ABSTRACT
Tissue factor (TF) is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa). Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that, together with PPARα and PPARß/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARß/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway.
ABSTRACT
BACKGROUND: Atherosclerosis is an inflammatory disease in which macrophages play a crucial role. Macrophages are present in different phenotypes, with at the extremes of the spectrum the classical M1 pro-inflammatory and the alternative M2 anti-inflammatory macrophages. The neuron-derived orphan receptor 1 (NOR1), together with Nur77 and Nurr1, are members of the NR4A orphan nuclear receptor family, expressed in human atherosclerotic lesion macrophages. However, the role of NOR1 in human macrophages has not been studied yet. OBJECTIVES: To determine the expression and the functions of NOR1 in human alternative macrophages. METHODS AND RESULTS: In vitro IL-4 polarization of primary monocytes into alternative M2 macrophages enhances NOR1 expression in human but not in mouse macrophages. Moreover, NOR1 expression is most abundant in CD68+MR+ alternative macrophage-enriched areas of human atherosclerotic plaques in vivo. Silencing NOR1 in human alternative macrophages decreases the expression of several M2 markers such as the Mannose Receptor (MR), Interleukin-1 Receptor antagonist (IL-1Ra), CD200 Receptor (CD200R), coagulation factor XIII A1 polypeptide (F13A1), Interleukin 10 (IL-10) and the Peroxisome Proliferator-Activated Receptor (PPAR)γ. Bioinformatical analysis identified F13A1, IL-1Ra, IL-10 and the Matrix Metalloproteinase-9 (MMP9) as potential target genes of NOR1 in human alternative macrophages. Moreover, expression and enzymatic activity of MMP9 are induced by silencing and repressed by NOR1 overexpression in M2 macrophages. CONCLUSIONS: These data identify NOR1 as a transcription factor induced during alternative differentiation of human macrophages and demonstrate that NOR1 modifies the alternative macrophage phenotype.
Subject(s)
Carotid Artery Diseases/metabolism , DNA-Binding Proteins/metabolism , Macrophage Activation , Macrophages/metabolism , Receptors, Steroid/metabolism , Receptors, Thyroid Hormone/metabolism , Animals , Biomarkers/metabolism , Carotid Artery Diseases/genetics , Carotid Artery Diseases/immunology , Carotid Artery Diseases/pathology , Cell Differentiation , Cells, Cultured , DNA-Binding Proteins/genetics , Gene Silencing , Humans , Interleukin 1 Receptor Antagonist Protein/genetics , Interleukin 1 Receptor Antagonist Protein/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Interleukin-4/pharmacology , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/immunology , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice, Inbred C57BL , Phenotype , Plaque, Atherosclerotic , Primary Cell Culture , RNA Interference , Receptors, Steroid/genetics , Receptors, Thyroid Hormone/genetics , STAT6 Transcription Factor/genetics , STAT6 Transcription Factor/metabolism , Signal Transduction , Time Factors , TransfectionABSTRACT
Coronary artery disease (CAD) is a major cause of morbidity and mortality. Mutations in C6ORF105, associated with decreased gene expression, positively correlate with the risk of CAD in Chinese populations. Moreover, the C6ORF105-encoded protein may play a role in coagulation. Here, we report that C6ORF105 gene expression is lower in circulating mononuclear cells from obese diabetic than lean subjects. Moreover, C6ORF105 is expressed in human macrophages and atherosclerotic lesions, where its expression positively correlates with expression of the transcription factor Peroxisome Proliferator-Activated Receptor (PPAR)γ. Activation of PPARγ increases, in a PPARγ-dependent manner, the expression of C6ORF105 in human macrophages and atherosclerotic lesions.
