Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing.

OBJECTIVES: The aim of this study was to combine clinical criteria and next-generation sequencing (pyrosequencing) to establish a diagnosis of familial hypercholesterolaemia (FH). DESIGN, SETTING AND SUBJECTS: A total of 77 subjects with a Dutch Lipid Clinic Network score of ≥ 3 (possible, probable or definite FH clinical diagnosis) were recruited from the Lipid Clinic at Sahlgrenska Hospital, Gothenburg, Sweden. Next-generation sequencing was performed in all subjects using SEQPRO LIPO RS, a kit that detects mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLR adapter protein 1 (LDLRAP1) genes; copy-number variations in the LDLR gene were also examined. RESULTS: A total of 26 mutations were detected in 50 subjects (65% success rate). Amongst these, 23 mutations were in the LDLR gene, two in the APOB gene and one in the PCSK9 gene. Four mutations with unknown pathogenicity were detected in LDLR. Of these, three mutations (Gly505Asp, Ile585Thr and Gln660Arg) have been previously reported in subjects with FH, but their pathogenicity has not been proved. The fourth, a mutation in LDLR affecting a splicing site (exon 6-intron 6) has not previously been reported; it was found to segregate with high cholesterol levels in the family of the proband. CONCLUSIONS: Using a combination of clinical criteria and targeted next-generation sequencing, we have achieved FH diagnosis with a high success rate. Furthermore, we identified a new splicing-site mutation in the LDLR gene.

Glucose impairs B-1 cell function in diabetes.

B-1 lymphocytes produce natural immunoglobulin (Ig)M, among which a large proportion is directed against apoptotic cells and altered self-antigens, such as modified low-density lipoprotein (LDL). Thereby, natural IgM maintains homeostasis in the body and is also protective against atherosclerosis. Diabetic patients have an increased risk of developing certain infections as well as atherosclerosis compared with healthy subjects, but the underlying reason is not known. The aim of this study was to investigate whether diabetes and insulin resistance affects B-1 lymphocytes and their production of natural IgM. We found that diabetic db/db mice had lower levels of peritoneal B-1a cells in the steady state-condition compared to controls. Also, activation of B-1 cells with the Toll-like receptor (TLR)-4 agonist Kdo2-Lipid A or immunization against Streptococcus pneumoniae led to a blunted IgM response in the diabetic db/db mice. In-vitro experiments with isolated B-1 cells showed that high concentrations of glucose, but not insulin or leptin, caused a reduced secretion of total IgM and copper-oxidized (CuOx)-LDL- and malondialdehyde (MDA)-LDL-specific IgM from B-1 cells in addition to a decreased differentiation into antibody-producing cells, proliferation arrest and increased apoptosis. These results suggest that metabolic regulation of B-1 cells is of importance for the understanding of the role of this cell type in life-style-related conditions.

LDL-associated apolipoprotein J and lysozyme are associated with atherogenic properties of LDL found in type 2 diabetes and the metabolic syndrome.

OBJECTIVES: Exchangeable low-density lipoprotein (LDL)-associated proteins can affect the atherogenic properties of LDL. Our aim was to analyse the protein composition of LDL from individuals with or without type 2 diabetes and the metabolic syndrome (T2DM) in relation to other LDL particle characteristics, to assess whether certain proteins associate more with certain subclasses of LDL typical for T2DM, such as small, apoCIII-rich LDL. DESIGN: Low-density lipoprotein from two cohorts of 61-year-old men (n = 19 and 64) with or without T2DM was isolated using size-exclusion chromatography or deuterium oxide-based ultracentrifugation. LDL-associated proteins were identified using mass spectrometry and quantified using two-dimensional gel electrophoresis or enzyme-linked immunosorbent assay. Differently expressed LDL-associated proteins apolipoprotein (apo)J and lysozyme were also measured in serum from a third cohort of women (n = 71) with or without T2DM. Lysozyme binding to advanced glycation end product (AGE)-LDL was examined in vitro. RESULTS: ApoJ and lysozyme were increased in LDL particles with increased apoCIII content and decreased cholesterol content. When isolated with size-exclusion chromatography, LDL from individuals with T2DM contained more apoJ and lysozyme and less apoA1 than LDL from control individuals. LDL content of apoJ correlated with a smaller LDL particle size. Serum levels of lysozyme, but not apoJ, were increased in individuals with T2DM. In vitro, lysozyme associated more with AGE-LDL than with unmodified LDL. CONCLUSIONS: Our results indicate that apoJ and lysozyme are increased in LDL with characteristics of small dense LDL in T2DM. Small dense LDL is easily glycated, and the increased affinity of lysozyme for AGE-LDL provides a possible partial explanation for an increase lysozyme in LDL from those with type 2 diabetes.

