Bruno Derudas

Human Atherosclerotic Plaque Alternative Macrophages Display Low Cholesterol Handling but High Phagocytosis Because of Distinct Activities of the PPARγ and LXRα Pathways

Rationale: A crucial step in atherogenesis is the infiltration of the subendothelial space of large arteries by monocytes where they differentiate into macrophages and transform into lipid-loaded foam cells. Macrophages are heterogeneous cells that adapt their response to environmental cytokines. Th1 cytokines promote monocyte differentiation into M1 macrophages, whereas Th2 cytokines trigger an “alternative” M2 phenotype. Objective: We previously reported the presence of CD68+ mannose receptor (MR)+ M2 macrophages in human atherosclerotic plaques. However, the function of these plaque CD68+MR+ macrophages is still unknown. Methods and Results: Histological analysis revealed that CD68+MR+ macrophages locate far from the lipid core of the plaque and contain smaller lipid droplets compared to CD68+MR− macrophages. Interleukin (IL)-4–polarized CD68+MR+ macrophages display a reduced capacity to handle and efflux cellular cholesterol because of low expression levels of the nuclear receptor liver x receptor (LXR)&agr; and its target genes, ABCA1 and apolipoprotein E, attributable to the high 15-lipoxygenase activity in CD68+MR+ macrophages. By contrast, CD68+MR+ macrophages highly express opsonins and receptors involved in phagocytosis, resulting in high phagocytic activity. In M2 macrophages, peroxisome proliferator-activated receptor (PPAR)&ggr; activation enhances the phagocytic but not the cholesterol trafficking pathways. Conclusions: These data identify a distinct macrophage subpopulation with a low susceptibility to become foam cells but high phagocytic activity resulting from different regulatory activities of the PPAR&ggr;-LXR&agr; pathways.

Regulation of Bile Acid Synthesis by the Nuclear Receptor Rev-erbα

BACKGROUND & AIMS Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erbalpha is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erbalpha in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. METHODS Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erbalpha. RESULTS Rev-erbalpha-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erbalpha-deficient mice, whereas adenovirus-mediated hepatic Rev-erbalpha overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erbalpha-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erbalpha-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erbalpha target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erbalpha-deficient mice. CONCLUSIONS These data identify a role for Rev-erbalpha in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression.

SUMOylation of Human Peroxisome Proliferator-activated Receptor alpha Inhibits Its Trans-activity through the Recruitment of the Nuclear Corepressor NCoR

The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a key regulator of genes implicated in lipid homeostasis and inflammation. PPARα trans-activity is enhanced by recruitment of coactivators such as SRC1 and CBP/p300 and is inhibited by binding of corepressors such as NCoR and SMRT. In addition to ligand binding, PPARα activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. In this report, we demonstrate that hPPARα is SUMOylated by SUMO-1 on lysine 185 in the hinge region. The E2-conjugating enzyme Ubc9 and the SUMO E3- ligase PIASy are implicated in this process. In addition, ligand treatment decreases the SUMOylation rate of hPPARα. Finally, our results demonstrate that SUMO-1 modification of hPPARα down-regulates its trans-activity through the specific recruitment of corepressor NCoR but not SMRT leading to the differential expression of a subset of PPARα target genes. In conclusion, hPPARα SUMOylation on lysine 185 down-regulates its trans-activity through the selective recruitment of NCoR.

PPARα gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis

BACKGROUND & AIMS Peroxisome proliferator-activated receptors (PPARs) have been implicated in non-alcoholic steatohepatitis (NASH) pathogenesis, mainly based on animal data. Gene expression data in NASH patients are scarce. We studied liver PPARα, β/δ, and γ expression in a large cohort of obese patients assessed for presence of NAFLD at baseline and 1 year follow-up. METHODS Patients presented to the obesity clinic underwent a hepatic work-up. If NAFLD was suspected, liver biopsy was performed. Gene expression was studied by mRNA quantification. Patients were reassessed after 1 year. RESULTS 125 patients were consecutively included in the study, of which 85 patients had paired liver biopsy taken at 1 year of follow-up. Liver PPARα expression negatively correlated with the presence of NASH (p=0.001) and with severity of steatosis (p=0.003), ballooning (p=0.001), NASH activity score (p=0.008) and fibrosis (p=0.003). PPARα expression was positively correlated to adiponectin (R(2)=0.345, p=0.010) and inversely correlated to visceral fat (R(2)=-0.343, p<0.001), HOMA IR (R(2)=-0.411, p<0.001) and CK18 (R(2)=-0.233, p=0.012). Liver PPARβ/δ and PPARγ expression did not correlate with any histological feature nor with glucose metabolism or serum lipids. At 1 year, correlation of PPARα expression with liver histology was confirmed. In longitudinal analysis, an increase in expression of PPARα and its target genes was significantly associated with histological improvement (p=0.008). CONCLUSION Human liver PPARα gene expression negatively correlates with NASH severity, visceral adiposity and insulin resistance and positively with adiponectin. Histological improvement is associated with an increase in expression of PPARα and its target genes. These data might suggest that PPARα is a potential therapeutic target in NASH.

