Corinne Copin

Modulation of Lipoprotein B Binding to the LDL Receptor by Exogenous Lipids and Apolipoproteins CI, CII, CIII, and E

We have recently shown that apo B-containing lipoproteins isolated by immunoaffinity chromatography bind to the LDL receptor with an affinity dependent on their apo E or apo CIII content. However, these lipoproteins--LpB:E, LpB:CIII, and LpB:CIII:E--isolated from whole plasma have variable lipid and apolipoprotein contents, and it is difficult to consider each parameter separately, particularly because an increase in the apo CIII content is always associated with an increase in the content of other C apolipoproteins. Therefore, we used affinity-purified LpB free of other apolipoproteins. Lipid content of LpB was increased by incubation with a lipid emulsion, and this triglyceride-enriched LpB was named TG-LpB. Free apo CI, apo CII, apo CIII, and apo E were added to LpB and TG-LpB and their associations to the lipoprotein were assessed by gel filtration, nondenaturing electrophoresis, and immunoblotting. Molar ratios of 6 (apo E), 30 (apo CII), 20 (apo CIII), and 30 (apo CI) for 1 apo B were obtained. The association of apo CII to LpB and TG-LpB induced modifications to the LpB structure and a redistribution of lipids and apolipoproteins on the lipoprotein particles. The binding of these LpBs and TG-LpBs with and without added apo CI, CII, CIII, and E was tested at 4 degrees C on the LDL receptors of HeLa cells. The increased content of lipids reduced TG-LpB binding to the LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipophilin Enhances Lipid Accumulation and Prevents Lipid Efflux From THP-1 Macrophages: Potential Role in Atherogenesis

Objective—Uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors results in lipid droplets accumulation and foam cell formation. Excess lipid deposition in macrophages has been reported to modulate expression of several genes including adipophilin. In this study, we investigated the function of adipophilin in lipid accumulation and cholesterol efflux in THP-1 macrophages. Methods and Results—Adipophilin mRNA expression was 3.5-fold higher in human atherosclerotic plaques compared with healthy areas of the same arteries. Moreover, in the presence of acetylated LDL (AcLDL), triglycerides and cholesteryl esters were increased in macrophages overexpressing adipophilin by 40% and 67%, respectively, whereas their accumulation was reduced when endogenous cellular adipophilin was depleted using siRNA approach. In addition, neither overexpression nor downregulation of adipophilin altered expression of genes involved in lipid efflux. However, the affinity and the number of AcLDL receptors were not affected. After 24-hour incubation of lipid-loaded macrophages with apolipoprotein A-I, cholesterol efflux was reduced by 47% in adipophilin transfected cells versus control cells. Conclusion—Our results showed that stimulation of adipophilin expression in macrophages by modified LDL promotes triglycerides and cholesterol storage and reduces cholesterol efflux. Therefore, adipophilin might contribute, in vivo, to lipid accumulation in the intima of the arterial wall.

Antiinflammatory and Antiatherogenic Effects of the NF-κB Inhibitor Acetyl-11-Keto-β-Boswellic Acid in LPS-Challenged ApoE−/− Mice

Objective—In this article, we studied the effect of acetyl-11-keto-β-boswellic acid (AKβBA), a natural inhibitor of the proinflammatory transcription factor NF-&kgr;B 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 ≈100%, and treatment with AKβBA significantly reduced it by ≈50%. Moreover, the activity of NF-&kgr;B was also reduced in the atherosclerotic plaques of LPS-injected apoE−/− mice treated with AKβBA. As a consequence, AKβBA treatment led to a significant downregulation of several NF-&kgr;B–dependent genes such as MCP-1, MCP-3, IL-1α, MIP-2, VEGF, and TF. By contrast, AKβBA did not affect the plasma concentrations of triglycerides, total cholesterol, antioxidized LDL antibodies, and various subsets of lymphocyte-derived cytokines. Moreover, AKβBA potently inhibited the I&kgr;B kinase (IKK) activity immunoprecipitated from LPS-stimulated mouse macrophages and mononuclear cells leading to decreased phosphorylation of I&kgr;Bα and inhibition of p65/NF-&kgr;B activation. Comparable AKβBA-mediated inhibition was also observed in LPS-stimulated human macrophages. Conclusion—The inhibition of NF-&kgr;B 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. (Arterioscler Thromb Vasc Biol. 2008;28:272-277)

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.