15-Lipoxygenase-2 is expressed in macrophages in human carotid plaques and regulated by hypoxia-inducible factor-1alpha.

BACKGROUND: Macrophages are prominent in hypoxic areas of atherosclerotic lesions and their secreted cytokines, growth factors and activity of enzymes are involved in atherogenesis. Previously, we showed that 15-lipoxygenase (LOX)-2 is expressed in human monocyte-derived macrophages and that hypoxia increases 15-LOX-2 expression and secretion of pro-inflammatory molecules. Here we investigated whether human carotid plaque macrophages express 15-LOX-2 and whether its expression in macrophages is regulated by hypoxia through hypoxia-inducible factor 1alpha (HIF-1alpha). MATERIALS AND METHODS: Carotid plaques from 47 patients with high-grade symptomatic carotid artery stenosis were analysed using immunohistochemistry, and stained areas were quantified by digital image analysis. Carotid plaque macrophages were isolated with anti-CD14 immunobeads using an immunomagnetic bead technique. Primary macrophages were transfected with HIF-1alpha siRNA or control siRNA before extraction of RNA and medium analysis. RESULTS: In paired tissue sections, the extent of staining for CD68 correlated with staining for 15-LOX-2 but not for 15-LOX-1. In carotid plaque macrophages isolated with anti-CD14 immunobeads, 15-LOX-2 mRNA was expressed at high levels. In primary macrophages, 15-LOX-2 expression was significantly increased by incubation with the HIF-1alpha stabilizer dimethyloxalylglycine. Knockdown of HIF-1alpha significantly decreased production of the 15-LOX-2 enzyme products 12- and 15-hydroxyeicosatetraenoic acid. In carotid plaques, HIF-1alpha staining correlated with staining for 15-LOX-2. CONCLUSIONS: These results demonstrate that 15-LOX-2 is highly expressed in human plaques and is correlated with the presence of macrophages and HIF-1alpha. 15-LOX-2 enzyme activity can be modulated by HIF-1alpha. Thus, increased expression of 15-LOX-2 in macrophages in hypoxic atherosclerotic plaque may enhance inflammation and the recruitment of inflammatory cells.

Circulating matrix metalloproteinase 9 levels in relation to sampling methods, femoral and carotid atherosclerosis.

OBJECTIVES: To examine whether circulating levels of matrix metalloproteinase 9 (MMP-9) were associated with ultrasound-assessed intima-media thickness (IMT) and echolucent plaques in the carotid and femoral arteries. To examine preanalytical sources of variability in MMP-9 concentrations related to sampling procedures. SUBJECTS AND DESIGN: Plasma and serum MMP-9 levels were compared with ultrasound assessed measures of femoral and carotid atherosclerosis, in a cross-sectional study of 61-year-old men (n = 473). Preanalytical sources of variability in MMP-9 levels were examined in 10 healthy subjects. Main outcome measures were circulating levels of MMP-9 in serum and plasma, IMT of the carotid and femoral arteries, and plaque status based on size and echolucency. SETTING: Research unit at university hospital. RESULTS: Plasma concentrations of total and active MMP-9 were associated with femoral artery IMT independently of traditional cardiovascular risk factors, and were higher in subjects with moderate to large femoral plaques. Plasma MMP-9 concentration was higher in men with echolucent femoral plaques (P = 0.006) compared with subjects without femoral plaques. No similar associations were found for carotid plaques. MMP-9 concentrations were higher in serum than in plasma, and higher when sampling was performed with Vacutainer than with syringe. MMP-9 levels in serum were more strongly associated with peripheral neutrophil count compared with MMP-9 levels in plasma. CONCLUSIONS: Plasma MMP-9 levels were associated with atherosclerosis in the femoral artery, and total MMP-9 concentration was higher in men with echolucent femoral plaques. The choice of sample material and sampling method affect the measurements of circulating MMP-9 levels.