The nuclear receptor FXR is expressed in pancreatic β-cells and protects human islets from lipotoxicity

Farnesoid X receptor (FXR) is highly expressed in liver and intestine where it controls bile acid (BA), lipid and glucose homeostasis. Here we show that FXR is expressed and functional, as assessed by target gene expression analysis, in human islets and β‐cell lines. FXR is predominantly cytosolic‐localized in the islets of lean mice, but nuclear in obese mice. Compared to FXR+/+ mice, FXR−/− mice display a normal architecture and β‐cell mass but the expression of certain islet‐specific genes is altered. Moreover, glucose‐stimulated insulin secretion (GSIS) is impaired in the islets of FXR−/− mice. Finally, FXR activation protects human islets from lipotoxicity and ameliorates their secretory index.

Liver X Receptor Activation Stimulates Iron Export in Human Alternative Macrophages

Rationale: In atherosclerotic plaques, iron preferentially accumulates in macrophages where it can exert pro-oxidant activities. Objective: The objective of this study was, first, to better characterize the iron distribution and metabolism in macrophage subpopulations in human atherosclerotic plaques and, second, to determine whether iron homeostasis is under the control of nuclear receptors, such as the liver X receptors (LXRs). Methods and Results: Here we report that iron depots accumulate in human atherosclerotic plaque areas enriched in CD68 and mannose receptor (MR)-positive (CD68+MR+) alternative M2 macrophages. In vitro IL-4 polarization of human monocytes into M2 macrophages also resulted in a gene expression profile and phenotype favoring iron accumulation. However, M2 macrophages on iron exposure acquire a phenotype favoring iron release, through a strong increase in ferroportin expression, illustrated by a more avid oxidation of extracellular low-density lipoprotein by iron-loaded M2 macrophages. In line, in human atherosclerotic plaques, CD68+MR+ macrophages accumulate oxidized lipids, which activate LXR&agr; and LXR&bgr;, resulting in the induction of ABCA1, ABCG1, and apolipoprotein E expression. Moreover, in iron-loaded M2 macrophages, LXR activation induces nuclear factor erythroid 2-like 2 expression, thereby increasing ferroportin expression, which, together with a decrease of hepcidin mRNA levels, promotes iron export. Conclusions: These data identify a role for M2 macrophages in iron handling, a process regulated by LXR activation.

Peroxisome Proliferator-Activated Receptor-α Activation Protects Brain Capillary Endothelial Cells from Oxygen-Glucose Deprivation-Induced Hyperpermeability in the Blood-Brain Barrier

That promising neuroprotectants failed to demonstrate benefit against stroke highlights the great difficulties to translate preclinical pharmacological effects in clinical outcomes. Part of this hurdle implies the complex response to injury of the neurovascular unit increasing the cerebrovascular permeability at the level of the blood-brain barrier (BBB). Previous studies reported neuroprotection in animal models upon activation of the nuclear receptor PPARα (peroxisome proliferator-activated receptor) α, but the cellular targets at the BBB level remain largely unexplored. Here, to study whether PPAR-α activation acts on BBB permeability, we adapted a mouse BBB cell model to ischaemic conditions at the stage of occlusion defined in vitro as oxygen-glucose deprivation (OGD). This model consists of a co-culture of brain capillary endothelial cells (ECs) on a filter insert placed upon a rat glial cell culture. The EC monolayer permeability increase induced by 4 h of OGD was significantly restricted after treatment with the PPAR-α agonist fenofibric acid (FA) 24 h before or at the onset of OGD. Treatments of separated ECs or glial cells showed that this protective effect was conferred by BBB ECs but not glial cells. Furthermore, co-cultures with ECs from PPAR-α-deficient mice revealed that FA had no effect on OGD-induced hyperpermeability. No transcriptional modulation of classical PPAR-α target genes such as SOD, ICAM-1, VCAM-1, ACO, CPT-1, PDK-4 or ET-1 was observed in wild type mouse ECs. In conclusion, these results suggest that part of the preventive PPAR-α-mediated protection may occur via BBB ECs by limiting hyperpermeability.

Human Adipose Tissue Macrophages Display Activation of Cancer-related Pathways

Background: Obesity increases the risk for cancer development, suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages infiltrate adipose tissue as well as tumors. Results: Human adipose tissue macrophages (ATM) resemble human tumor-associated macrophages (TAM). Conclusion: ATM may modulate cancer cell function. Significance: ATM may be potential contributors to cancer development in obese subjects. Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.

Impaired alternative macrophage differentiation of peripheral blood mononuclear cells from obese subjects

Visceral obesity is a chronic, low-grade inflammatory disease that predisposes people to the metabolic syndrome, type 2 diabetes and its cardiovascular complications. Adipose tissue is not a passive storehouse for fat, but an endocrine organ synthesizing and releasing a variety of bioactive molecules, some of which are produced by infiltrated immune-inflammatory cells including macrophages. Two different subpopulations of macrophages have been identified in adipose tissue: pro-inflammatory ‘classical’ M1 and anti-inflammatory ‘alternative’ M2 macrophages, and their ratio is suggested to influence the metabolic complications of obesity. These macrophages derive primarily from peripheral blood mononuclear cells (PBMCs). We hypothesised that obesity and the metabolic syndrome modulate PBMC functions. Therefore, alteration of the monocyte response, and more specifically their ability to differentiate toward alternative anti-inflammatory macrophages, was assessed in PBMCs isolated from lean and obese subjects with or without alterations in glucose homeostasis. Our results indicate that PBMCs from obese subjects have an altered expression of M2 markers and that their monocytes are less susceptible to differentiate toward an alternative phenotype. Thus PBMCs in obesity are programmed, which may contribute to the inflammatory dysregulation and increased susceptibility to inflammatory diseases in these patients.