Nerve growth factor in the equine joint.

Nerve growth factor (NGF) is a neurotrophin with many functions. In humans, it is involved in inflammation, nerve growth, apoptosis and pain signalling. Increased concentrations of NGF in synovial fluid has been shown in humans and dogs with osteoarthritis. Despite osteoarthritis being a common problem in horses, no studies have previously been published on NGF in the equine joint. The aim of this study was to quantify NGF in equine synovial fluid from healthy joints, acutely inflamed septic joints and joints with structural changes associated with osteoarthritis. A secondary aim was to identify the localisation of NGF and its two receptors, TrkA and p75NTR, in healthy and osteoarthritic articular cartilage. NGF concentrations in synovial fluid from osteoarthritic joints (n = 27), septic joints (n = 9) and healthy joints (n = 16) were determined by ELISA. In addition, articular cartilage from osteoarthritic and healthy joints was examined for NGF, TrkA and p75NTR using immunohistochemistry staining. NGF was present in equine synovial fluid and articular cartilage. Compared to synovial fluid from healthy joints, NGF concentration was higher in synovial fluid from joints with structural osteoarthritic changes (P = 0.032) or acute septic inflammation (P = 0.006). In articular cartilage with severe osteoarthritic changes, there was more abundant positive immunohistochemistry staining for NGF and its receptors than in normal articular cartilage. Further studies should focus on identifying precursor forms of NGF, and on receptor expression and downstream signalling of TrkA and P75NTR in health and disease.

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.

Oxysterols present in atherosclerotic tissue decrease the expression of lipoprotein lipase messenger RNA in human monocyte-derived macrophages.

The presence of oxysterols in macrophages isolated from atherosclerotic tissue and the effect of oxysterols on the regulation of lipoprotein lipase (LPL) mRNA were studied. Both rabbit and human macrophages, freshly isolated from atherosclerotic aorta, show about the same distribution of oxysterols, analyzed by isotope dilution mass spectrometry, except that all three preparations of human arterial-derived macrophages contained high levels of 27-hydroxycholesterol, which was not found in rabbit macrophages. To determine if oxysterols regulate LPL expression, human monocyte-derived macrophages were incubated with different oxysterols. Incubation with 7 beta-hydroxycholesterol and 25-hydroxycholesterol resulted in a 70-75% reduction of LPL mRNA, analyzed by quantitative RT-PCR. Cholesterol and other tested oxysterols showed no effect on macrophage LPL mRNA expression compared with control. LPL activity in the medium was also reduced after exposure of the macrophages to 7 beta-hydroxycholesterol and 25-hydroxycholesterol. In conclusion, we have demonstrated accumulation of oxysterols in macrophage-derived foam cells isolated from atherosclerotic aorta. There was suppression of LPL mRNA in human monocyte-derived macrophages after incubation with 7 beta-hydroxycholesterol and 25-hydroxycholesterol. It is tempting to suggest that an exposure to oxysterols may explain our earlier observation of a low level of LPL mRNA in arterial foam cells.

Harmful singlet oxygen can be helpful.

Highly reactive harmful singlet oxygen O2(1delta(g)) can be helpful while relaxing to its triplet ground state O2(3sigma(g)-). The energy emitted during this relaxation from the excited energy state is discernable at 634 nm. We report here on the effect of this energy as photon illumination and as energy transfer in air on the production of reactive oxygen species (ROS) by human monocytes, measured as isoluminol-enhanced chemiluminescence. We demonstrate up to 60% decrease in the secretion of ROS after 2-min illumination of the monocytes stimulated with phorbol myristate acetate (PMA). The results provide in vitro documentation of the utility of singlet oxygen energy in modifying cellular behaviour.

Low level expression of hormone-sensitive lipase in arterial macrophage-derived foam cells: potential explanation for low rates of cholesteryl ester hydrolysis.

Conversion of arterial macrophages into foam cells is a key process involved in both the initiation and progression of atherosclerotic lesions. Foam cell formation involves the progressive accumulation and storage of lipoprotein-derived cholesteryl esters. The resulting imbalance in cholesterol metabolism in arterial foam cells may be due in part to an inadequately low level of cytoplasmic neutral cholesteryl ester hydrolase (NCEH) activity. In this study, we have demonstrated that hormone-sensitive lipase (HSL) mRNA is expressed at very low levels in macrophage-derived foam cells, using the unique approach of extracting mRNA from macrophage-derived foam cells purified from human and rabbit atherosclerotic plaques coupled with reverse transcriptase polymerase chain reaction (RT-PCR). We also demonstrate that macrophage-derived foam cells isolated from rabbit atherosclerotic lesions exhibit a resistance to high density lipoprotein (HDL)-mediated cholesterol efflux along with reduced levels of NCEH activity compared to lipid-loaded mouse peritoneal macrophages. Thus, low level expression of HSL may partially account for the reduced NCEH activity observed in arterial foam cells isolated from atherosclerosis-susceptible species.