Increased lipolysis by secretory phospholipase A(2) group V of lipoproteins in diabetic dyslipidaemia.

BACKGROUND: Lipolysis of lipoproteins by secretory phospholipase A(2) group V (sPLA(2)-V) promotes inflammation, lipoprotein aggregation and foam cell formation--all considered as atherogenic mechanisms. OBJECTIVE: In this study, we compared the susceptibility to sPLA(2)-V lipolysis of VLDL and LDL from individuals with type 2 diabetes and the metabolic syndrome (T2D-MetS) and from healthy controls. Design. VLDL and LDL were isolated from 38 T2D-MetS subjects and 38 controls, treated pair-wise. Extent of sPLA(2)-V lipolysis was measured as release of nonesterified free fatty acids (NEFA). In a subset of the subjects, lipoprotein composition was determined as a relationship between lipid and apolipoprotein components. RESULTS: Mean paired increase in sPLA(2)-V lipolysis after 1 h for T2D-MetS versus control was 2.0 micromol NEFA l(-1) for VLDL (P = 0.004) and 0.75 micromol NEFA l(-1) for LDL (P = 0.001). There were also substantial differences in lipoprotein composition between the groups. T2D-MetS VLDL had higher triglyceride and cholesterol contents than control VLDL. T2D-MetS LDL was smaller and contained more triglycerides and less cholesterol than control LDL. Both VLDL and LDL from T2D-MetS subjects also contained more apolipoprotein CIII per particle. CONCLUSION: VLDL and LDL from T2D-MetS individuals were more susceptible to sPLA(2)-V lipolysis than those from control individuals. This may result in elevated levels of NEFA and lysophosphatidylcholine, both in circulation and in LDL, possibly contributing to the elevated inflammatory state and increased risk of cardiovascular diseases seen in these individuals.

C-reactive protein, interleukin-6, secretory phospholipase A2 group IIA and intercellular adhesion molecule-1 in the prediction of late outcome events after acute coronary syndromes.

OBJECTIVE: We investigated whether levels of C-reactive protein (CRP), interleukin-6 (IL-6), secretory phospholipase A(2) group IIA (sPLA(2)-IIA) and intercellular adhesion molecule-1 (ICAM-I) predict late outcomes in patients with acute coronary syndromes (ACS). DESIGN: Prospective longitudinal study. CRP (mg L(-1)), IL-6 (pg mL(-1)), sPLA(2)-IIA (ng mL(-1)) and ICAM-1 (ng mL(-1)) were measured at days 1 (n = 757) and 4 (n = 533) after hospital admission for ACS. Their relations to mortality and rehospitalization for myocardial infarction (MI) and congestive heart failure (CHF) were determined. SETTING: Coronary Care Unit at Sahlgrenska University Hospital, Gothenburg, Sweden. SUBJECTS: Patients with ACS alive at day 30; median follow-up 75 months. RESULTS: Survival was related to day 1 levels of all markers. After adjustment for confounders, CRP, IL-6 and ICAM-1, but not sPLA(2)-IIA, independently predicted mortality and rehospitalization for CHF. For CRP, the hazard ratio (HR) was 1.3 for mortality (95% confidence interval (CI): 1.1-1.5, P = 0.003) and 1.4 for CHF (95% CI: 1.1-1.9, P = 0.006). For IL-6, HR was 1.3 for mortality (95% CI: 1.1-1.6, P < 0.001) and 1.4 for CHF (95% CI: 1.1-1.8, P = 0.02). For ICAM-1, HR was 1.2 for mortality (95% CI: 1.0-1.4, P = 0.04) and 1.3 for CHF (95% CI: 1.0-1.7, P = 0.03). No marker predicted MI. Marker levels on day 4 provided no additional predictive value. CONCLUSIONS: In patients with ACS, CRP, IL-6, sPLA(2)-IIA and ICAM-1 are associated with long-term mortality and CHF, but not reinfarction. CRP, IL-6 and ICAM-1 provide prognostic information beyond that obtained by clinical variables.

Release of protein as well as activity of MMP-9 from unstable atherosclerotic plaques during percutaneous coronary intervention.

