Inhibition of SYK kinase does not confer a pro-proliferative or pro-invasive phenotype in breast epithelium or breast cancer cells.

SYK has been reported to possess both tumour promotor and repressor activities and deletion has been linked to a pro-proliferative / pro-invasive phenotype in breast tumours. It is unclear whether this is a consequence of protein deletion or loss of kinase activity. The SYK inhibitor, BI 1002494, caused no increase in proliferation in breast cancer cells or primary mammary epithelial cells in 2D or 3D cultures, nor changes in proliferation (CD1/2, CDK4, PCNA, Ki67) or invadopodia markers (MMP14, PARP, phospho-vimentin Ser56). BI 1002494 did not alter SYK protein expression. There was no change in phenotype observed in 3D cultures after addition of BI 1002494. Thirteen weeks of treatment with BI 1002494 resulted in no ductal branching or cellular proliferation in the mammary glands of mice. An in silico genetic analysis in breast tumour samples revealed no evidence that SYK has a typical tumour suppressor gene profile such as focal deletion, inactivating mutations or lower expression levels. Furthermore, SYK mutations were not associated with reduction in survival and disease-free period in breast cancer patients. In conclusion, small molecule inhibition of the kinase function of SYK does not contribute to a typical tumour suppressor profile.

Opposing effects of in vitro differentiated macrophages sub-type on epithelial wound healing.

Inappropriate repair responses to pulmonary epithelial injury have been linked to perturbation of epithelial barrier function and airway remodelling in a number of respiratory diseases, including chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. We developed an in vitro mechanical scratch injury model in air-liquid interface differentiated primary human small airway epithelial cells that recapitulates many of the characteristics observed during epithelial wound injury in both human tissue and small animal models. Wound closure was initially associated with de-differentiation of the differentiated apical cells and rapid migration into the wound site, followed by proliferation of apical cells behind the wound edge, together with increases in FAK expression, fibronectin and reduction in PAI-1 which collectively facilitate cell motility and extracellular matrix deposition. Macrophages are intimately involved in wound repair so we sought to investigate the role of macrophage sub-types on this process in a novel primary human co-culture model. M1 macrophages promoted FAK expression and both M1 and M2 macrophages promoted epithelial de-differentiation. Interestingly, M2a macrophages inhibited both proliferation and fibronectin expression, possibly via the retinoic acid pathway, whereas M2b and M2c macrophages prevented fibronectin deposition, possibly via MMP expression. Collectively these data highlight the complex nature of epithelial wound closure, the differential impact of macrophage sub-types on this process, and the heterogenic and non-delineated function of these macrophages.

Neutralization of both IL-1α/IL-1ß plays a major role in suppressing combined cigarette smoke/virus-induced pulmonary inflammation in mice.

Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations, which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1ß are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α, IL-1ß or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells, neutrophils, and macrophages. Furthermore, it reduced KC levels significantly. Blocking of IL-1ß did not provide significant activity. In primary human bronchial epithelial air-liquid-interface cell cultures infected with H1N1, IL-1α Abs but not IL-1ß Abs reduced levels of TNF-α and IL-6. Concomitant usage of Abs against IL-1α/IL-1ß revealed strong effects in vivo and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1ß were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1ß Ab treatment. Blocking the IL-1R1 provided significant effects on total cells, neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1ß. Our results suggest that combined inhibition of IL-1α/IL-1ß might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover, combined targeting of both IL-1α/IL-1ß might be more efficient compared to individual neutralization IL-1α or IL-1ß or inhibition of the IL-1R1.

BI 1002494, a Novel Potent and Selective Oral Spleen Tyrosine Kinase Inhibitor, Displays Differential Potency in Human Basophils and B Cells.

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

The Novel Oral Syk Inhibitor, Bl1002494, Protects Mice From Arterial Thrombosis and Thromboinflammatory Brain Infarction.