Subject(s)
Coronary Artery Disease/genetics , Macrophages/metabolism , Membrane Proteins/genetics , PPAR gamma/physiology , Atherosclerosis/metabolism , Cells, Cultured , Diabetes Mellitus, Type 2/metabolism , Female , Gene Expression , Humans , Membrane Proteins/biosynthesis , Obesity/metabolism , Transcriptional ActivationABSTRACT
AIMS/HYPOTHESIS: Human adipose tissue macrophages (ATMs) display an alternatively activated (M2) phenotype, but are still able to produce excessive inflammatory mediators. However, the processes driving this particular ATM phenotype are not understood. Genome-wide association studies associated the CDKN2A locus, encoding the tumour suppressor p16(INK4A), with the development of type 2 diabetes. In the present study, p16(INK4A) levels in human ATMs and the role of p16(INK4A) in acquiring the ATM phenotype were assessed. METHODS: Gene expression of p16 ( INK4A ) in ATMs was analysed and compared with that in monocyte-derived macrophages (MDMs) from obese patients or with macrophages from human atherosclerotic plaques (AMs). Additionally, p16(INK4A) levels were studied during macrophage differentiation and polarisation of monocytes isolated from healthy donors. The role of p16(INK4A) in MDMs from healthy donors was investigated by small interfering (si)RNA-mediated silencing or adenovirus-mediated overproduction of p16(INK4A). RESULTS: Compared with MDMs and AMs, ATMs from obese patients expressed lower levels of p16 ( INK4A ). In vitro, IL-4-induced M2 polarisation resulted in lower p16(INK4A) protein levels after differentiation of monocytes from healthy donors in macrophages. Silencing of p16(INK4A) in MDMs mediated by siRNA increased the expression of M2 marker genes and enhanced the response to lipopolysaccharide (LPS), to give a phenotype resembling that of ATM. By contrast, adenovirus-mediated overproduction of p16(INK4A) in MDMs diminished M2 marker gene expression and the response to LPS. Western blot analysis revealed that p16(INK4A) overproduction inhibits LPS- and palmitate-induced Toll-like receptor 4 (TLR4)-nuclear factor of κ light polypeptide gene enhancer in B cells (NF-κB) signalling. CONCLUSIONS/INTERPRETATION: These results show that p16(INK4A) inhibits the acquisition of the ATM phenotype. The age-related increase in p16(INK4A) level may thus influence normal ATM function and contribute to type 2 diabetes risk.
Subject(s)
Adipose Tissue/metabolism , Cell Polarity , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Macrophages/metabolism , Cyclin-Dependent Kinase Inhibitor p16/genetics , Diabetes Mellitus, Type 2/metabolism , Down-Regulation , Female , Gene Silencing , Humans , Male , NF-kappa B/metabolism , Obesity/metabolism , Plaque, Atherosclerotic/metabolism , RNA, Small Interfering/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
Fibrates and glitazones are two classes of drugs currently used in the treatment of dyslipidemia and insulin resistance (IR), respectively. Whereas glitazones are insulin sensitizers acting via activation of the peroxisome proliferator-activated receptor (PPAR) gamma subtype, fibrates exert their lipid-lowering activity via PPARalpha. To determine whether PPARalpha activators also improve insulin sensitivity, we measured the capacity of three PPARalpha-selective agonists, fenofibrate, ciprofibrate, and the new compound GW9578, in two rodent models of high fat diet-induced (C57BL/6 mice) or genetic (obese Zucker rats) IR. At doses yielding serum concentrations shown to activate selectively PPARalpha, these compounds markedly lowered hyperinsulinemia and, when present, hyperglycemia in both animal models. This effect relied on the improvement of insulin action on glucose utilization, as indicated by a lower insulin peak in response to intraperitoneal glucose in ciprofibrate-treated IR obese Zucker rats. In addition, fenofibrate treatment prevented high fat diet-induced increase of body weight and adipose tissue mass without influencing caloric intake. The specificity for PPARalpha activation in vivo was demonstrated by marked alterations in the expression of PPARalpha target genes, whereas PPARgamma target gene mRNA levels did not change in treated animals. These results indicate that compounds with a selective PPARalpha activation profile reduce insulin resistance without having adverse effects on body weight and adipose tissue mass in animal models of IR.