Butylated hydroxytoluene and N-acetylcysteine attenuates tumor necrosis factor-alpha (TNF-alpha) secretion and TNF-alpha mRNA expression in alveolar macrophages from human lung transplant recipients in vitro.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide cytokine principally produced by macrophages/monocytes and commonly associated with inflammatory conditions. The present study was designed to investigate whether the antioxidants butylated hydroxytoluene (BHT) and N-acetylcysteine (NAC) modified TNF-alpha production in stimulated and unstimulated alveolar macrophages from lung transplant recipients in vitro. METHODS: The effects of BHT and NAC on TNF-alpha production were studied both with and without lipopolysaccharide (LPS) activation of alveolar macrophages from bronchoalveolar lavage fluid. TNF-alpha was quantitated in cell culture medium using an enzyme-linked immunosorbent assay. TNF-alpha mRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction on total RNA extracted from the incubated alveolar macrophages. RESULTS: In unstimulated alveolar macrophages, TNF-alpha levels were significantly reduced by incubation with BHT or NAC. When alveolar macrophages from patients with cytomegalovirus infection were incubated with BHT, TNF-alpha secretion was significantly lowered. A significant reduction of TNF-alpha levels in LPS-stimulated alveolar macrophages was obtained in the presence of BHT or NAC. Our data from quantitative reverse transcription-polymerase chain reaction showed that the observed decrease in protein levels of TNF-alpha was associated with a decrease in TNF-alpha mRNA expression. CONCLUSIONS: Our results indicate that antioxidant treatment may be an effective step to lower the inflammatory process caused by cytomegalovirus infection or in endotoxin (LPS)-activated macrophages. The therapeutic use of antioxidant compounds could, therefore, be of interest in conditions such as lung transplantation, in which oxidative stress and inflammation can contribute significantly to the loss of allograft function.

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.

Macrophages isolated from human atherosclerotic plaques produce IL-8, and oxysterols may have a regulatory function for IL-8 production.

Oxysterols are biologically active molecules generated during oxidation of LDL. Several of these oxysterols were found in macrophage-derived foam cells from human atherosclerotic tissue (eg, 7-hydroxycholesterol, 7-ketocholesterol, 5-epoxycholesterol, and 25-hydroxycholesterol). A specific stimulation of interleukin-8 (IL-8) production by oxidized LDL (oxLDL) has been shown by other investigators. In foam cells from human atherosclerotic tissue, we found high levels of IL-8 (183.1 pg/10(6) cells) compared with monocytes (23.2 pg/10(6) cells) or monocyte-derived macrophages in culture (1.5 pg/10(6) cells). When monocytes and monocyte-derived macrophages, in vitro, were exposed to a series of different oxysterols, we found that all oxysterols tested had a tendency to stimulate IL-8 production but that 25-hydroxycholesterol was the most potent one. This stimulation of IL-8 production was time and dose dependent and could be blocked by cycloheximide. These results indicate that oxysterols in oxLDL may have a regulatory effect on IL-8 production. IL-8, a potent chemoattractant, may play a role in the recruitment of T lymphocytes and smooth muscle cells into the subendothelial space and may contribute to the formation of atherosclerotic lesions.

Oxysterols induce interleukin-1beta production in human macrophages.

BACKGROUND: Oxysterols are biologically active molecules generated during the oxidation of low-density lipoprotein or formed enzymatically in vivo. In the atherosclerotic plaque newly recruited macrophages may be exposed to oxysterols present in the plaque. How these oxysterols affect the expression and secretion of inflammatory cytokines such as interleukin-1beta (IL-1beta) in macrophages is not known. Therefore the aim of the present study was to investigate how oxysterols regulate the expression and secretion of IL-1beta in human monocyte-derived macrophages. METHODS: The IL-1beta messenger RNA (mRNA) expression was analysed by reverse transcription-polymerase chain reaction, and the IL-1beta protein secretion was measured by enzyme-linked immunosorbent assay. RESULTS: A significant, dose-dependent increase in the secretion of IL-1beta was given by 25-hydroxycholesterol without the addition of lipopolysaccharide (LPS). At a concentration of 2.5 microg mL(-1) this increase was similar to that obtained by endotoxin (LPS, 1 microg mL(-1)). A transient increase in IL-1beta mRNA expression was found in macrophages incubated with 25-hydroxycholesterol compared with untreated controls. In addition, 25-hydroxycholesterol dramatically increased the IL-1beta secretion induced by LPS. At a concentration of 5 microg mL(-1) of 25-hydroxycholesterol the LPS-induced IL-1beta secretion was increased by about 25-fold. A similar tendency, but not so consistent, was found for 27-hydroxycholesterol. CONCLUSIONS: Our results show that oxysterols, and 25-hydroxycholesterol in particular, may modulate the inflammatory response in human macrophages. Consequently the presence of oxysterols in atherosclerotic tissue may dramatically influence the effect of inflammation.

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.

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.

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.