OBJECTIVE: Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is the second leading cause of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein VI (GPVI) is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of not only GPVI but also other platelet and immune cell receptors. We sought to assess whether Syk might be an effective antithrombotic target. APPROACH AND RESULTS: We demonstrate that mice lacking Syk in platelets specifically are protected from arterial thrombus formation and ischemic stroke but display unaltered hemostasis. Furthermore, we show that mice treated with the novel, selective, and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 hours after transient middle cerebral artery occlusion, also when BI1002494 was administered therapeutically, that is, after ischemia. CONCLUSIONS: These results provide direct evidence that pharmacological Syk inhibition might provide a safe therapeutic strategy to prevent arterial thrombosis and to limit infarct progression in acute stroke.

Differences in respiratory syncytial virus and influenza infection in a house-dust-mite-induced asthma mouse model: consequences for steroid sensitivity.

A significant number of clinical asthma exacerbations are triggered by viral infection. We aimed to characterize the effect of virus infection in an HDM (house dust mite) mouse model of asthma and assess the effect of oral corticosteroids. HDM alone significantly increased eosinophils, lymphocytes, neutrophils, macrophages and a number of cytokines in BAL (bronchoalveolar lavage), all of which were sensitive to treatment with prednisolone (with the exception of neutrophils). Virus infection also induced cell infiltration and cytokines. RSV (respiratory syncytial virus) infection in HDM-treated animals further increased all cell types in BAL (except eosinophils, which declined), but induced no further increase in HDM-elicited cytokines. However, while HDM-elicited TNF-α (tumour necrosis factor-α), IFN-γ (interferon-γ), IL (interleukin)-2, IL-5 and IL-10 were sensitive to prednisolone treatment, concomitant infection with RSV blocked the sensitivity towards steroid. In contrast, influenza infection in HDM- challenged animals resulted in increased BAL lymphocytes, neutrophils, IFN-γ, IL-1ß, IL-4, IL-5, IL-10 and IL-12, but all were attenuated by prednisolone treatment. HDM also increased eNO (exhaled NO), which was further increased by concomitant virus infection. This increase was only partially attenuated by prednisolone. RSV infection alone increased BAL mucin. However, BAL mucin was increased in HDM animals with virus infection. Chronic HDM challenge in mice elicits a broad inflammatory response that shares many characteristics with clinical asthma. Concomitant influenza or RSV infection elicits differing inflammatory profiles that differ in their sensitivity towards steroids. This model may be suitable for the assessment of novel pharmacological interventions for asthmatic exacerbation.

Design and synthesis of long acting inhaled corticosteroids for the treatment of asthma.

In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.

Serum zinc and copper status in dyslipidaemic patients with and without established coronary artery disease.

INTRODUCTION: Aspects of trace element status have previously been investigated as possible contributory factors to atherosclerosis. In this present study a more comprehensive approach has been taken, looking at the relationship between dietary macro- and micronutrient intake, serum concentrations of zinc and copper, and markers of inflammation in dyslipidaemic patients with or without established coronary artery disease (CAD) and healthy controls, so that a clearer understanding of the potential relationship between copper and zinc status and coronary disease may be ascertained. METHODS AND MATERIALS: Dyslipidaemic patients (n = 238) were recruited from the local General Hospital in Guildford, UK. Fifty-five of these patients had established CAD. Control subjects (n = 135) were recruited from among employees at the local University and Hospital. A validated food frequency questionnaire was used for estimating the dietary intake of zinc and copper. RESULTS: Serum copper, copper/caeruloplasmin ratio, zinc/copper ratio, and C-reactive protein (CRP) were significantly different in the patient groups compared to controls [serum copper: 17.20 +/- 0.2 v 15.91 +/- 0.29 micromol/L, p < 0.001; copper/caeruloplasmin ratio: 111.37 +/- 2.18 v 100.63 +/- 2.93 micromol/g, p < 0.01; zinc/copper ratio: 0.85 +/- 0.01 v 0.90 +/- 0.01, p < 0.05; and CRP: 1.25 (0.42-3.26) v 0.58 (0.17-1.42) mg/L, p < 0.001]. Dietary protein, total fat, starch, fibre, monounsaturated fat, zinc, and zinc/copper ratio were also significantly higher in the patients compared to controls. Patients with established CAD had significantly higher serum CRP (p < 0.05) and lower serum zinc (p < 0.01) and zinc/copper ratio (p < 0.01) compared to both patients without CAD and healthy controls. CONCLUSION: Significant differences in copper and zinc status, dietary intake and markers of inflammation were observed in patients with dyslipidaemia, with or without established CAD, compared with control subjects. Differences in serum CRP, copper and caeruloplasmin may be related to a heightened state of inflammation. The imbalance in zinc/copper metabolism may either contribute to the CAD risk or be a consequence of an acute phase response.

