epidermal-growth-factor and Arteriosclerosis

To elucidate the biological characteristics of adipose tissue, we analyzed the gene expression profile of visceral and subcutaneous fat. Unexpectedly, adipose tissue, especially visceral fat, expressed a variety of genes for secretory proteins. About 30% of the genes expressed in visceral adipose tissue encoded secretory proteins and most were biologically active molecules, which we called adipocytokines. We found plasminogen activator inhibitor type 1 and heparin binding EGF-like growth factor. Production of these atherogenic adipocytokines was shown to increase with the accumulation of visceral fat, which may be one of the mechanisms of vascular disease in visceral obesity. We found a unique and novel collagen-like protein, adiponectin, encoded by the most abundantly expressed gene in adipose tissue, termed APM1 (adipose most abundant gene transcript-1). Plasma levels of adiponectin ranged from 0.3 to approximately 3 mg/dl but were decreased in patients with visceral obesity, type 2 diabetes and coronary artery disease (CAD). Screening for mutations in the adiponectin gene revealed that patients carrying a missense mutation showed markedly decreased plasma levels of adiponectin and had CAD. These data suggest that hypoadiponectinemia may be considered an important risk factor for CAD. Cell biology studies revealed that adiponectin has a potent inhibitory effect on the expression of adhesion molecules in endothelial cells and an inhibitory effect on the expression in macrophages. In order to confirm these antidiabetic and antiatherogenic functions of adiponectin, we developed adiponectin knockout mice. Adiponectin knockout mice showed severe insulin resistance and impaired glucose metabolism when fed a high-fat, high-sucrose diet. Knockout mice also developed intimal thickening in response to endothelial injury.

Topics: Adiponectin; Adipose Tissue; Animals; Arteriosclerosis; Coronary Artery Disease; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Obesity; Plasminogen Activator Inhibitor 1; Proteins; Risk Factors

3-deoxyglucosone (3-DG) is accumulated not only in uremic serum but also in uremic erythrocytes. 3-DG rapidly reacts with protein amino groups to form advanced glycation end products (AGEs) such as imidazolone, pyrraline, and N(epsilon)-(carboxymethyl)lysine, among which imidazolone is the AGE that is most specific for 3-DG. In diabetes, hyperglycemia enhances the synthesis of 3-DG via the Maillard reaction and the polyol pathway and thereby leads to its high plasma and erythrocyte levels. In uremia, however, the decreased catabolism of 3-DG that may be due to the loss of 3-DG reductase activity in the end-stage kidneys may lead to a high plasma 3-DG level. The elevated 3-DG levels in uremic patients may promote the formation of AGEs such as imidazolone in erythrocytes, aortas, and dialysis-related amyloid deposits. Treatment with an aldose reductase inhibitor reduced the erythrocyte levels of 3-DG and AGEs such as imidazolone in diabetic uremic patients. This finding demonstrates an important role of the polyol pathway in the formation of erythrocyte 3-DG and AGEs. The erythrocyte levels of 3-DG are elevated in not only diabetic uremic but also nondiabetic uremic patients. 3-DG showed some cytotoxicities by inducing intracellular oxidative stress. In contrast, oxidative stress was demonstrated to cause accumulation of intracellular 3-DG. Recently, we have demonstrated that 3-DG inactivates intracellular enzymes such as glutathione peroxidase, a key enzyme in the detoxification of hydrogen peroxide. Thus, intracellular accumulation of 3-DG may enhance oxidative stress by inactivating the antioxidant enzymes. In conclusion, 3-DG may play a principal role in the development of uremic complications, such as dialysis-related amyloidosis, atherosclerosis, and enhanced oxidative stress.

Topics: Amyloidosis; Animals; Apoptosis; Arteriosclerosis; Cell Division; Deoxyglucose; Epidermal Growth Factor; Erythrocytes; Glucose; Glutathione Peroxidase; Glutathione Reductase; Glycation End Products, Advanced; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Oxidative Stress; Uremia

HB-EGF is a heparin-binding member of the EGF family that was initially identified in the conditioned medium of human macrophages. Soluble mature HB-EGF is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells but not endothelial cells. HB-EGF activates two EGF receptor subtypes, HER1 and HER4 and binds to cell surface HSPG. The transmembrane form of HB-EGF is a juxtacrine growth and adhesion factor and is uniquely the receptor for diphtheria toxin. HB-EGF gene expression is highly regulated, for example by cytokines, growth factors, and transcription factors such as MyoD. HB-EGF has been implicated as a participant in a variety of normal physiological processes such as blastocyst implantation and wound healing, and in pathological processes such as tumor growth, SMC hyperplasia and atherosclerosis.

