epidermal-growth-factor and Atherosclerosis

Topics: Animals; Antineoplastic Agents; Aorta; Atherosclerosis; Biomechanical Phenomena; Chondroitin Sulfate Proteoglycans; Chondroitin Sulfates; Cytokines; Disease Progression; Epidermal Growth Factor; Extracellular Matrix; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lipoproteins, LDL; Neoplasms; Signal Transduction; Versicans

Sushi, von Willebrand factor type A, EGF pentraxin domain-containing 1 (SVEP1), an extracellular matrix protein, is a human coronary artery disease locus that promotes atherosclerosis. We previously demonstrated that SVEP1 induces vascular smooth muscle cell (VSMC) proliferation and an inflammatory phenotype in the arterial wall to enhance the development of atherosclerotic plaque. The only receptor known to interact with SVEP1 is integrin α9β1, a cell surface receptor that is expressed by VSMCs and myeloid lineage-derived monocytes and macrophages. Our previous in vitro studies suggested that integrin α9β1 was necessary for SVEP1-induced VSMC proliferation and inflammation; however, the underlying mechanisms mediated by integrin α9β1 in these cell types during the development of atherosclerosis remain poorly understood.. Here, using cell-specific gene targeting, we investigated the effects of the integrin α9β1 receptor on VSMCs and myeloid cells in mouse models of atherosclerosis. Interestingly, we found that depleting integrin α9β1 in either VSMCs or myeloid cells did not affect the formation or complexity of atherosclerotic plaque in vessels after either 8 or 16 weeks of high fat diet feeding.. Our results indicate that integrin α9β1 in these two cell types does not mediate the in vivo effect of SVEP1 in the development of atherosclerosis. Instead, our results suggest either the presence of other potential receptor(s) or alternative integrin α9β1-expressing cell types responsible for SVEP1 induced signaling in the development of atherosclerosis.

Topics: Animals; Atherosclerosis; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Extracellular Matrix Proteins; Humans; Macrophages; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Plaque, Atherosclerotic; von Willebrand Factor

Topics: Adaptive Immunity; Atherosclerosis; CD4-Positive T-Lymphocytes; Epidermal Growth Factor; Humans

Toll-like receptor 4 (TLR4)-tumor necrosis factor receptor 6 (TRAF6) signaling is activated in atherosclerosis (AS), inducing inflammatory mediators. Because miR-146a, a TLR4 microRNA (miRNA), can regulate TLR4 signaling during inflammatory responses, this study investigated the effects of aerobic exercise on TLR4-targeted miRNAs in AS. Apolipoprotein E-null mice fed a high-fat diet for 12 weeks were separated into 3 groups: (i) no treatment (AS), (ii) statin treatment (AD), or (iii) aerobic exercise (AE). Plaques and foam cells were observed in the untreated control and statin groups, respectively, but not in the AE group. Reduced angiotensin II (Ang II) and endothelin 1 (ET1) levels were observed in the AE group. Both treatment groups significantly altered the expression of inflammatory cytokine expression and reduced vascular TLR4 levels. Increased miR-146a and miR-126 and reduced miR-155 levels were observed in both treatment groups (all, P<0.001). miR-146a interacted with the 3' untranslated region of the TRAF6 gene, reducing its expression. Thus, aerobic exercise and statins may induce miR-146a expression, thereby reducing vascular TRAF and TLR4 signaling and vascular inflammatory injury in AS. Further analysis of this pathway may provide insight into the protective effects of aerobic exercise on vascular disease as well as new therapeutic targets.

Topics: Angiotensin II; Animals; Atherosclerosis; Disease Models, Animal; Endothelin-1; Epidermal Growth Factor; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Male; Mice; MicroRNAs; NF-kappa B; Physical Conditioning, Animal; Signal Transduction; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4

Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE(-/-) mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE(-/-) mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE(-/-) mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α(1)-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α(1)-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE(-/-) mice on HFD. Urinary excretion levels of collagen and α(1)-antitrypsin fragments also significantly correlated with intraplaque collagen and α(1)-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α(1)-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.