Subject(s)
Adipose Tissue/drug effects , Butyrates/pharmacology , Clofibrate/pharmacology , Fenofibrate/pharmacology , Insulin Resistance , Phenylurea Compounds/pharmacology , Receptors, Cytoplasmic and Nuclear/agonists , Transcription Factors/agonists , Animals , Butyrates/therapeutic use , Clofibrate/therapeutic use , Fenofibrate/therapeutic use , Hypolipidemic Agents/pharmacology , Hypolipidemic Agents/therapeutic use , Male , Mice , Mice, Inbred C57BL , Obesity/drug therapy , Phenylurea Compounds/therapeutic use , Rats , Rats, ZuckerABSTRACT
Plasma fibrinogen levels have been identified as an important risk factor for cardiovascular diseases. Among the few compounds known to lower circulating fibrinogen levels in humans are certain fibrates. We have studied the regulation of fibrinogen gene expression by fibrates in rodents. Treatment of adult male rats with fenofibrate (0.5% [wt/wt] in the diet) for 7 days decreased hepatic Aalpha-, Bbeta-, and gamma-chain mRNA levels to 52% +/- 7%, 46% +/- 8%, and 81% +/- 19% of control values, respectively. In parallel, plasma fibrinogen concentrations were decreased to 63% +/- 7% of controls. The suppression of fibrinogen expression was dose-dependent and was already evident after 1 day at the highest dose of fenofibrate tested (0.5% [wt/wt]). Nuclear run-on experiments showed that the decrease in fibrinogen expression after fenofibrate occurred at the transcriptional level, as exemplified for the gene for the Aalpha-chain. Other fibrates tested showed similar effects on fibrinogen expression and transcription. The effect of fibrates is specific for peroxisome proliferator-activated receptor-alpha (PPARalpha) because a high-affinity ligand for PPARgamma, the thiazolidinedione BRL 49653, lowered triglyceride levels, but was unable to suppress fibrinogen expression. Direct evidence for the involvement of PPARalpha in the suppression of fibrinogen by fibrates was obtained using PPARalpha-null (-/-) mice. Compared with (+/+) mice, plasma fibrinogen levels in (-/-) mice were significantly higher (3.20 +/- 0.48 v 2.67 +/- 0.42 g/L). Also, hepatic fibrinogen Aalpha-chain mRNA levels were 25% +/- 11% higher in the (-/-) mice. On treatment with 0.2% (wt/wt) fenofibrate, a significant decrease in plasma fibrinogen to 77% +/- 10% of control levels and in hepatic fibrinogen Aalpha-chain mRNA levels to 65% +/- 12% of control levels was seen in (+/+) mice, but not in (-/-) mice. These studies show that PPARalpha regulates basal levels of plasma fibrinogen and establish that fibrate-suppressed expression of fibrinogen in rodents is mediated through PPARalpha.
Subject(s)
Fenofibrate/pharmacology , Fibrinogen/genetics , Gene Expression Regulation/drug effects , Hypolipidemic Agents/pharmacology , Liver/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Animals , Cells, Cultured , Clofibrate/pharmacology , Clofibric Acid/analogs & derivatives , Clofibric Acid/pharmacology , DNA-Binding Proteins/physiology , Fenofibrate/analogs & derivatives , Fibric Acids , Fibrinogen/metabolism , Kinetics , Liver/cytology , Liver/drug effects , Male , Mice , Mice, Knockout , Peroxisome Proliferators/pharmacology , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/deficiency , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription, Genetic , Triglycerides/bloodABSTRACT
We analysed the distribution of LpA-I particles according to their molecular weight in 34 men with symptomatic coronary artery disease (CAD) and 11 men with no symptoms of CAD (control group). Using an original rapid and reproducible gradient gel electrophoresis technique, three LpA-I subclasses were defined: large (L-LpA-I), intermediate (I-LpA-I) and small LpA-I (S-LpA-I). The proportion of L-LpA-I was significantly lower in the CAD group (37.5 +/- 18.5%) than in the control group (58.9 +/- 15.0%) (P < 0.01). Conversely, a significantly (P < 0.05) higher proportion of I-LpA-I (31.9 +/- 20.7%) was observed in the CAD group compared with the control group (14.2 +/- 8.2%). Also, in the CAD group, the proportion of L-LpA-I was positively associated with the plasma level of LpA-I (P < 0.05) and, conversely, the proportion of S-LpA-I was negatively associated with LpA-I levels (P < 0.01). L-LpA-I and I-LpA-I from CAD patients and from control subjects were most effective in promoting cholesterol efflux from Fu5AH rat hepatoma cells, whereas S-LpA-I was ineffective in this regard. In conclusion, the decreased ratio in CAD patients of L-LpA-I, lipoprotein subspecies that are required for cholesterol efflux from cells, suggests a potential anti-atherogenic effect of these particles associated with the larger LpA-I subfractions.
Subject(s)
Apolipoprotein A-I/classification , Cholesterol/metabolism , Coronary Disease/metabolism , Aged , Animals , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Male , Middle Aged , Rats , Tumor Cells, CulturedABSTRACT
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.