Topics: Amino Acid Sequence; Animals; Arteriosclerosis; Binding Sites; Cell Adhesion; Cell Division; Cell Membrane; Chromosome Mapping; Epidermal Growth Factor; Gene Expression Regulation; Genes; Heparan Sulfate Proteoglycans; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Hyperplasia; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Muscular Diseases; Neoplasms; Promoter Regions, Genetic; Protein Binding; Receptors, Cell Surface; Reproduction; Signal Transduction; Wound Healing

Diabetes mellitus is associated with typical patterns of long term vascular complications which vary with the organ involved. The microvascular kidney disease (Olgemoller and Schleicher, 1993) is characterized by thickening of the capillary basement membranes and increased deposition of extracellular matrix components (ECM), while loss of microvessels with subsequent neovascularisation is predominant in the eye and peripheral nerves. On the other hand macrovascular disease is characterized by accelerated atherosclerosis. These complications are dependent on long term hyperglycemia. Specific biochemical pathways linking hyperglycaemia to microvascular changes were proposed: the polyol pathway (Greene et al., 1987), non-enzymatic glycation of proteins (Brownlee et al., 1988), glucose autooxidation and oxidative stress (Hunt et al., 1990), hyperglycemic pseudohypoxia (Williamson et al., 1993) enhanced activation of protein kinase C by de novo-synthesis of diacyl glycerol (Lee et al., 1989; DeRubertis and Craven 1994) and others. These pathways are not mutually exclusive (Larkins and Dunlop, 1992; Pfeiffer and Schatz, 1992). They may be linked to alterations in the synthesis of growth factors particularly since atherosclerosis and angioneogenesis are associated with increased proliferation of endothelial and smooth muscle cells. Increased synthesis of ECM components is stimulated by growth factors like transforming growth factor beta (TGF beta) (Derynck et al., 1984) and insulin-like growth factor I (IGF-I) (Moran et al., 1991). This review will summarize some of the recent evidence for an involvement of growth factors in diabetic vascular complications and will attempt to assign their emergence in the sequence of events leading to vascular complications.

Topics: Animals; Arteriosclerosis; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Retinopathy; Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Hormone; Growth Substances; Humans; Hyperglycemia; Insulin Resistance; Insulin-Like Growth Factor I; Receptors, Somatotropin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Arteries; Arteriosclerosis; Blood Platelets; Cell Communication; Child; Diet, Atherogenic; Disease Models, Animal; Endothelium; Epidermal Growth Factor; Humans; Hyperlipoproteinemia Type II; Macrophages; Middle Aged; Monocytes; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; RNA, Messenger

Endothelium, platelets and macrophages can each provide growth factors that may participate in atherosclerotic lesion initiation or progression, or both. These mitogens, coupled with alternations in endothelial integrity or function resulting from a variety of different risk-associated factors, such as hyperlipidaemia, hypertension, tobacco smoke, antibodies, infections, or homocystinaemia, may provide the basis for the intimal proliferative smooth muscle cell response of atherosclerosis. Platelets, endothelium or macrophages may be important in lesion initiation and progression in some circumstances but not in others, depending upon the extent and type of endothelial "injury'.

Topics: Animals; Arteriosclerosis; Blood Platelets; Cell Division; Endothelium; Epidermal Growth Factor; Humans; Lipoproteins, LDL; Macrophages; Muscle, Smooth

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.

Topics: Animals; Aorta; Arteriosclerosis; Cell Division; Chemotaxis, Leukocyte; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Macrophages; Mitogens; Monocytes; Rabbits; RNA, Messenger

Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3alpha/beta, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear beta-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3'-kinase/Akt/GSK/FoxO/beta-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.

Topics: Agammaglobulinaemia Tyrosine Kinase; Amphiregulin; Arteriosclerosis; beta Catenin; Betacellulin; Cell Cycle; Cell Cycle Proteins; Cell Nucleus; Cells, Cultured; Cyclin D1; Cytoskeletal Proteins; DNA; DNA-Binding Proteins; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; ets-Domain Protein Elk-1; Forkhead Box Protein O1; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; RNA, Messenger; Signal Transduction; Trans-Activators; Transcription Factors; Up-Regulation