Anti-inflammatory modulation of chronic airway inflammation in the murine house dust mite model.

Asthma affects 300 million people worldwide and continues to be a major cause of morbidity and mortality. Disease relevant animal models of asthma are required for benchmarking of novel therapeutic mechanisms in comparison to established clinical approaches. We demonstrate that chronic exposure of mice to house dust mite (HDM) extract results in allergic airway inflammation, that can be significantly attenuated by therapeutic intervention with phosphodiesterase 4 inhibition and corticosteroid treatment. Female BALB/c mice were administered intranasally with HDM (Dermatophagoides pteronyssinus) extract daily for five weeks, and therapeutic intervention with anti-inflammatory treatment (dexamethasone 1 mg/kg subcutaneous once daily, prednisolone 10mg/kg orally twice daily, fluticasone 3, 10 and 30 microg intranasally twice daily, roflumilast 10 mg/kg orally twice daily and intranasally 10 and 30 microg twice daily) was initiated after three weeks of exposure. Chronic HDM extract exposure resulted in significant airway inflammation, demonstrated by bronchoalveolar lavage cell infiltration and lung tissue inflammatory gene expression by TaqMan low density array. Chronic steroid treatment significantly inhibited these parameters. In addition, roflumilast caused a significant reduction in airway inflammatory cell infiltration. We have demonstrated that chronic HDM-induced allergic inflammation can be significantly ameliorated by steroid treatment, and that phosphodiesterase 4 inhibition modulates inflammatory cell infiltration. Therefore, the murine HDM model may be a useful tool for evaluating new targets for the treatment of asthma.

Efficacy of two alcohol-free cetylpyridinium chloride mouthwashes - a randomized double-blind crossover study.

AIM: (1) To determine the plaque inhibition properties of two formulations of alcohol-free mouthwash [0.1% w/w cetylpyridinium chloride (CPC) (B) and 0.05% w/w CPC (A)] versus a placebo mouthwash (C). (2) To compare the plaque-inhibiting activity between these two new CPC mouthwashes. MATERIAL AND METHODS: A double-blind, crossover study with three 1-week periods was used. Subjects were randomly assigned to one of the following groups. Group 1 (n=10) received the mouthwashes A, C and B in the periods 1, 2 and 3, respectively, group 2 (n=11) received the mouthwashes in the order B, A, C, while group 3 (n=11) received the mouthwashes in the order C, B, A. Mean plaque areas and Quigley & Hein plaque index scores were analysed using anova (analysis of variance). Measurements were made at the start of each period (baseline) and at 16, 24 and 40 h. RESULTS: Mean plaque scores were similar across the groups at baseline. At all time points thereafter, volunteers using mouthwash A or B had significantly lower plaque areas and plaque index scores than those using mouthwash C (p<0.05), but there were no significant differences between the test formulations. At 16 h, the reduction in plaque area relative to mouthwash C was 22% for mouthwash A and 18% for mouthwash B; at 24 h, 11% for mouthwash A and 15% for mouthwash B; and at 40 h, 15% for mouthwash A and 16% for mouthwash B. CONCLUSIONS: The use of both CPC mouthwashes resulted in less plaque accumulation compared with the control. There was no statistically significant difference in plaque accumulation between the two CPC mouthwashes.

Relationship between plasma cholesterol, von Willebrand factor concentrations, extent of atherosclerosis and antibody titres to heat shock proteins-60, -65 and -70 in cholesterol-fed rabbits.

