Detection and Cellular Localization of Heparin-Binding Epidermal Growth Factor-like Growth Factor mRNA and Protein in Human Atherosclerotic Tissue
ABSTRACT 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.
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ABSTRACT: Monocyte recruitment and their differentiation into macrophages are both early events in native and accelerated atherosclerosis that follows angioplasty. We have investigated the putative functional role of the epidermal growth factor receptor (EGFR) present on rabbit monocytes/macrophages. The impact of periadventitial delivery of an EGFR-specific, blocking monoclonal antibody (ICR62, which inhibits EGF-binding to its receptor) was investigated in a rabbit model of accelerated atherosclerosis induced by a combination of carotid injury and 4 weeks of a 2% cholesterol-diet. Two weeks after the initiation of the diet, a balloon-catheter angioplasty of the left common carotid artery was performed and a collar placed around the injured carotid artery immediately, for the delivery of ICR62 antibody, isotype-matched antibody or saline control. Monocyte/macrophage accumulation, cell proliferation and neointimal thickening were determined 2 weeks after the delivery of the antibodies. The function of the EGFR on rabbit monocytes was also investigated in vitro, using chemotaxis assays. Treatment with ICR62 was associated with a significant reduction in macrophage accumulation and neointimal thickening and a 76% reduction in neointimal area of the vessel wall compared with controls. In vitro ICR62 inhibited macrophage and smooth muscle cell migration towards EGFR ligands including EGF and HB-EGF. These findings suggest that EGFR ligation may be important in the development of early atherosclerotic lesions following balloon-catheter angioplasty, and periadventitial delivery may provide a feasible approach for administration of the inhibitors of EGFR-binding such as ICR62.International Journal of Experimental Pathology 12/2009; 91(3):224-34. DOI:10.1111/j.1365-2613.2009.00700.x · 2.05 Impact Factor
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ABSTRACT: Heparin-binding EGF-like growth factor (HB-EGF) is a 22 kDa, O-glycosylated protein that is mitogenic for fibroblasts, smooth muscle cells (SMC) and epithelial cells. This review describes the primary structure of HB-EGF, as well as its processing. The structure of the mouse and human HB-EGF genes is also discussed. Finally, this review summarizes HB-EGF expression patterns, receptor-mediated signaling, and role in several important biological systems.Frontiers in Bioscience 04/1998; 3:d288-99. · 4.25 Impact Factor
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ABSTRACT: Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.Circulation Research 08/2000; 87(2):92-8. DOI:10.1161/01.RES.87.2.92 · 11.09 Impact Factor