Topics: alpha 1-Antitrypsin; Animals; Apolipoproteins E; Atherosclerosis; Biomarkers; Collagen Type I; Diet, High-Fat; Disease Progression; Electrophoresis, Capillary; Epidermal Growth Factor; Humans; Mass Spectrometry; Mice; Mice, Knockout; Peptides; Plaque, Atherosclerotic; Proteome; Sensitivity and Specificity; Sequence Analysis, Protein

To characterize the relationship between the expression of epidermal growth factor (EGF)-like ligands and vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and activity in a primate model of atherosclerosis.. Adult male Cynomolgus monkeys were fed a normal or atherogenic (AS) diet for 45 months, after which animals from the AS group were placed on a normal diet for 8 months (regression). The expression of membrane-associated EGF-like ligands was increased in arteries from animals on the AS diet and normalized in the regression group. EGF-like ligands were distributed throughout atherosclerotic vessels but predominantly colocalized with macrophages. Consistent with ligand shedding, circulating heparin-bound EGF was elevated in the plasma of AS monkeys but not in those on regression diet. Atherosclerosis was associated with the activation of EGF receptor signaling. Expression of NADPH oxidase subunits Nox1 and Nox2 but not Nox4 or Nox5 was increased in arteries from monkeys on the AS diet and returned to normal with regression. Levels of Nox1 and Nox2 positively correlated with EGF-like ligands. In cultured monkey smooth muscle cells, treatment with EGF-like ligands increased Nox1 expression and activity.. These data identify EGF-like ligands as potential modulators of atherogenesis, resulting in part from increased vascular NADPH oxidase activity.

Topics: Animals; Arteries; Atherosclerosis; Cells, Cultured; Diet, Atherogenic; Disease Models, Animal; Epidermal Growth Factor; Ligands; Macaca fascicularis; Male; Muscle, Smooth, Vascular; NADPH Oxidases; Signal Transduction

iNKT cells are a unique T cell subset, which is CD1d-restricted and specific for glycolipid antigens. In advanced atherosclerotic plaques, focal collections of inflammatory cells correlate with areas of intraplaque neovascularization. We reported recently that iNKT cells might facilitate intraplaque neovascularization by enhancing EC migration and sprouting in an IL-8-dependent manner. This study investigated the participating effector mechanisms. In ECs, CM, derived from antigen-stimulated human iNKT cells (CM+), induced up-regulation of IL-8R CXCR2 and the phosphorylation of EGFR and of multiple intracellular signaling effectors, including FAK, Src, Erk, Jnk, p38-MAPK, and STAT1 and -3. We found that a cascade of events, which were IL-8-dependent and involved EGFR activation, was responsible for signaling through FAK and Src kinases and necessary for acquisition of angiogenic morphology, migration in a two-dimensional wound assay, and sprout outgrowth in a three-dimensional model of angiogenesis in vitro. The data support that IL-8-dependent activation of angiogenic behavior in ECs, in response to activated iNKT, involves CXCR2, transactivation of EGFR, and subsequent FAK/Src signaling. We found too that activated iNKT increased VEGFR2 expression in ECs. Functional studies confirmed that EGF is the motogenic-enhancing factor in CM+ and is necessary, together with an exogenous source of VEGF, for iNKT-promoted sprout formation. EGFR inhibition may represent a novel therapeutic modality aimed at plaque stabilization through control of neovascularization within developing atherosclerotic plaques.

Topics: Antigens; Atherosclerosis; Cell Communication; Cell Line; Cell Movement; Endothelial Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Inflammation; Interleukin-8; Lipids; Lymphocyte Activation; Natural Killer T-Cells; Neovascularization, Pathologic; Phosphorylation; Receptors, Interleukin-8B; Signal Transduction; Up-Regulation

Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved.. CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1.. We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

Topics: Apoptosis; Atherosclerosis; Cell Division; Cells, Cultured; Chemokine CX3CL1; Coronary Vessels; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Humans; In Vitro Techniques; Mitogens; Muscle, Smooth, Vascular; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