Epidemiological studies have shown an association between atherosclerosis, Heat shock protein (Hsp) expression, and Hsp antibody titres. We aimed to investigate the time course of appearance of Hsp-60, -65 and -70 antibodies in the cholesterol-fed rabbit and to relate antibody titres to serum concentrations of von Willebrand factor (vWF), a marker of endothelial injury. Rabbits were fed with 0.25-1.0% cholesterol diet for 13 weeks. Plasma levels of anti Hsp-60, -65 and -70 IgG titres, were measured using in-house enzyme-linked immunosorbent assays (ELISAs) together with plasma vWF concentrations. Plasma titres of anti-Hsp-60, -65 and -70 antibodies were all significantly increased by weeks 5, 7 and 9 following commencement of the experimental diet compared with baseline (P < 0.05 for all). In non-cholesterol-fed rabbits, plasma levels of anti-Hsp titres were unchanged over this period. Increased plasma vWF concentrations were also found in the cholesterol-fed rabbits, reaching a maximum at approximately week 8, and falling thereafter. Furthermore, plasma vWF concentrations at 13 weeks correlated strongly with antibody titres to all three Hsps (r = 0.90, P = 0.002; r = 0.80, P = 0.017; r = 0.86, P = 0.006 for Hsp 60, -65 and -70 respectively) and titres were also strongly correlated with final plasma cholesterol concentrations in cholesterol-fed animals (r = 0.95, P = 0.002; r = 0.8, P = 0.001; r = 0.84, P = 0.01 respectively). In cholesterol-fed rabbits, antibody titres to Hsp-60, -65 and -70 appear to rise in association with a marker of endothelial injury, peaking at approximately the same time (8 weeks) after starting a high cholesterol diet.

Magnetic retention for obturators.

Prosthetic rehabilitation can be challenging in cases of maxillectomy or developmental defect. This article describes a case in which a magnetically retained, implant-supported denture was used to restore the maxilla following hemi-maxillectomy. Use of the Oral Health Impact Profile before and after treatment showed a marked diminution in the number of adverse impacts 2 weeks post-placement and during review 6 months later.

Association between serum CRP concentrations with dietary intake in healthy and dyslipidaemic patients.

Serum CRP concentrations are elevated in subjects at risk of coronary events and in subjects with metabolic syndrome. Although dietary fat and antioxidants are known for their immune-modulating actions, their reported effects on CRP concentrations have been inconsistent. In the present study we have investigated whether dietary constituents are associated with serum CRP concentrations in healthy subjects and patients with dyslipidaemic. Dyslipidaemic subjects (n=238) were recruited from Hospital Outpatient Clinics in Guilford, UK. Apparently healthy subjects (n=188) were recruited from amongst adjacent University and Hospital employees. A validated food frequency questionnaire was used to estimate dietary intake. Dyslipidaemic patients had higher serum CRP [1.25 (0.42-3.26) mg/L] than control subjects [0.50 (0.17-1.42) mg/L] (p<0.001). In the dyslipidaemic patients, approximately 4% of the variation in serum CRP could be explained by dietary cholesterol intake (p = 0.015, 2.8%), and weakly by dietary vitamin C intake (p = 0.06, 1.2%). No relationship between dietary constituents and serum CRP concentrations was found among the healthy subjects. Hence the present study shows that serum CRP concentrations are increased in patients with classical coronary risk factors, and that they may be modulated by dietary cholesterol.

The epidermal growth factor receptors and their family of ligands: their putative role in atherogenesis.

The epidermal growth factor receptor is a member of type-I growth factor receptor family with tyrosine kinase activity that is activated following the binding of multiple cognate ligands. Several members of the EGF family of ligands are expressed by cells involved in atherogenesis. EGF receptor mediated processes have been well characterised within epithelial, smooth muscle and tumour cell lines in vitro, and the EGF receptor has been identified immunocytochemically on intimal smooth muscle cells within atherosclerotic plaques. There is also limited evidence for the expression of the EGF receptor family on leukocytes, although their function has yet to be clarified. In this review, we will discuss the biological functions of this receptor and its ligands and their potential to modulate the function of cells involved in the atherosclerotic process.