The coordinated activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) is critical for the induction of vascular and visceral smooth muscle cell (SMC) dedifferentiation. We previously reported that on the forced activation of both MAPKs, visceral SMCs secrete a non-heparin-binding protein factor(s) that is involved in the dedifferentiation of neighboring SMCs. In this study, we sought to identify the dedifferentiation factor(s) derived from vascular SMCs (VSMCs).. We fractionated the VSMC dedifferentiation factor(s) in the conditioned medium obtained from differentiated VSMCs in which both ERK and p38MAPK were forcedly activated and identified epiregulin as a major autocrine/paracrine factor for VSMC dedifferentiation. The epiregulin-induced VSMC dedifferentiation was mediated through the coordinated activation of ERK and p38MAPK. Unsaturated lysophosphatidic acid and platelet-derived growth factor-BB, which are potent VSMC dedifferentiation factors, rapidly upregulated epiregulin mRNA expression in an ERK- and p38MAPK-dependent manner. Reverse transcriptase-polymerase chain reaction and/or immunohistological analyses revealed the restricted expression of epiregulin in human atherosclerotic and balloon-injured rat arteries, in which the phenotypic modulation of medial VSMCs occurred in vivo.. Epiregulin is released from VSMCs primed by atherogenic factors and acts as a major autocrine/paracrine factor for VSMC dedifferentiation. It may be involved in the progression of vascular remodeling such as atherosclerosis.

Topics: Animals; Arteriosclerosis; Autocrine Communication; Becaplermin; Biomarkers; Carotid Arteries; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Epidermal Growth Factor; Epiregulin; Gene Expression Regulation; Humans; Lysophospholipids; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; p38 Mitogen-Activated Protein Kinases; Paracrine Communication; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; RNA, Messenger

Remnant lipoproteins (RLPs) have been shown to play a causative role during atherosclerosis. Furthermore, it is known that vascular smooth muscle cell (SMC) proliferation is crucial for the development of atherosclerosis and restenosis after percutaneous coronary intervention. We examined the direct effect of RLPs on the proliferation and signal transduction of SMCs.. Incubation in the presence of RLPs (20 mg cholesterol per dL) for 48 hours induced rat aortic SMC proliferation (2.3-fold over medium alone). RLPs also induced the phosphorylation of epidermal growth factor (EGF) receptor in SMCs, which was followed by the activation of mitogen-activated protein kinases. Moreover, the activation of protein kinase C (PKC) as well as the shedding of membrane-bound soluble heparin-binding EGF-like growth factor (HB-EGF) was observed after RLP treatment of SMCs, whereas PKC inhibitors and metalloprotease inhibitors inhibited RLP-induced EGF receptor transactivation and HB-EGF shedding in SMCs. Furthermore, anti-HB-EGF neutralizing antibody inhibited RLP-induced EGF receptor transactivation. Phosphorylation of EGF receptor and HB-EGF shedding were also observed in the aortas of apolipoprotein E-knockout mice but not in those of C57BL6 mice.. These results suggest that RLPs transactivate EGF receptor via PKC and HB-EGF shedding from SMCs, resulting in SMC proliferation.

Topics: Animals; Apolipoproteins E; Arteriosclerosis; Bromodeoxyuridine; Cell Division; Cells, Cultured; Cholesterol; Epidermal Growth Factor; ErbB Receptors; Genes, Dominant; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lipoproteins; Lipoproteins, VLDL; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Phosphorylation; Proto-Oncogene Proteins c-raf; Rats; Receptors, G-Protein-Coupled; Signal Transduction; Transcriptional Activation; Triglycerides

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF family, has a potent mitogenic activity for vascular smooth muscle cells (SMCs). We previously reported that HB-EGF is involved in atherogenesis of human aorta and coronary arteries. ProHB-EGF (the membrane-anchored form of HB-EGF) has also been demonstrated to possess a mitogenic activity, which is approximately 30-fold increased when coexpressed with CD9 in mouse L cells. Thus, in the process of atherogenesis, CD9 may be involved in the proliferation of SMCs. We immunohistochemically investigated the localization of CD9 and proHB-EGF in the human aorta and coronary arteries. In normal aorta and coronary arteries, CD9 immunostaining was virtually negative, whereas proHB-EGF immunostaining was positive, especially in the arteries of babies. In contrast, in atherosclerotic lesions, some intimal SMCs were strongly positive for CD9 and proHB-EGF immunostaining. The juxtacrine growth activities of human aortic SMCs were inhibited in vitro by adding neutralization antibodies for CD9 or adding the specific inhibitor of HB-EGF. Besides, coexpressed CD9 and proHB-EGF cells markedly incorporated [(3)H]thymidine into the SMCs. CD9 is localized immunohistochemically in the SMCs of the atherosclerotic aorta and coronary arteries. CD9, when coexpressed with proHB-EGF, enhances proHB-EGF activities for SMC growth in a so-called juxtacrine manner in vitro and may be involved in atherogenesis.