Previous in vitro studies have shown a relationship between epidermal growth factor (EGF) and lipid metabolism. Indeed, EGF is able to modulate lipoprotein fractions in human fetal intestine and hepatic-derived cell lines. The aim of this study was to search for potential associations between EGF concentrations and lipid parameters in both plasma and peripheral blood mononuclear cells (PBMCs) among healthy individuals.. EGF concentrations were quantified in plasma of 273 men and 279 women and in PBMCs of 57 men and 62 women from the STANISLAS cohort. In addition, basic blood constituents including lipid parameters (apolipoproteins A1, and B, total and high-density lipid (HDL)-cholesterols and triglycerides) were measured.. Plasma EGF concentrations were significantly and positively correlated with apolipoprotein A1 and HDL-cholesterol concentrations after adjustment for age, sex, and body mass index (r=0.155, P=0.0003; and r=0.112, P=0.0086, respectively). They were also negatively correlated with the apolipoprotein B/A1 ratio (r=-0.107, P=0.012). In addition, EGF concentrations in PBMCs were negatively correlated with apolipoprotein B, total and low-density lipid-cholesterols, and triglyceride concentrations, and apolipoprotein B/A1 ratio (P=0.0018, P=0.0137, P=0.0142, P=0.0162, and P=0.0077, respectively, with r ranging between -0.287 and -0.222). No interactions were found with gender.. New associations between EGF and lipid concentrations have been reported, providing new perspectives with regard to the role of EGF in atherosclerosis.

Topics: Adult; Atherosclerosis; Cohort Studies; Epidermal Growth Factor; Female; Humans; Leukocytes, Mononuclear; Lipid Metabolism; Lipids; Male; Middle Aged

We have investigated the functional significance of conserved sequences within the 9p21.3 risk locus for coronary artery disease (CAD) and determined the relationship of 9p21.3 to expression of ANRIL and to whole genome gene expression.. We demonstrate that a conserved sequence within the 9p21.3 locus has enhancer activity and that the risk variant significantly increases reporter gene expression in primary aortic smooth muscle cells. Whole blood RNA expression of the short variants of ANRIL was increased by 2.2-fold whereas expression of the long ANRIL variant was decreased by 1.2-fold in healthy subjects homozygous for the risk allele. Expression levels of the long and short ANRIL variants were positively correlated with that of the cyclin-dependent kinase inhibitor, CDKN2B (p15) and TDGF1 (Cripto), respectively. Relevant to atherosclerosis, genome-wide expression profiling demonstrated upregulation of gene sets modulating cellular proliferation in carriers of the risk allele.. These findings are consistent with the hypothesis that the 9p21.3 risk allele contains a functional enhancer, the activity of which is altered in carriers of the risk allele. 9p21.3 may promote atherosclerosis by regulating expression of ANRIL, which in turn is associated with altered expression of genes controlling cellular proliferation pathways.

Topics: Aged; Alleles; Atherosclerosis; Cell Proliferation; Chromosome Mapping; Chromosomes, Human, Pair 9; Coronary Artery Disease; Cyclin-Dependent Kinase Inhibitor p15; Epidermal Growth Factor; Female; Gene Expression Regulation; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Luciferases; Male; Membrane Glycoproteins; Neoplasm Proteins; Regulatory Elements, Transcriptional; Risk; RNA, Untranslated