Effect of statin therapy on serum trace element status in dyslipidaemic subjects.

Patients previously not treated with a lipid-lowering agent (n = 20; mean age 49.15 +/- 3.28 years) were treated with either 10 mg/day of Simvastatin (n = 11), or Atorvastatin (n = 9) for 4 months. Fourteen additional patients were recruited from the same clinic at the same hospital as a control group. The medication of these latter patients was unaltered for 4 months and the same parameters were measured as for the statin groups. Serum concentrations of zinc, copper, caeruloplasmin, selenium, glutathione peroxidase (GPx) and C-reactive protein (CRP) were measured together with their lipid profiles pre- and post-treatment. In addition to reducing serum total and low-density lipoprotein (LDL) cholesterol (p < 0.0001), statin treatment was associated with a significant reduction in mean serum zinc (9%, p = 0.03), copper (9%, p < 0.01), caeruloplasmin (24%, p < 0.05), and median CRP (45%, p < 0.03). Similar changes were not observed in the control patients. No significant effects were observed for serum selenium, copper/caeruloplasmin ratio, or GPx (p > 0.05) in either statin or control groups. These changes may be related to the known anti-inflammatory properties of the statin class of drugs.

Dietary copper supplements modulate aortic superoxide dismutase, nitric oxide and atherosclerosis.

The objective was to test the hypothesis that dietary copper inhibits atherosclerosis by inducing superoxide dismutase (SOD) and potentiating nitric oxide (NO). New Zealand White rabbits were fed either a cholesterol diet (n = 8) or a cholesterol diet containing 0.02% copper acetate (n = 8) for 13 weeks. We found that the intimal area was significantly smaller in the animals supplemented with copper (P < 0.005), although integrated plasma cholesterol levels were not significantly different. This was associated with a significant increase in aortic copper content (P < 0.05), SOD activity (P < 0.05) and Cu/Zn SOD mRNA (P < 0.05) and a significant decrease in nitrotyrosine content (P < 0.05). Furthermore, there was a positive correlation between aortic copper content and SOD activity (P < 0.005, R(2) = 0.83) and a negative correlation between aortic superoxide dimutase activity and nitrotyrosine content (P < 0.005, R(2) = 0.93). In organ bath experiments, the relaxation of precontracted carotid artery rings to calcium ionophore was greater in animals supplemented with copper. No difference in response to sodium nitroprusside was observed. These data suggest that in the cholesterol-fed rabbit, copper supplements inhibit the progression of atherosclerosis by increasing SOD expression, thereby reducing the interaction of NO with superoxide, and hence potentiating NO-mediated pathways that may protect against atherosclerosis.

Dietary antioxidants and fat are associated with plasma antibody titers to heat shock proteins 60, 65, and 70 in subjects with dyslipidemia.

BACKGROUND: The heat shock proteins (HSPs) are protein chaperones. Higher titers of antibody to HSPs (anti-HSPs) have been reported in atherosclerosis, which may contribute to immunoactivation in this process. OBJECTIVE: We investigated whether dietary antioxidants and fat intake are associated with changes in anti-HSP titers in dyslipidemic subjects. DESIGN: Patients (n = 238) were recruited from hospital lipid clinics. Control subjects (n = 188) were recruited from university and hospital employees. Food-frequency questionnaires were used to estimate dietary antioxidants and fat. RESULTS: Dyslipidemic patients had significantly higher titers of anti-HSPs than did control subjects; expressed in medians and interquartile ranges of absorbance units, anti-HSP-60 titers were 0.27 (0.18-0.37) and 0.22 (0.16-0.30), anti-HSP-65 titers were 0.45 (0.28-0.79) and 0.31 (0.22-0.50), and anti-HSP-70 titers were 0.22 (0.17-0.30) and 0.19 (0.13-0.27), respectively. Median and interquartile ranges of serum concentrations of C-reactive protein [1.25 (0.42-3.26) and 0.58 (0.17-1.42)] and mean (+/-SEM) concentrations of vitamin E (16.36 +/- 0.31 and 14.08 +/- 0.38) were also significantly higher in patients than in control subjects, respectively. In dyslipidemic patients, the major dietary predictors of the variability in anti-HSP-60 titers were vitamin C (P = 0.005), vitamin E (P = 0.04), and total fat (P = 0.009) intakes; for anti-HSP-65 titers, vitamin C was the major predictor (P = 0.002). These findings remained significant after adjustment for confounding factors. CONCLUSIONS: Anti-HSP-60, -65, and -70 titers are significantly higher in dyslipidemic patients with or without established coronary disease. Our data indicate an association between dietary constituents and the immune response to HSPs in dyslipidemic subjects.