OBJECTIVES: Few studies have investigated the composition of unstable coronary plaques in vivo in humans. The aims of this study were to investigate if substances released from plaques during percutaneous coronary intervention (PCI) under distal protection could give information about plaque composition and also indicate possible biomarkers in plasma that may be used to identify patients at risk. METHODS AND RESULTS: Twenty patients with acute coronary syndromes undergoing PCI with distal protection were included. Plasma samples were taken before, during, and after the PCI in the aortic root, locally in the culprit vessel and intravenously. Plasma was analysed for possible markers of plaque instability. During PCI, local increases were observed for matrix metalloproteinase 9 (MMP-9), protein (P < 0.001) as well as activity (P < 0.001), interleukin 6 (IL-6; P < 0.01) and oxidized low-density lipoprotein (oxLDL; P = 0.01) in the culprit coronary artery. A systemic inflammatory response was also seen with increased levels of IL-10, MMP-3, serum amyloid A and C-reactive protein, but with no increase in MMP-9. CONCLUSIONS: Our study shows that local sampling of blood under distal protection may be used to analyse coronary plaques and to identify biomarkers for unstable plaques. Our results suggest that MMP-9 is a potential biomarker, and that IL-6, MMP9 and possibly oxLDL are released from plaques.

How can we prevent cardiovascular disease in diabetes.

Evidence based goals for the treatment and prevention of atherosclerosis in diabetes are given in international and national guidelines. The importance of optimal control of lipids and blood pressure has been shown in several studies. With available drugs and behavioural modifications the treatment goals can be reached in most cases. However, only a few patients with diabetes are treated optimally today. A major possibility to reduce cardiovascular disease in diabetes is to treat patients according to guidelines. New treatment targets may include specific treatment of the dyslipidaemia, manifested in high levels of small dense LDL and low HDL, active anti-inflammatory treatments, specific reduction of inflammatory activity in adipose tissue, reduced volume of adipose tissue, antioxidants and reduction of advanced glycosylation endproducts production. Possible strategies for these treatments are available, and should be evaluated in clinical trials.

Pathogenesis and treatment of diabetic vascular disease - illustrated by two cases.

This publication is a summary of the presentations given at the First JIM Grand Round held at the Sahlgrenska University Hospital on 15 March 2006. The Grand Round was based on two case reports; a patient with type 2 diabetes and pronounced macrovascular disease and another patient with early microvascular disease combined with the macrovascular complications. The pathogenesis of the vascular complications and the current treatment regimens were discussed in relation to the history and examinations performed in these patients.

Composition of coronary plaques obtained by directional atherectomy in stable angina: its relation to serum lipids and statin treatment.

OBJECTIVES: The stability and inflammatory activity in atherosclerotic plaques may be modulated by lipids and lipoproteins as well as the pleiotropic effects of statins. The aim of this study was to analyse the effect of statin treatment as well as the relation of plasma lipids and lipoproteins to tissue composition in atherosclerotic plaques. DESIGN: Patients with stable angina and coronary plaques suitable for directional coronary atherectomy (DCA) were randomized to atorvastatin (80 mg once daily) or placebo (29 randomized, 22 underwent DCA, 11/group). After an average treatment of 10 weeks, patients underwent DCA, tissue specimens were obtained, and the tissue composition was determined by immunohistochemistry. RESULTS: Atorvastatin reduced the T-cell content, but did not change lipid, collagen, smooth muscle cell, or macrophage content. Plasma levels of apolipoprotein AI (apoAI) correlated positively with tissue collagen and inversely with metalloproteinase-9 and macrophage content. About half the specimens contained neutrophil granulocytes. CONCLUSIONS: Short-term atorvastatin treatment tended to reduce the T-cell content of atherosclerotic plaques, indicating modulation of cell-mediated immunity. High plasma levels of apoAI correlated with increased collagen content and reduced inflammation, supporting the notion that plasma apoAI stabilizes atherosclerotic plaques. The significance of neutrophils in the lesions merits further study.

Monocytic expression of CD14 and CD18, circulating adhesion molecules and inflammatory markers in women with diabetes mellitus and impaired glucose tolerance.

