Veroffentlichungen aus der Geomedizinischen Forschungsstelle (Leiter: Professor Dr. Dres. h. c. G. Schettler) der Heidelberger Akademie der Wissenschaften Supplement zu den Sitzungsberichten der Mathematisch-naturwissenschaftlichen Klasse J ahrgang 1987 G. Schettler (Ed.) Molecular Biology of the Arterial Wall Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Prof. Dr. Ores. h. c. Ootthard Schettler Prasident der Heidelberger Akademie der Wissenschaften Karlstrasse 4, 6900 Heidelberg, FRO ISBN-13:978-3-540-17899-6 e-ISBN-13:978-3-642-83118-8 DOl: 10.1007/978-3-642-83118-8 Library of Congress Cataloging-in-Publication Data Molecular biology of the arterial wall. (VerOffentlichungen aus der Geomedizinischen Forschungsstelle der Heidelberger Akademie der Wissenschaften) (Supplement zu den Sitzungsberichten der Mathematisch-Naturwissenschaftlichen KJasse; Jahrg.) Based on a conference held in Heidelberg, Germany, Sept_ 27 -Oct. 4, 1986 and organized by the Heidelberg Academy of Sciences. I. Arteriosclerosis Etiology-Congresses. 2. Arteries-Congresses. 3. Pathology, Molecular-Con gresses. 4. Molecular biology-Congresses. I. Schettler, Gotthard. II. Heidelber ger Akademie der Wissenschaften. III. Series. IV. Series: Supplement zu den Sitzungsberichten der Mathematisch-Naturwissenschaftlichen Klasse; Jahrg. 1987. (DNLM: 1. Arteries-congresses. 2. Molecular Biology-congresses. WG 510 M718) RC692.M6 1987 612'.133 87-12843 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9, 1965, in its version of June 24, 1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1987 The use of general descriptive names, trademarks, etc.. in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may ac cordingly be used freely by anyone. Product Liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. "fYpesetting: K +V Fotosatz GmbH, Beerfelden 2125/3140-543210 Table of Contents Opening Address .....•........................... o. ... IX 0.0 •• 0.0. 0... •• •• •• •• Workshop I lipoproteIns and Lipoprotein Receptors 0 ••• 0 0 •••••••••••••• 0 •••••••• , •• 0 0 0 0 •• LDL Receptor Mutations in Patients with Familial Hypercholesterolemia M.A. Lehrman, H.H. Hobbs, M.S. Brown, J.L. Goldstein, DoW. Russel ..... 2 Lipoprotein Receptors and Their Ligands: Structure and Function of Apoproteins E and B R.W. Mahley ... ,., .................................................... ,. 5 The Role of HDL in Cholesterol Homeostasis of Macrophages and Reversed Cholesterol Transport G. Schmitz, H. Robenek .................................... ,............ 8 Lipoproteins, Macrophages and Atherosclerosis 0....... D. Steinberg ........ , ................... , ....................... 10 Arterial Lipoprotein Metabolism: Site-Specific Differences Related to Susceptibi Iity to Atherosclerotic Lesions T.E. Carew, D.C. Schwenke ............................................. 13 Lipoprotein Disposition by the Liver: A Concerted Action of Various Cell Types T.J.C. van Berkel, L. Harkes, J. F. Nagelkerke, J. K. Kruijt.............. 17 The Macrophage and Endothelial Cell Scavenger Receptor D.P. Via, A. Fanslow, W.E. Koff, H.A. Dresel. ........................... 19 Characterization of the Hepatic Scavenger Receptor H.A. Dresel, E. Friedrich, H.J. Sinn, G. Schettler, D.P. Via ......... 22 0 •••• Workshop II Growth Factors .......................................... 25 0 ••••••••••••• 0 • • •• The Role of PDGF in Health and Disease R. Ross .......................................... 26 0 •••••••••••••••••••••• Role of PDGF-Iike Growth Factors in Autocrine Stimulation of Cell Growth C .-H. Heldin ............................•.....•.......... 30 0 •••••• 0 •••• ••• The PDGF Receptor as a Mediator of Mitogenesis LoT, Williams, J .A. Escobedo, M.T. Keating, T ,0. Daniel, S.R. Coughlin .. 32 VI Alpha- and Beta-Heparin Binding Growth Factors: Chemistry and Biology of Acidic and Basic Fibroblast Growth Factors A. Baird, P. Cuevas, N. Ueno, F. Esch, N. Ling ............•.............. 36 Structure, Homologies and Activities of Acidic Fibroblast Growth Factor K.A. Thomas, G. Gimenez-Gallego, J. DiSalvo, D. Linemeyer, L. Kelly, J. Menke................................................................ 41 Endothelial Cell Growth Factor and its Receptor T. Maciag, W.H. Burgess ................................., ............... 44 Workshop III Endocytosis and Exocytosis .................................................. 47 Intracellular Transport of Human Class I Antigens and an Adenoviral Glycoprotein P.A. Peterson, M. Andersson, I. Martens, S. Piiiibo, L. Severinsson .......• 48 Molecular Mechanisms of Endocytic Sorting I. Mellman, S. Schmid, R. Fuchs, M. Marsh, V. Lewis, T. Koch, S. Green, H. Plutner, A. Helenius. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51 Characterisation of a Fusion Event from the Endocytic Pathway J. Davey, G. Warren. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . .. 56 Membrane Boundaries Involved in the Selective Trafficking of Cell Surface Receptors C.R. Hopkins ............................................................ 59 The Molecular Basis of Human Cellular Iron Metabolism R.D. Klausner, J.B. Harford ..........................•..........•....... 61 Workshop IV ONC Genes and Proliferative Disease........................................ 