The effects of coadministration of dietary copper and zinc supplements on atherosclerosis, antioxidant enzymes and indices of lipid peroxidation in the cholesterol-fed rabbit.

It has previously been shown that dietary copper can modulate the extent of atherosclerosis in the thoracic aorta of cholesterol-fed rabbits. The metabolism of copper and zinc are closely related, and it has been hypothesized that the balance of dietary copper to zinc may be important in determining coronary risk. Hence, we have investigated the interaction between dietary copper and zinc in atherogenesis in the New Zealand White rabbit. Juvenile male rabbits were randomly allocated to eight groups. Four groups were fed a normal chow diet with zinc (0.5%, w/w), copper (0.2%, w/w), copper plus zinc or neither in their drinking water for 12 weeks. Four other groups were fed a diet containing 0.25-1% (w/w) cholesterol plus zinc, copper, both or neither. Serum cholesterol of individual animals was maintained at approximately 20 mmol/l. Integrated plasma cholesterol levels were similar for all groups receiving cholesterol and significantly higher than those in the chow-fed groups (P < 0.001). Aortic copper concentrations were higher in the animals receiving cholesterol diets with copper compared to rabbits receiving normal chow and copper (P < 0.001). Aortic zinc content was significantly higher in cholesterol-fed rabbits supplemented with zinc alone or with copper than in those fed cholesterol alone (P < 0.001). Plasma ceruloplasmin concentrations were significantly higher in groups receiving cholesterol, irrespective of their trace element supplementation (P < 0.001). However, trace element supplementation increased the level significantly (P < 0.05). Trace element supplements did not appear to affect erythrocyte superoxide dismutase in the cholesterol-fed animals; however, zinc supplementation was associated with a significant increase in the enzyme in chow-fed animals (P < 0.05). The activity of the enzyme per mg of protein in aortic tissue was higher in animals receiving copper in the presence of cholesterol (P < 0.05) but not significantly so in its absence. Dietary trace element supplementation in cholesterol-fed animals was associated with a significant reduction in aortic lesion area. Plasma thiobarbituric acid-reactive substances and FOX concentrations were both significantly higher in the cholesterol-fed rabbits compared with the animals that fed on a chow diet (P < 0.001), and these were reduced significantly by dietary copper or zinc supplementation (P < 0.001). Hence, dietary supplements of copper or zinc at the doses used both inhibited aortic atherogenesis in the cholesterol-fed rabbits, although there was no significant additional effect when given in combination.

EGF mediates monocyte chemotaxis and macrophage proliferation and EGF receptor is expressed in atherosclerotic plaques.

The recruitment of peripheral monocytes to the sub-endothelial space, their development into macrophages and subsequent proliferation are critical events during atherosclerosis. Receptors for epidermal growth factor (EGF) have been identified on cells of the myeloid lineage, but a role for them in atherogenesis has yet to be described. We have identified functional EGF receptors (EGFR, ErbB1/HER-1) on peripheral blood monocytes and monocyte-derived macrophages. Uniquely, these receptors were found to mediate both chemotaxis in monocytes and macrophages and proliferation in macrophages. EGFR mRNA was detected in atherosclerotic plaques, but not in morphologically normal aortae and EGFR receptor staining co-localised with macrophage staining in these plaques. The identification of receptors for EGF on peripheral blood monocytes, macrophages and atherosclerotic lesions, together with their transduction of two functionally important cellular events, heightens the potential importance of members of the EGF super-family in atherogenesis and other chronic inflammatory processes.