AIMS/HYPOTHESIS: Type 2 diabetes is a major risk factor for cardiovascular disease. Monocyte recruitment and inflammatory activation are crucial steps in the development of atherosclerosis and several receptors are involved in these processes. The aim of this study was to investigate levels of CD14 and the beta(2)-integrin subunits CD11b and CD18 on monocytes from women with diabetes or impaired glucose tolerance. METHODS: A population-based sample of 112 Swedish women, who were aged 64 years and had diabetes mellitus or impaired or normal glucose tolerance, was investigated. Cell surface receptors were analysed with flow cytometry and serum inflammation markers and soluble adhesion molecules with enzyme-linked methods. RESULTS: The monocytic CD14 expression and serum levels of C-reactive protein, IL-6 and soluble adhesion molecules were higher in the diabetes group than in the group with normal glucose tolerance. Monocytic CD18 was elevated both in the diabetes and in the impaired glucose tolerance groups. The levels of monocytic surface markers correlated with BMI and to a lesser extent with glycaemic control. CONCLUSIONS/INTERPRETATION: The increased monocytic expression of important surface receptors together with elevated serum inflammation markers supports the concept of increased inflammation in type 2 diabetes and may be an important factor for the risk of atherosclerosis.

A common polymorphism in the interleukin-6 gene promoter is associated with overweight.

OBJECTIVE: Human body fat mass is to a large extent genetically determined, but little is known about the susceptibility genes for common obesity. Interleukin-6 (IL-6) suppresses body fat mass in rodents, and IL-6 treatment increases energy expenditure in both rodents and humans. The -174 G/C single-nucleotide polymorphism (SNP) in the IL-6 gene promoter is common in many populations, and -174 C-containing promoters have been found to be weaker enhancers of transcription. Moreover, a SNP at position -572 in the IL-6 promoter has recently been reported to affect transcription. The objective was to investigate the association between the IL-6 gene promoter SNPs and obesity. DESIGN: Trans-sectional association study of IL-6 gene promoter SNPs and indices of obesity. SUBJECTS: Two study populations, the larger one consisting of hypertensive individuals (mean age 57 y, 73% males, n=485) and the other consisting of 20 y younger nonobese healthy females (n=74). MEASUREMENTS: Genotyping for the -174 IL-6 G/C and the -572 G/C SNPs, body mass index (BMI), serum leptin levels, serum IL-6 levels, C-reactive protein, fasting blood glucose and various blood lipids. RESULTS: The common -174 C allele (f(C)=0.46), but not any -572 allele, was associated with higher BMI and higher serum leptin levels in both study populations. In the larger population, there were significant odds ratios for the association of CC (2.13) and GC (1.76) genotypes with overweight (BMI>25 kg/m(2)). Moreover, as the C allele was common, it accounted for a significant population-attributable risk of overweight (12%; CI 2-21%), although its average effect was modest in this sample. CONCLUSION: Genetically determined individual differences in production of IL-6 may be relevant for the regulation of body fat mass.

Low-grade inflammation, endothelial activation and carotid intima-media thickness in type 2 diabetes.

OBJECTIVES: The objective of this study was to assess the relationship between inflammation, endothelial activation and incipient atherosclerosis in type 2 diabetes. DESIGN: Cross-sectional study. Setting and subjects. We studied 239 type 2 diabetic patients [71 with clinical cardiovascular disease (CVD)] and 78 healthy control subjects, aged 50-75 in a single research centre. METHODS: Carotid intima-media thickness (IMT) was determined by ultrasound. Circulating intracellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, ultra-sensitive C-reactive protein, human serum amyloid A, interleukin-6, monocyte colony-stimulating factor, secretory nonpancreatic phospholipase A(2) type IIA, glucose, HbA1c, and lipid/lipoprotein variables were measured. RESULTS: Carotid IMT was significantly thicker in diabetic patients than healthy controls across the whole age range. IMT was also thicker in diabetic patients with, than without, CVD, but this difference disappeared after controlling for confounding factors. Concentrations of the inflammatory and endothelial markers except IL-6 were significantly higher in the diabetic patients than in healthy controls, but comparable in diabetic patients with and without CVD. The main determinants of IMT in the diabetic patients were blood pressure, age and diabetes duration. CONCLUSIONS: Low-grade inflammation and endothelial activation are increased in diabetic patients but do not associate with IMT or clinical CVD. The inflammatory reaction seems to be rather a feature of the metabolic syndrome than a direct determinant of atherosclerosis.

