Christophe Furman

Oxidized phospholipids activate PPARα in a phospholipase A2‐dependent manner

The peroxisome proliferator‐activated receptor α (PPARα) is a transcription factor belonging to the PPAR subfamily of nuclear receptors. Fatty acids and eicosanoids are natural PPARα ligands. Here, we show using transient transfection assays that oxidized (oxLDL) but not native low‐density lipoproteins (LDL) dose‐dependently activate PPARα in endothelial cells without affecting PPARα protein expression. Fractioning of oxLDL lipids followed by transactivation experiments demonstrated that the oxidized phospholipid component in oxLDL is responsible for PPARα activation. Using specific inhibitors, it is shown that oxLDL‐mediated PPARα activation requires phospholipase A2 activity and that the oxidized fatty acids 9‐ and 13‐HODE activate PPARα directly. Finally, we found that, similar to the synthetic PPARα ligand Wy‐14643, oxLDL induced expression of the fatty acid transport protein‐1 in human primary endothelial cells. Our findings define a novel group of PPARα activators and provide a molecular basis for certain effects of these biologically active phospholipids on gene transcription.

Adipophilin increases triglyceride storage in human macrophages by stimulation of biosynthesis and inhibition of β‐oxidation

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 β‐oxidation and acyl‐coenzyme A synthetase, results were obtained which suggest that adipophilin elevates cellular lipids by inhibition of β‐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.

Systemic tissue inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured rat carotid artery

Metalloproteinases (MMP)‐2 and MMP‐9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase‐1 (TIMP‐1) into balloon‐injured rat arteries (3×1011 viral particles/rat; n=7) resulted in a transient expression of TIMP‐1 and a significant inhibition of neointima thickening within 16 days (∼40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by ∼98% in TIMP‐1‐treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP‐1 is a promising approach in early restenosis treatment.

Enhanced VDUP-1 Gene Expression by PPARγ Agonist Induces Apoptosis in Human Macrophage

The fate and phenotype of lesion macrophages is regulated by cellular oxidative stress. Thioredoxin‐1 (Trx‐1) plays a major role in the regulation of cellular redox balance, with resultant effects on gene expression and cellular responses including cell growth and death. Trx‐1 activity is inhibited by interaction with vitamin D‐upregulated protein‐1 (VDUP‐1). Peroxisome proliferator‐activated receptor gamma (PPARγ) is expressed by human monocyte‐derived macrophages (HMDM) and PPARγ agonism has been reported to decrease expression of inflammatory genes and to promote apoptosis of these cells. To determine whether VDUP‐1 may be involved in regulating the effects of PPARγ agonists in macrophages, we investigated the effect of a synthetic PPARγ agonist (GW929) on the expression of VDUP‐1 in HMDM. GW929 concentration‐dependently increased HMDM expression of VDUP‐1 (mRNA and protein). Transfection of different fragments of the VDUP‐1 promoter as well as gel shift analysis revealed the presence of functional PPARγ response elements (PPRE) in the promoter. Under conditions in which PPAR agonism altered levels of VDUP‐1, caspase‐3 activity, and macrophage apoptosis were also elevated. The results suggest that PPARγ activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of VDUP‐1. J. Cell. Physiol. 214:183–191, 2008.

Daily melatonin supplementation in mice increases atherosclerosis in proximal aorta.

Considerable evidence supports the hypothesis that LDL oxidation plays an important role in atherosclerosis. Even though high melatonin doses inhibit LDL oxidation in vitro, the effect of melatonin on atherosclerosis has never been studied. We have demonstrated that the feeding of hypercholesterolemic mice with an atherogenic diet supplemented with melatonin highly increases the surface of atherosclerotic lesions in the proximal aorta. These observations occur without detectable lipidic or glucidic phenotype alteration. Melatonin treatment increased highly the sensitivity of atherogenic lipoprotein to Cu(2+) and gamma-radiolysis generated oxyradical ex vivo oxidation during the fasting period. Moreover, these altered lipoproteins were less recognized by the LDL receptor metabolic pathway of murine fibroblasts while they transferred many more cholesteryl esters to murine macrophages. This study suggests that caution should be taken as regards high melatonin dosage in hypercholesterolemic patients.

REDD2 Gene Is Upregulated by Modified LDL or Hypoxia and Mediates Human Macrophage Cell Death

Objective—Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. Methods and Results—The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. Conclusion—We showed that stimulation of REDD2 expression in macrophages increases oxLDL-induced cell death, suggesting that REDD2 gene might play an important role in arterial pathology.

Apolipoprotein E Knockout Mice Over-Expressing Human Tissue Inhibitor of Metalloproteinase 1 Are Protected against Aneurysm Formation but Not against Atherosclerotic Plaque Development

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.

Ozonized Low Density Lipoprotein (ozLDL) Inhibits NF-κB and IRAK-1–Associated Signaling

Objective—Recent studies have provided strong evidence for the presence of ozone in atherosclerotic lesions. In addition, modification of LDL has been suggested to be involved in atherosclerosis. In the present study we wanted to investigate whether LDL exposed to ozone (ozLDL) is able to modulate the NF-&kgr;B system, as a paradigm for inflammatory signaling. Methods and Results—We showed that activation of NF-&kgr;B by lipopolysaccharide (LPS), a prototypic inducer of innate immunity, was reversibly inhibited by ozLDL in monocytic THP-1 cells in a dose-dependent manner, whereas tumor necrosis factor (TNF) signaling was not affected. This was not attributable to a direct ozone effect or solely the presence of lipoprotein, and neither required direct contact to LPS nor was accompanied by a change in LPS binding. Comparable inhibitory effects of ozLDL were observed in human monocyte/macrophages and endothelial cells. The presence of ozLDL led to a decrease in LPS-induced I&kgr;&Bgr;&agr; proteolysis and a reduction of &kgr;B-dependent transcription/target-gene expression. Furthermore, ozLDL markedly lowered stimulus-induced I&kgr;&Bgr; kinase (IKK) activity and phosphorylation/proteolysis of interleukin (IL)-1 receptor-associated kinase-1 (IRAK-1). Finally, cholesterol ozonization products were identified as effective ozLDL inhibitory compounds. Conclusion—Our study demonstrated that ozLDL inhibited NF-&kgr;B and IRAK-1–associated signaling which may impair immune function and promote apoptosis.

Differential toxicities of air (mO-LDL) or copper-oxidized LDLs (Cu-LDL) toward endothelial cells.

In vivo low density protein (LDL) oxidation is a progressive phenomenon leading to the presence of minimally and highly oxidized LDLs in the subendothelial arterial space. Oxidized LDLs have been reported to be cytotoxic against endothelial cells. The goal of this study was to determine which of the minimally and highly oxidized LDLs were the most cytotoxic against bovine aortic endothelial cells (BAEC). Both the morphological aspect of the cells themselves, and LDH or MTT tests revealed that mO‐ or Cu‐LDLs had similar cytotoxicity with up to 8 hours of oxidation, showing no relation with the level of LDL oxidation; for longer oxidation times, Cu‐LDL cytotoxicity decreased. This phenomenon is linked to their different oxidation kinetics.