The lifespan of patients with chronic renal failure (CRF) is reduced, and coronary artery disease is the leading cause of morbidity and mortality in these patients. The progression of atherosclerosis is accelerated and angiogenesis is impaired in CRF. Risk factors that could contribute to further understanding of vascular pathology include markers of inflammation and growth factors. The purpose of this study was to determine the levels of cytokines (IL-2, IL4, IL-6, IL-8, IL-10, IL-1 alpha, IL-1 beta), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interferon-gamma (IFN gamma), tumor necrosis factor-alpha (TNFalpha) and monocyte chemotactic protein-1 (MCP-1) in patients on chronic hemodialysis (HD; n=75), and to compare values with those of control subjects (n=113).. Evidence((R)) biochip array analyzer was used for quantification of plasma concentrations in samples.. Significant differences were found between the control subjects and HD patients. IL-2 (p<0.001), IL-4 (p<0.001) and EGF (p<0.001) levels were higher in controls than in HD patients, while IL-6 (p<0.001), IL-8 (p=0.081), IL-10 (p=0.008), TNFalpha (p<0.001), IL-1 beta (p<0.001) and MCP-1 (p<0.001) levels were higher in HD patients. We also found IL-2 (p=0.015) and IL-1 alpha (p=0.035) levels to be significantly higher in males than females, while IL-4 (p=0.025) and IL-1 beta (p=0.049) levels were significantly higher in females. Among HD patients, IL-2 levels were higher in patients under the age of 50 years (p<0.048). It was also higher in female than in male patients (p<0.035) and in patients on HD for more than 10 years (p<0.009). IL-6 levels were higher in patients over the age of 50 years (p<0.047). Patients with previous glomerulonephritis had the highest level of IL-6 compared to patients with previous pyelonephritis and diabetes mellitus (p<0.063). IL-6 levels were higher in patients with concomitant hepatitis C virus (HCV) infection (p<0.036) and in patients with developed atherosclerosis (p<0.003). IL-8 levels were higher in patients over the age of 50 years (p<0.003) and in the group with previous glomerulonephritis (p<0.031). IL-10 levels were higher in the group with developed atherosclerosis (p<0.045). EGF was the highest in the group of patients with previous diabetes mellitus compared to pyelonephritis and glomerulonephritis groups (p<0.073). TNFalpha levels were higher in the patient population on HD for more than 10 years (p<0.032) and in the concomitant HCV group (p<0.073). IL-1 beta levels were higher in the HCV group (p<0.088).. Plasma concentrations of some cytokines and growth factors could serve as useful diagnostic and prognostic parameters for patients with CRF on HD.

Topics: Atherosclerosis; Biomarkers; Case-Control Studies; Chemokine CCL2; Cytokines; Disease Progression; Epidermal Growth Factor; Female; Humans; Interferon-gamma; Male; Middle Aged; Protein Array Analysis; Reference Values; Renal Dialysis; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Endothelial membrane-bound thrombomodulin is a high affinity receptor for thrombin to inhibit coagulation. We previously demonstrated that the thrombin-thrombomodulin complex restrains cell proliferation mediated through protease-activated receptor (PAR)-1. We have now tested the hypothesis that thrombomodulin transduces a signal to activate the endothelial nitric-oxide synthase (NOS3) and to modulate G protein-coupled receptor signaling. Cultured human umbilical vein endothelial cells were stimulated with thrombin or a mutant of thrombin that binds to thrombomodulin and has no catalytic activity on PAR-1. Thrombin and its mutant dose dependently activated NO release at cell surface. Pretreatment with anti-thrombomodulin antibody suppressed NO response to the mutant and to low thrombin concentration and reduced by half response to high concentration. Thrombin receptor-activating peptide that only activates PAR-1 and high thrombin concentration induced marked biphasic Ca2+ signals with rapid phosphorylation of PLC(beta3) and NOS3 at both serine 1177 and threonine 495. The mutant thrombin evoked a Ca2+ spark and progressive phosphorylation of Src family kinases at tyrosine 416 and NOS3 only at threonine 495. It activated rapid phosphatidylinositol-3 kinase-dependent NO synthesis and phosphorylation of epidermal growth factor receptor and calmodulin kinase II. Complete epidermal growth factor receptor inhibition only partly reduced the activation of phospholipase Cgamma1 and NOS3. Prestimulation of thrombomodulin did not affect NO release but reduced Ca2+ responses to thrombin and histamine, suggesting cross-talks between thrombomodulin and G protein-coupled receptors. This is the first demonstration of an outside-in signal mediated by the cell surface thrombomodulin receptor to activate NOS3 through tyrosine kinase-dependent pathway. This signaling may contribute to thrombomodulin function in thrombosis, inflammation, and atherosclerosis.

Topics: Atherosclerosis; Blotting, Western; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Catalysis; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Epidermal Growth Factor; ErbB Receptors; Humans; Inflammation; Models, Biological; Mutation; Nitric Oxide; Nitric Oxide Synthase Type III; Peptides; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Structure, Tertiary; Receptor, PAR-1; Receptors, Thrombin; Reverse Transcriptase Polymerase Chain Reaction; Serine; Signal Transduction; src-Family Kinases; Threonine; Thrombin; Thrombomodulin; Time Factors; Tyrosine; Umbilical Veins