Topics: Animals; Antigens, CD; Aorta; Arteriosclerosis; Cell Division; Cells, Cultured; Coronary Vessels; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Infant; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Muscle, Smooth, Vascular; Tetraspanin 29

A hallmark of vascular lesions is the phenotypic modulation of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a more primitive, proliferative phenotype with a more fetal pattern of gene expression. Using subtraction hybridization to identify genes that may regulate this transition, we cloned a novel gene named EVEC, an acronym for its expression in the embryonic vasculature and the presence of Ca2+ binding epidermal growth factor-like repeats contained in the predicted protein structure. Although these repeats are characteristic of the extracellular matrix proteins, fibrillin, fibulin, and the latent transforming growth factor-beta binding proteins, EVEC most closely resembles the H411 and T16/S1-5 gene products, the latter of which are believed to regulate DNA synthesis in quiescent fibroblasts. Using in situ hybridization, we demonstrated that EVEC is expressed predominantly in the VSMCs of developing arteries in E11.5 through E16.5 mouse embryos. Lower levels of expression are also observed in endothelial cells, perichondrium, intestine, and mesenchyme of the face and kidney. EVEC mRNA expression is dramatically downregulated in adult arteries, except in the uterus, where cyclic angiogenesis continues; however, EVEC expression is reactivated in 2 independent rodent models of vascular injury. EVEC mRNA is observed in cellular elements of atherosclerotic plaques of LDL receptor-deficient, human apolipoprotein B transgenic mice and in VSMCs of the media and neointima of balloon-injured rat carotid arteries. These data suggest that EVEC may play an important role in the regulation of vascular growth and maturation during development and in lesions of injured vessels.

Topics: Age Factors; Animals; Arteriosclerosis; Blotting, Northern; Cells, Cultured; Cloning, Molecular; COS Cells; Cytoplasmic Granules; Elastin; Epidermal Growth Factor; Extracellular Matrix Proteins; Fetus; Gene Expression Regulation, Developmental; In Situ Hybridization; Mice; Microsomes; Molecular Sequence Data; Muscle, Smooth, Vascular; Phenotype; Rats; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; RNA, Messenger; Sequence Homology, Amino Acid; Tunica Intima; Up-Regulation

Both oxidized low density lipoprotein (ox-LDL) and platelet-derived growth factor (PDGF) have been implicated in the genesis of various inflammatory responses, including atherosclerosis. We demonstrate here a novel interaction between specific oxidized lipids derived from ox-LDL and PDGF. The lipid moieties of ox-LDL caused concentration-dependent inactivation of PDGF as measured by loss of its mitogenic activity and its binding to high affinity receptors. Reverse-phase and normal-phase HPLC were used to purify the inactivating component in the lipid mixture. By fast atom bombardment mass spectrometry and infrared spectroscopy, we identified the inactivating lipids as the 9- and 13-hydroperoxy derivatives of cholesteryl linoleate, cholesteryl hydroperoxyoctadecadienoate. When a series of cholesteryl esters were subjected to oxidizing conditions, only those containing two or more double bonds caused inactivation of PDGF; the extent of inactivation increased with increased levels of oxidation. Exposing PDGF to cumene hydroperoxide, t-butyl hydroperoxide, or hydrogen peroxide did not affect the activity of the mitogen. The oxidized lipid had no effect on the mitogenic activity of epidermal growth factor but did abolish the mitogenic activity of basic fibroblast growth factor and the antiproliferative activity of transforming growth factor beta1. The inactivation of PDGF and other cytokines by lipid hydroperoxides may occur in such processes as vascular disease, inflammation, and wound healing.

Topics: Arteriosclerosis; Cholesterol Esters; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Inflammation; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Mass Spectrometry; Peroxides; Platelet-Derived Growth Factor; Spectrum Analysis

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2',7'-dichlorofluorescin diacetate. N-Acetyl-L-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC. DL-Buthionine-(S, R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

Topics: Acetylcysteine; Animals; Aorta, Thoracic; Arteriosclerosis; Cell Nucleus; Cells, Cultured; Cycloheximide; Dactinomycin; Deoxyglucose; Diabetic Angiopathies; Epidermal Growth Factor; Gene Expression; Guanidines; Heparin; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Kinetics; Muscle, Smooth, Vascular; Peroxides; Pyruvaldehyde; Rats; Rats, Wistar; Reactive Oxygen Species; RNA, Messenger; Transcription, Genetic

