP)-1 on arterial lesion development and aneurysm formation in apolipoprotein-E-deficient mice (ApoE(-/-))., Methods: Control and transgenic mice were fed either a chow diet or a high-fat diet for 90 and 180 days., Results: hTIMP-1 has a tendency to decrease atherosclerotic lesions, but did not attain significance (approximately 6% reduction in hTIMP-1(+/+), p = 0.075, and approximately 4% in hTIMP-1(+/0), p = 0.088 vs. control). Immunohistological and histological analyses revealed a reduction in macrophage accumulation (23% of control in hTIMP(+/0), p = 0.065, and 49% of control in hTIMP(+/+), p < 0.05) but not in collagen degradation within the lesion in transgenic mice. Moreover, elastin degradation in sites of pseudo-microaneurysms was reduced in transgenic mice (37% of control in hTIMP-1(+/0), p < 0.05, and 50% of control in hTIMP-1(+/+), p < 0.05). DNA array analysis of matrix metalloproteinase (MMP) expression followed by real-time PCR quantification revealed a significant up-regulation of MMP-3, MMP-12 and MMP-13 in arterial lesions of ApoE(-/-) mice fed a high-fat diet in comparison with the same mice fed a chow diet., Conclusion: These data show that hTIMP-1 reduces aneurysm formation in ApoE(-/-) mice but does not protect them against the development of arterial lesions.