Extracellular human thioredoxin-1 inhibits lipopolysaccharide-induced interleukin-1β expression in human monocyte-derived macrophages

Oxidative stress plays an important role in atherosclerotic vascular disease, and several recent studies were focused on thioredoxin-1 (Trx-1) and its potential protective role against oxidative stress. Since human monocyte-derived macrophages (HMDM) are important cells in several inflammatory diseases including atherosclerosis, we conducted this study to evaluate the impact of extracellular recombinant human Trx-1 (rhTrx-1) on gene expression in lipopolysaccharide-activated HMDM. Our results showed that rhTrx-1 was capable of reducing interleukin (IL)-1β mRNA and protein synthesis in a dose-dependent manner. This effect was partly mediated through a reduction of NF-κB activation as analyzed by transient transfection and gel shift assays. In addition, we showed that the attenuation of NF-κB activity was the result of the reduction of both p50 and p65 subunit mRNA and protein synthesis on one hand and of the induction of I-κBα mRNA and protein expression on the other hand. Moreover, inhibition of endogenous Trx-1 mRNA was also observed, suggesting a contribution to the diminution of NF-κB activity since endogenous Trx-1, in contrast to the exogenous Trx-1, activates the NF-κB system. Finally, H2O2-oxidized rhTrx-1 reduced IL-1β mRNA synthesis in lipopolysaccharide-activated HMDM. This result highly suggested that the rhTrx-1 used in this study could be oxidized in the culture medium and, in turn, reduced IL-1β mRNA and protein synthesis. Taken together, these data indicated a potential new mechanism through which extracellular rhTrx-1 exerts an anti-inflammatory function in HMDM.

Apolipoprotein AII Enrichment of HDL Enhances Their Affinity for Class B Type I Scavenger Receptor but Inhibits Specific Cholesteryl Ester Uptake

Apolipoproteins of high density lipoprotein (HDL) and especially apolipoprotein (apo)AI and apoAII have been demonstrated as binding directly to the class B type I scavenger receptor (SR-BI), the HDL receptor that mediates selective cholesteryl ester uptake. However, the functional relevance of the binding capacity of each apolipoprotein is still unknown. The human adrenal cell line, NCI-H295R, spontaneously expresses a high level of SR-BI, the major apoAI binding protein in these cells. As previously described for murine SR-BI, free apoAI, palmitoyl-oleoyl-phosphatidylcholine (POPC)-AI, and HDL are good ligands for human SR-BI. In vitro displacement of apoAI by apoAII in HDLs or in Lp AI purified from HDL by immunoaffinity enhances their ability to compete with POPC-AI to bind to SR-BI and also enhances their direct binding capacity. The next step was to determine whether the higher affinity of apoAII for SR-BI correlated with the specific uptake of cholesteryl esters from these HDLs. Free apoAII and, to a lesser extent, free apoAI that were added to the cell medium during uptake experiments inhibited the specific uptake of [(3)H]cholesteryl esters from HDL, indicating that binding sites on cells were the same as cholesteryl ester uptake sites. In direct experiments, the uptake of [(3)H]cholesteryl esters from apoAII-enriched HDL was highly reduced compared with the uptake from native HDL. These results demonstrate that in the human adrenal cell line expressing SR-BI as the major HDL binding protein, efficient apoAII binding has an inhibitory effect on the delivery of cholesteryl esters to cells.

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.

Peroxisome Proliferator–Activated Receptor-γ Activation Induces 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Human Alternative Macrophages

Objective—11&bgr;-Hydroxysteroid dehydrogenase type 1 (11&bgr;-HSD1) catalyzes the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome proliferator– activated receptor-&ggr; (PPAR&ggr;) is a nuclear receptor controlling inflammation, lipid metabolism, and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11&bgr;-HSD1 and the role of PPAR&ggr; therein. Methods and Results—11&bgr;-HSD1 gene expression is higher in proinflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Interestingly, PPAR&ggr; activation induces 11&bgr;-HSD1 enzyme activity in M2 macrophages but not in resting macrophages or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11&bgr;-HSD1 substrate cortisone, an effect amplified by PPAR&ggr; induction of 11&bgr;-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusion—Our data identify a positive cross-talk between PPAR&ggr; and GR in human M2 macrophages via the induction of 11&bgr;-HSD1 expression and activity.

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.