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family which binds to and activates the epidermal growth factor (EGF) receptor. HB-EGF mRNA is expressed by monocytes and vascular smooth muscle cells (VSMC) in culture, and has been shown to be a potent VSMC mitogen in vitro. The aim of this study was to screen normal and human atherosclerotic arteries and SMC cultured from these arteries for expression of HB-EGF, and to determine its cellular localization in human lesions. Using the highly sensitive technique of reverse transcription polymerase chain reaction (RT-PCR), we screened biopsies taken from normal human vessel walls and atherosclerotic tissue, for expression of HB-EGF mRNA. Northern blotting and RT-PCR were employed to determine levels of HB-EGF gene expression in SMC, cultured from normal and atherosclerotic arteries. Cellular localization of mRNA and protein, within human atherosclerotic plaques, was assessed using in situ hybridization with 35S labelled riboprobes, and immunohistochemistry with polyclonal antibodies specific for human HB-EGF. HB-EGF mRNA was found to be expressed in human atherosclerotic lesions and in VSMC cultured from these lesions. Expression of HB-EGF could not be detected in quiescent aortic VSMC using Northern blotting, but was highly up-regulated in these cells after treatment with basic fibroblast growth factor (bFGF) for 24 h. Although HB-EGF mRNA was detected in all vascular tissue examined using RT-PCR, in situ hybridization and immunohistochemistry revealed expression of HB-EGF in small portions of diseased arteries only. Immunohistochemistry showed strong staining for macrophages in all areas of HB-EGF expression. No association of HB-EGF with SMC was observed in any of the specimens examined. In conclusion, HB-EGF, a potent mitogen for VSMC, is expressed by macrophages in human.

Topics: Arteries; Arteriosclerosis; Carotid Arteries; Cells, Cultured; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Macrophages; Muscle, Smooth; Muscle, Smooth, Vascular; Polymerase Chain Reaction

Under diabetic conditions, the Maillard reaction facilitates the production of reactive oxygen species, and the activity of antioxidant enzymes such as Cu,Zn-superoxide dismutase is decreased, resulting in a remarkable increase of oxidative stress. The oxidative stress attacks DNA, lipids, and proteins and is also thought to be involved in the pathogenesis of diabetic complications, including the progression of macroangiopathy. Proliferation of smooth muscle cells (SMCs) is known to be associated with progression of macroangiopathy and is modulated by several growth factors. At least three mitogens for SMCs, platelet-derived growth factor (PDGF), fibroblast growth factor, and heparin-binding epidermal growth factor-like growth factor (HB-EGF), are known to be produced by SMCs themselves and are considered to be the most potent growth factors in the progression of macroangiopathy as seen in diabetes. HB-EGF, but not PDGF, is regulated at the transcriptional level by 3-deoxyglucosone (3-DG), a major and highly reactive intermediate in the glycation reaction. The induction seems to be triggered by the increase of reactive oxygen species produced by 3-DG. Taken together, glycation reactions under diabetic conditions may be highly associated with the pathogenesis of diabetic macroangiography by enhancing the gene expression of HB-EGF.

Topics: Animals; Aorta; Arteriosclerosis; Deoxyglucose; Diabetes Mellitus; Diabetic Angiopathies; DNA Damage; Epidermal Growth Factor; Gene Expression; Glycation End Products, Advanced; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Muscle, Smooth, Vascular; Oxidation-Reduction; Oxidative Stress; Platelet-Derived Growth Factor; Rats; RNA, Messenger

When injured, vascular endothelial cells produce growth factors that cause smooth muscle cells (SMC) to migrate from the media to the intima of the vessel wall, replicate in the intima, and stimulate arteriosclerotic changes. Interference with the actions of growth factors in allograft arteriosclerosis was explored. The somatostatin analog angiopeptin was administered to allograft-recipient rats after transplantation of aortic allografts between major and minor histoincompatible rat strains. Levels of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and platelet-derived growth factor (PDGF) in grafts from angiopeptin-treated recipients were 35% to 75% of levels in grafts from nontreated recipients. Replication of SMC in the media and intima was reduced by 30% to 90% and intimal thickening by approximately 50%. The effect of blockade of IGF-1 receptors (IGF-1R) on the intimal response was also investigated. SMC cultures were serum-deprived of growth factors, then stimulated to replicate by addition of PDGF-B and EGF. Anti-IGF-1 and anti-IGF-1R antibodies reduced SMC replication by 50% and 90%, respectively. A D-amino acid analog of IGF-1, JB3, inhibited SMC replication and dose-dependently inhibited insulin receptor substrate 1 (IRS-1) and IGF-1R phosphorylation in vitro. Infusion of JB3 into rats undergoing balloon dilatation injury inhibited SMC replication in the injured vascular area by nearly 70%, but inhibited intimal thickening by only 30%. In conclusion, interference in the growth factor response may be one way of reducing/preventing vascular injury. However, blockade of more than one growth factor may be needed to achieve an optimal effect.