Mapping of ATP, glucose, glycogen, and lactate concentrations within the arterial wall.

OBJECTIVE: In large- and medium-sized arteries, the diffusion distances for oxygen and nutrients are long. This has been suggested to make these vessels prone to develop local energy metabolic deficiencies that could contribute to atherogenesis. To validate this hypothesis, we introduced a new method to measure energy metabolites within the arterial wall at high spatial resolution. METHODS AND RESULTS: Bioluminescence imaging was used to quantify local metabolite concentrations in cryosections of snap frozen (in vivo) and incubated pig carotid artery rings. Incubation at hypoxia resulted in increased lactate concentrations, whereas ATP, glucose, and glycogen concentrations were decreased, especially in the mid media, where concentrations of these metabolites were close to zero. In snap frozen arteries, glycogen concentrations were markedly higher in deep layers of the media than toward the lumen. ATP, glucose, and lactate were more homogenously distributed. CONCLUSIONS: Bioluminescence imaging is a new and powerful tool to assess arterial wall energy metabolism at high spatial resolution. Our experiments demonstrate heterogeneous distributions of energy metabolites under hypoxic in vitro conditions. Furthermore, we show that glycogen concentrations are higher in deep medial layers in vivo. This might represent a local adaptation to a low-oxygen microenvironment.

Increased Interleukin-6 levels in pituitary-deficient patients are independently related to their carotid intima-media thickness.

OBJECTIVE: Increased cardiovascular morbidity and mortality has been observed in patients with pituitary deficiency and untreated growth hormone deficiency (GHD). We investigated peripheral inflammatory and fibrinolytic markers and their associations with arterial intima-media thickness (IMT) in GHD. DESIGN: Cross-sectional study. PATIENTS: Thirty-four patients with GHD, but without cardiovascular disease, were compared to healthy controls matched for age, sex, body mass index (BMI) and smoking habits. MEASUREMENTS: IMT of the common carotid artery, C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) activity and tissue plasminogen activator antigen (tPA-ag) were measured. RESULTS: Median IL-6 concentrations were increased by 208% and 248% in GHD patients compared to BMI-matched and nonobese controls, respectively. Median CRP and tPA-ag levels were increased by 237% and 167% in patients compared to nonobese controls, but not significantly different compared to BMI-matched controls. Plasma levels of fibrinogen and PAI-1 activity did not differ between groups. Age, low-density lipoprotein (LDL) cholesterol, tPA-ag and IL-6 were positively correlated, and IGF-I was negatively correlated to IMT in the patient group, but only age and IL-6 were independently related to IMT. CONCLUSIONS: IL-6 concentrations were increased in GHD patients compared to controls and independently related to IMT in patients. This finding may help to explain the variance in IMT and the increased vascular morbidity and mortality in hypopituitary patients with GHD.

Fatty acids modulate the effect of darglitazone on macrophage CD36 expression.

BACKGROUND: Scavenger receptor-mediated uptake of cholesterol by macrophages in the arterial wall is believed to be proatherogenic. Thiazolidinediones are peroxisome proliferator-activated receptor gamma (PPARgamma)-agonists, which are used in the treatment of type II diabetes. They reduce atherogenesis in LDL receptor deficient and ApoE knockout mice, but up-regulate CD36, which may contribute to foam cell formation. The dyslipidaemia in type II diabetes is characterized by high levels of nonesterified fatty acids. Therefore we tested the effect of fatty acids and how fatty acids and the thiazolidinedione darglitazone interact in their effect on CD36 expression in human monocytes and macrophages. MATERIALS AND METHODS: Flow cytometry and reverse transcription-polymerase chain reaction were used to study CD36 expression. Cellular lipids were analyzed with high performance liquid chromatography. RESULTS: Darglitazone increased CD36 mRNA and protein expression in human macrophage cells. In the presence of 5% human serum, darglitazone increased the accumulation of triglycerides, but did not affect cholesterol ester levels. In the presence of albumin-bound oleic or linoleic acid, darglitazone did not increase CD36 mRNA, cell-surface CD36 protein or triglyceride content. Fatty acids per se increased CD36 mRNA and protein. DISCUSSION: The increase in CD36 in macrophages suggests a role for fatty acids in the regulation of foam cell formation. The results also suggest that the potentially proatherogenic CD36 up-regulating effect of thiazolidinediones in macrophages might not be present when the cells have access to physiological levels of albumin-bound fatty acids.

