epidermal-growth-factor and Hypercholesterolemia

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.

Topics: Angioplasty, Balloon; Animals; Antibodies, Monoclonal; Carotid Arteries; Cell Movement; Chemotaxis, Leukocyte; Connective Tissue; Diet, Atherogenic; Disease Models, Animal; Epidermal Growth Factor; Hypercholesterolemia; Image Processing, Computer-Assisted; Immunohistochemistry; Macrophages; Male; Muscle, Smooth, Vascular; Rabbits; Tunica Intima

The low density lipoprotein (LDL) receptor is a modular protein involved in the endocytosis of cholesterol-rich lipoproteins from the circulation. Mutations to the receptor result in familial hypercholesterolemia, and over 60 of these occur in the calcium-binding epidermal growth factor-like domain pair. Two selected mutations in this region (G322S and R329P) were introduced into the domain pair and analyzed by in vitro refolding. Both exhibited differing levels of protein misfolding with R329P being the most pronounced. Solution NMR studies of the mutant domain pairs after purification established that a fraction of protein maintains a native-like fold and that this fraction contains two intact calcium-binding sites. An in vivo analysis of intact receptors containing these binding sites showed significantly reduced cell-surface expression compared with the native LDL receptor levels, again with R329P showing the most severe decrease. The sum of these results suggests that either local changes in structure or domain misfolding may be associated with the mutations. There is also the possibility that the misfolding of the calcium-binding epidermal growth factor-like pair region is propagated to other regions of the intact receptor, resulting in more global defects. Surprisingly, for both mutants, those full-length receptors that fold and reach the cell surface retain the ability to bind LDL and release the ligand upon exposure to low pH. This analysis provides significant insight into the protein defect resulting from each of the two mutations and allows their classification to be 2B (partially transport-defective). The results also highlight a range of misfolding defects that may be associated with familial hypercholesterolemia and may enable the prediction of the consequences of homologous disease-causing mutations to other proteins.

Topics: Binding Sites; Blotting, Western; Calcium; Cell Line; Cell Membrane; Chelating Agents; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Flow Cytometry; Humans; Hydrogen-Ion Concentration; Hypercholesterolemia; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Models, Molecular; Mutation; Plasmids; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, LDL; Time Factors

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase blocks the mevalonate metabolic pathway, which is necessary for the isoprenylation of a number of small guanosine triphosphatases. We examined the effects of HMG-CoA reductase inhibitors, fluvastatin and lovastatin, on human pancreatic cancer cell invasion in vitro and experimental liver metastasis in vivo.. Cell invasion was studied in a modified Boyden chamber assay. The translocation of RhoA was assessed by immunoblotting. Experimental liver metastases were induced in nude mice by intrasplenic inoculation of ASPC-1 human pancreatic cancer cells.. Fluvastatin and lovastatin inhibited the in vitro cancer cell invasion induced by epidermal growth factor (EGF) in a manner sensitive to C3 transferase, a specific inhibitor of Rho. Treatment of ASPC-1 cells with fluvastatin markedly attenuated the EGF-induced translocation of RhoA from the cytosol to the membrane fraction and caused cell rounding. The effects of fluvastatin could be reversed by the addition of all-trans-geranylgeraniol. Administration of fluvastatin to nude mice reduced both metastatic tumor formation in the liver and the growth of established liver metastases at doses recommended for the treatment of hypercholesterolemia in humans.. HMG-CoA reductase inhibitors can be antimetastatic agents with the potential for useful clinical applications.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Membrane; Cytosol; Epidermal Growth Factor; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Indoles; Liver Neoplasms, Experimental; Lovastatin; Mice; Mice, Nude; Neoplasm Invasiveness; Pancreatic Neoplasms; rhoA GTP-Binding Protein; Tumor Cells, Cultured

The effect of moderate elevation in serum cholesterol on vascular reactivity to epidermal growth factor (EGF), endothelin (ET-1) and thrombin, vasoactive peptides present at sites of vascular injury, was examined in isolated aortic rings from rabbits fed either a casein-rich or a control diet for 10-12 weeks. In rings from hypercholesterolemic rabbits, development of maximal isometric tension to all peptide agonists was increased 22 +/- 0.6% while the EC50 for contraction was decreased. Vasorelaxant responses to nitroprusside, an endothelium-independent dilator, were largely intact, while those to A231897, an endothelium-dependent agent, were attenuated. These data suggest that elevation in serum cholesterol in the absence of atherosclerotic lesions is sufficient to increase vascular reactivity to peptide vasoactive mediators, an effect which may predispose arteries to vasospasm.

Topics: Animals; Aorta, Abdominal; Calcimycin; Cholesterol; Endothelins; Endothelium, Vascular; Epidermal Growth Factor; Hypercholesterolemia; In Vitro Techniques; Isometric Contraction; Male; Muscle, Smooth, Vascular; Nitroprusside; Peptides; Rabbits; Thrombin