Topics: Animals; Aorta; Arteriosclerosis; Blood Vessels; Catheterization; Cell Division; Coronary Artery Bypass; Epidermal Growth Factor; Growth Inhibitors; Growth Substances; Insulin-Like Growth Factor I; Muscle, Smooth, Vascular; Oligopeptides; Peptides, Cyclic; Platelet-Derived Growth Factor; Rats; Receptors, Somatomedin; Somatomedins; Somatostatin; Transplantation, Homologous

Despite significant infiltration into tumors and atherosclerotic plaques, the role of T lymphocytes in these pathological conditions is still unclear. We have demonstrated that tumor-infiltrating lymphocytes (TILs) and plaque-infiltrating lymphocytes (PILs) produce heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF) in vitro under nonspecific conditions and in vivo in tumors by immunohistochemical staining. HB-EGF and bFGF derived from TILs and PILs directly stimulated tumor cells and vascular smooth muscle cells (SMCs) in vitro, respectively, while bFGF displayed angiogenic properties. Therefore, T cells may play a critical role in the SMC hyperplasia of atherosclerosis and support tumor progression by direct stimulation and angiogenesis.

Topics: 3T3 Cells; Animals; Arteriosclerosis; Biological Assay; Breast Neoplasms; Cell Division; Chromatography, Affinity; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lymphocytes, Tumor-Infiltrating; Mice; Ovarian Neoplasms; T-Lymphocytes

Heparin-binding EGF-like growth factor (HB-EGF) is a potent chemoattractant and mitogen for smooth muscle cells (SMC) in culture. To elucidate whether HB-EGF is implicated in the pathogenesis of human atherosclerosis, we examined immunohistochemical localization of HB-EGF in human aortic walls and atherosclerotic plaques. The medial SMC of the aorta in babies and children synthesized HB-EGF protein, while the number of SMC producing HB-EGF was dramatically decreased in young and middle-aged adults. In atherosclerotic plaques, however, marked production of HB-EGF protein was detected in SMC and macrophages of the plaques. Furthermore, EGF receptors, to which HB-EGF is known to bind, were detected in plaque SMC. These data suggest that HB-EGF may be implicated in the migration and proliferation of SMC that occurs in the normal development of arterial walls, and in the formation of atherosclerotic plaques.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Aorta; Arteriosclerosis; Child; Epidermal Growth Factor; ErbB Receptors; Female; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Infant; Intercellular Signaling Peptides and Proteins; Macrophages; Male; Middle Aged; Muscle, Smooth, Vascular

Vascular medial smooth muscle cells migrate, proliferate and transform to foam cells in the process of atherosclerosis. We have reported that the intimal smooth muscle cells express proto-oncogene c-fms, a characteristic gene of monocyte-macrophages, which is not normally expressed in medial smooth muscle cells. In the present study, we demonstrated that combinations of platelet-derived growth factor (PDGF)-BB and either epidermal growth factor (EGF) or fibroblast growth factor (FGF) induced high expression of c-fms in normal human medial smooth muscle cells to the level of intimal smooth muscle cells or monocyte-derived macrophages, whereas c-fms expression by PDGF-BB alone was 1/10 and both EGF and FGF had no independent effect on c-fms expression. By contrast, interferon (IFN)-gamma and macrophage colony-stimulating factor (M-CSF) suppressed the induction of c-fms expression. These results indicate that multiple growth factors and cytokines may play a role in the phenotypic transformation of medial smooth muscle cells to intimal smooth muscle cells in atherosclerotic lesions by altering c-fms expression.

Topics: Aorta; Arteriosclerosis; Cells, Cultured; Cytokines; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Genes, fms; Humans; Interferon-gamma; Macrophage Colony-Stimulating Factor; Membrane Proteins; Muscle Development; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Proto-Oncogene Mas; Receptor, Macrophage Colony-Stimulating Factor; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class B; Tunica Intima; Tunica Media

Increased proliferation of intimal smooth muscle cells (SMCs) plays an important role in the early phase of atherogenesis. To investigate growth mechanisms of these cells, we used intimal SMCs from rabbits fed an atherogenic diet and examined the sequential events that may facilitate induction of intimal SMC proliferation as well as the possible effects of growth-promoting factors secreted by these cells. In serum-free medium, epidermal growth factor (EGF) stimulated [3H]thymidine uptake by quiescent intimal SMCs at a rate six times higher than quiescent medial SMCs. There was no significant difference between the two cell types in terms of the number of specific EGF receptor per cell, the dissociation constant of EGF, and the time course of EGF binding and internalization. Furthermore, in both types of cells, c-fos, c-jun, and c-myc mRNAs were induced after 1, 1, and 4 hours of EGF treatment, respectively, whereas they required 8 hours of contact with EGF to induce proliferation. Growth response of medical SMCs to EGF was greatly enhanced when rabbit serum, deficient in lipoproteins and free of platelet-derived growth factor, was added to the medium. Moreover, EGF induced a twofold to fourfold increase in DNA synthesis in medial SMCs cocultured with intimal SMCs compared with medial SMCs incubated alone. Likewise, DNA synthesis of medial SMCs grown in medium conditioned by intimal SMCs was six times higher than that observed in medium conditioned by medical SMCs. Adding EGF to the medium conditioned by intimal SMCs increased their DNA synthesis even further.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aorta, Thoracic; Arteriosclerosis; Cell Division; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Genes, fos; Genes, jun; Genes, myc; Immunohistochemistry; Male; Muscle, Smooth, Vascular; Rabbits; RNA, Messenger