Circulating adiponectin levels are reduced in nonobese but insulin-resistant first-degree relatives of type 2 diabetic patients.

Adiponectin, one of the most abundant gene transcript proteins in human fat cells, has been shown to improve insulin action and is also suggested to exert antiatherogenic effects. We measured circulating adiponectin levels and risk factors for atherosclerosis in 45 healthy first-degree relatives of type 2 diabetic subjects (FDR) as well as 40 healthy control subjects (CON) without a known family history of diabetes. Insulin sensitivity (S(i)) was studied with the minimal model, and measurements of adiponectin, metabolic variables, inflammatory markers, and endothelial injury markers, as well as lipoprotein concentrations, were performed. FDR were insulin resistant (3.3 +/- 2.4 vs. 4.5 +/- 2.6 x 10(-4) x min(-1) per microU/ml [mean +/- SD], P < 0.01), and their circulating plasma adiponectin levels (6.6 +/- 1.8 vs. 8.1 +/- 3.0 microg/ml, P < 0.03) were decreased. After adjustments for age in FDR, adiponectin levels were negatively correlated with fasting proinsulin (r -0.64, P < 0.001), plasminogen activator inhibitor (PAI)-1 activity (r -0.56, P < 0.001), fasting insulin (r -0.55, P < 0.001), and acute insulin response (r -0.40, P < 0.05); they were positively related to HDL cholesterol (r 0.48, P < 0.01) and S(i) (r 0.41, P < 0.01). Furthermore, when adjusted for age, waist, and S(i), adiponectin was associated with HDL cholesterol and proinsulin, which explained 51% of the variation in adiponectin in multiple regression analyses in that group. In conclusion, circulating plasma adiponectin levels were decreased in nonobese but insulin-resistant FDR and, in addition, related to several facets of the insulin resistance syndrome (IRS). Thus, hypoadiponectinemia may be an important component of the association between cardiovascular disease and IRS.

Post-transcriptional regulation of VEGF expression by oxidised LDL in human macrophages.

BACKGROUND: Accumulation of oxidised low density lipoproteins (oxLDL) in macrophages and their subsequent transformation into lipid-filled foam cells is generally considered an early event in atherosclerosis. Vascular Endothelial Growth Factor (VEGF) may contribute to atherogenesis through increased vascular permeability, chemoattraction towards monocytes and intraplaque vessel formation. In this study we investigate the effect and regulation of VEGF expression in human macrophages stimulated with oxLDL. MATERIALS AND METHODS: Vascular Endothelial Growth Factor mRNA and protein expression was assayed using RT-PCR and ELISA, respectively. The activity of mitogen-activated protein kinases (MAPKs) was investigated using Western blots. RESULTS: In human monocyte-derived macrophages, oxLDL significantly increased VEGF mRNA expression and subsequent protein secretion in a concentration-dependent manner after 6 h and 18 h, respectively. Using an in vitro mRNA decay assay, we show that this oxLDL-induced VEGF expression partly is regulated through increased stability of the VEGF mRNA. Involvement of MAPKs has previously been implicated in the stabilisation of VEGF mRNA. Activity of the p38 MAPK, but not the c-jun-N-terminal kinase (JNK), increased in macrophages stimulated with oxLDL (50 micro g mL-1) for 5-15 min. Preincubation with SB202190 (20 micro M), a specific inhibitor of p38 MAPK, significantly decreased the oxLDL-induced VEGF mRNA expression by 40%. The prolonged half-life of VEGF mRNA, induced by oxLDL, was not inhibited by SB202190. CONCLUSIONS: OxLDL increases VEGF expression and p38 MAPK activity in human macrophages. The increased VEGF mRNA expression by oxLDL is mediated through at least two intracellular pathways, one involving p38 MAPK and another that independently of p38 MAPK activity increases VEGF mRNA stability.