Lysophosphatidylcholine is increased in the plasma of hypercholesterolemic patients, is a component of oxidatively modified low-density lipoprotein, and, as such, may play an important role in atherosclerosis. Here we demonstrate that in human monocytes, lysophosphatidylcholine increases the level of mRNA encoding the heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle mitogen. Lysophosphatidylcholine treatment also enhances the release of heparin-binding mitogenic activity by these cells in culture. The anti-inflammatory glucocorticoid dexamethasone inhibits the upregulation of HB-EGF mRNA induced by either lysophosphatidylcholine or bacterial lipopolysaccharide in cultured monocytes. However, the responses induced by lysophosphatidylcholine and by lipopolysaccharide differ in their kinetics. In addition, the response to lysophosphatidylcholine is resistant to the action of cycloheximide, whereas the response to lipopolysaccharide is not, suggesting that the activation mechanisms induced by these two stimuli are different. Since a nuclear run-on assay showed no effect of lysophosphatidylcholine on the transcription of the HB-EGF gene, we speculate that lysophosphatidylcholine may increase the level of HB-EGF mRNA by altering the processing or degradation of primary or mature transcripts. Lysophosphatidylcholine enhancement of monocyte production of HB-EGF may represent an important result of the interactions among oxidized low-density lipoprotein and monocyte-derived macrophages and may play a role in initiation of smooth muscle proliferation in atherogenesis.

Topics: Arteriosclerosis; Cells, Cultured; Cycloheximide; Epidermal Growth Factor; Fatty Acids; Gene Expression; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lipopolysaccharides; Lysophosphatidylcholines; Monocytes; Muscle, Smooth, Vascular; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Up-Regulation

Smooth muscle cell proliferation in the intima of arteries is a principal event associated with vascular narrowing after balloon angioplasty and bypass surgery. Techniques for limiting smooth muscle cell proliferation, however, have not as yet yielded any therapeutic benefit for these conditions. This may reflect the present lack of sufficiently potent and specific inhibitors of smooth muscle cell proliferation. DAB389 EGF is a genetically engineered fusion protein in which the receptor-binding domain of diphtheria toxin has been replaced by human epidermal growth factor. We evaluated the effect of this fusion toxin on human vascular smooth muscle cells in culture. Incubation of proliferating cells with DAB389 EGF yielded a dose-dependent inhibition of protein synthesis, as assessed by uptake of [3H]leucine, with an IC50 of 40 pM. The cytotoxic effect was inhibited in the presence of excess EGF or with monoclonal antibody to the EGF receptor. We further studied the effect of the fusion toxin on smooth muscle cell outgrowth from human atherosclerotic plaque. Outgrowth was markedly inhibited after as little as 1 h of exposure to the fusion protein. Furthermore, complete inhibition of proliferation of cells within the plaque could be attained. These results demonstrate that DAB389 EGF is highly cytotoxic to human smooth muscle cells proliferating in culture and can prevent smooth muscle cell outgrowth from "growth-stimulated" human atherosclerotic plaque. DAB389 EGF may therefore be of therapeutic value in vascular diseases characterized by smooth muscle cell accumulation.

Topics: Arteriosclerosis; Cell Division; Cells, Cultured; Diphtheria Toxin; Epidermal Growth Factor; ErbB Receptors; Humans; Muscle, Smooth, Vascular; Recombinant Fusion Proteins

The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.

Topics: Animals; Aorta; Arteriosclerosis; Binding Sites; Cattle; Cell Movement; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glycosylphosphatidylinositols; Hirudins; Muscle, Smooth, Vascular; Peptide Fragments; Plasminogen Activators; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Thrombin; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

Topics: Animals; Arteriosclerosis; Endothelial Growth Factors; Endothelium, Vascular; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Lymphokines; Muscle, Smooth, Vascular; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Arteriosclerosis; Cell Division; Cells, Cultured; Coronary Artery Disease; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor

Topics: Actin Cytoskeleton; Animals; Arteriosclerosis; Cell Division; Cells, Cultured; Epidermal Growth Factor; Humans; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Thymidine

We studied the effects of elastase on [3H]thymidine incorporation into aortic smooth muscle cells. When elastase was added to cultured aortic smooth muscle cells, [3H]thymidine incorporation was inhibited in a dose-dependent manner in the presence of fetal bovine serum. Elastase also inhibited this incorporation in cells treated with epidermal growth factor. Epidermal growth factor (50 ng/ml) stimulated thymidine incorporation, without elastase, but with the addition of 20 units/ml of elastase the incorporation was inhibited 70%. The incorporation of thymidine into cells treated with 50 ng/ml epidermal growth factor was also inhibited 50% by a low concentration of elastase (5 units/ml). These inhibitory effects on thymidine incorporation were also observed in cells stimulated with platelet-derived growth factor. Platelet-derived growth factor (20 units/ml) markedly stimulated thymidine incorporation into cells, and elastase inhibited its activity in a dose-dependent manner. These results suggest that elastase has the potential to prevent the development of atherosclerosis by inhibiting smooth muscle proliferation.

Topics: Animals; Aorta; Arteriosclerosis; Cell Division; Cells, Cultured; DNA; Elastin; Epidermal Growth Factor; Muscle, Smooth, Vascular; Pancreatic Elastase; Rats; Rats, Inbred Strains; Thymidine

Based on the findings presented in this study, we propose the hypothesis that calcium could be a mediator for the development of atherosclerosis. Figure 8 shows a schematic illustration of the hypothesis. The presence of risk factors such as hypertension, hyperlipidemia, and smoking may increase the influx of calcium into vascular ECs. We have shown that reactive oxygen species, which are considered to be a risk factor for the development of atherosclerosis, actually increase [Ca++]i in vascular ECs. Increased intracellular calcium may damage the function of ECs, resulting in platelet aggregation at the damaged site. Increased intracellular calcium may also increase uptake of macromolecules in plasma such as fibrinogen and LDL, eventually forming atherosclerotic plaque. We have also shown that the influx of calcium into vascular ECs is associated with LDL transport across vascular ECs. The pretreatment by nifedipine inhibited both the increase in [Ca++]i and the increase in LDL transport, suggesting that intracellular calcium modulates LDL transport across ECs. Growth factors released from platelets may provoke migration and proliferation of medial SMCs in the aterial intima. It has been reported that migration of SMCs from arterial media to intima is enhanced by the presence of calcium, and can be inhibited by the pretreatment of calcium antagonist. As demonstrated in this study, calcium also plays an important role in the proliferation of SMCs provoked by some kinds of growth factors such as EGF. On the other hand, we found that an increased amount of dietary Mg suppressed the development of atherosclerotic lesions in the aorta of cholesterol-fed rabbits without affecting plasma total cholesterol and HDL-cholesterol concentrations. The mechanism of action might also be related to the calcium entry blocking action. The clinical and nutritional implications of these phenomena should be investigated further. The evidences presented in this study, however, would not be sufficient to fully explain the etiological role of calcium in atherogenesis. Further studies are required to elucidate the mechanism of the contribution of calcium to atherogenesis. The efficacy of calcium antagonist for the prevention of atherosclerosis in humans should also be investigated further.

Topics: Animals; Arteriosclerosis; Calcium; Cells, Cultured; DNA; Epidermal Growth Factor; Female; Guinea Pigs; Humans; Lipoproteins, LDL; Magnesium; Male; Middle Aged; Nifedipine; Platelet-Derived Growth Factor; Rabbits; Xanthine Oxidase

The growth behavior of intimal smooth muscle cells (SMC) prepared from atheromatous plaques of the thoracic aorta in hyperlipidemic rabbits was studied in a culture system. Specimens of intimal and normal medial SMC were examined in terms of their proliferative response to various growth factors, polypeptide hormones or 12-O-teradecanoylphorbol-13-acetate (TPA). Intimal SMC showed lower rates of growth and DNA synthesis when the cells were exposed to TPA, but there was no difference in growth response between intimal and medial SMC to the other growth-promoting stimuli such as fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin or serotonin. 3H-phorbol-12,13-dibutyrate (3H-PDBu) binding assays showed the number of binding sites to phorbol esters in intimal SMC to be decreased by 65% as compared with that in medial SMC. These results suggested that intimal SMC have different growing characteristics, which seemed to be acquired during the process of intimal thickening.

Topics: Animals; Aorta; Arteriosclerosis; Cell Division; Cells, Cultured; Diet, Atherogenic; Epidermal Growth Factor; Insulin; Kinetics; Male; Muscle, Smooth, Vascular; Rabbits; Serotonin; Tetradecanoylphorbol Acetate