65 Molecular Features Involved in Cell Surface Receptor Function and Their Role in Oncogenesis H. Riedel, Y. Yarden, A. Gray, L. Coussens, T. Dull, J. Sch lessinger, A. Ullrich ........................... " . .. . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . 66 Transforming Activity of the fms Oncogene (CSF-1 Receptor) C.J. Sherr, M.F. Roussel~F. Wheeler, C.W. Rettenmier.... .•... ....... 69 Functional and Structural Analysis of the v-src and c-src Genes H. Hanafusa ...........................~ ......~ . . . . . . . . . • . . . • • • . . . 71 Ras Oncogenes and Cell Transformation C.J. Marshall ................................................... · .... ··. 73 Cis Regulatory Control of mos Oncogene Expression -M.L. McGeady, T.G. Wood, D.G. Blair, A. Seth, F. Propst, M. Oskarsson, M. Schmidt, G. Vande Woude. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Analysis of the Function of the fos Oncogene Product R. Muller, T. Jenuwein, M.Renz ........................................ 81 VII Cooperative Interactions Involving Cellular and Viral Oncogenes in the Development of Malignant Tumors E. Mougneau, C. Cerni, F. Cuzin ......................................... 84 Workshop V White Blood Cells........................................................... 85 The Motor of Leukocytes and Platelets: 1986 T.P. Stossel, J.H. Hartwig, P.A. Janmey, H.L. Yin, K.S. Zaner ........... 86 Heterogeneity of Murine and Human Fcy Receptors J . C. Unkeless........................................................... 88 Complement Receptors D.T. Fearon............................................................ 91 Cachectin: A Macrophage Protein that Induces a Catabolic State in Infected Animals A. Cerami. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 94 The Role of Cytosolic Free Calcium, (Ca2+)., and Phosphoinositides as Intracellu lar Signals I P.O. Lew •............................................................... 95 The Macrophage-Blood Interface: Cell Localisation and Recruitment in Bone Marrow, Liver and Brain S. Gordon, P.R. Crocker, L. Morris, H.V. Perry ......................... 97 Normal and Neoplastic Early Lymphocyte Maturation I. Weissman, C. Muller, G. Tidmarsh, C. Okada, C. Whitlock .............. 101 Molecular Regulation of Complement Gene Expression H.R. Colten ......................................................•...... 102 Workshop VI Prostaglandins and Leukotrienes ......................................•..... 105 The Role of GTP-Binding Proteins in Coupling Prostaglandin and Leukotriene Receptors to Intracellular Second Messenger Systems P. Gierschik, K.H. Jakobs ............................................... 106 Regulation of Vascular Tone by Prostaglandins and Endothelium-Derived Relaxing Factor U. Forstermann......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . • • . . . . . • . . . . .. 110 Endogenous Cysteinyl Leukotrienes as Mediators in Endotoxin Shock and Tissue Trauma D. Keppler, W. Hagmann, C. Denzl inger, S. Rapp, M. Huber, A. Guhlmann, S. Kastner.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • • . . . . . . .. 115 Source and Mechanism of Release of Arachidonic Acid in Blood Platelets J.B. Smith .............................................•.....••..•••.... 117 Eicosanoids and the Pulmonary Circulation W. Seeger, N. Suttorp, H. Neuhof ........................................ 119 VIII Regulation of the Production (Leukotriene All Hydrolase) and the Action (L~~:~r~~~~~t!fu'!:~:s~~.~I.t~~~ .~~ ~~~.~~~~~~~.~~ .......•....•.•.•..••....... 123 Enzymic Synthesis and Degradation of Prostaglandin D2 O. Hayalshi, Y. Urade, K. Watanabe ...................................... 126 Receptor-Dependent Regulation of Human Polymorphonuclear Leukocyte Responses to Leukotrienes . C.H. Koo, L. Baud, T. Marotti, M. Cheung, J.P. Harvey, E.J. Goetzl ...... 129 Leukotriene C -Binding Proteins K.F.Auste~ ..........................•.................••........•..... 132 Leukotriene C Metabolizing Enzymes L. tlrning, K. Bernstrom, M. SOderstrom, B. Mannervik, S. Hammarstrom......................................................... 134 Workshop VII Biology of Smooth Muscle and Endothelium ................................... 139 The Fibrinolytic System of Cultured Bovine Aortic Endothelial Cells D.J. Loskutoff.......................................................... 140 Tumor Necrosis Factor /Cachectin and the Modulation of Endothelial Cell Coagulant Properties D.M. Stern, P.P. Nawroth ...........................•...........•.•..... :145 Membrane Glycoproteins of Endothelial Cells and Platelets J.A. van Mourik, J.C. Giltay, O.C. Leeksma, J. Zandbergen-Spaargaren .. 148 The Molecular Biology of Endothelial Cell von Willebrand Factor D.C. Lynch ......................................................•...•.. 151 Induction and Regulation of Endothelial Gene Expression T. Collins, J.S. Pober ................................................... 155 Endothelium-Vascular Smooth Muscle Interactions in Culture R.W. Alexander, J. Leopold, K. Griendling, P. Ganz ...................... 159 Endothelial Morphogenesis S.M. Schwartz, R.L. Heimark .•.................................•....•.•. 163 Closing Remarks S.C. Silverstein ................................•..•....•.•.•.••.••.••... 166 Final Word G. Schettler ..................................•.....••...••..•.....•.•... 167 List of Participants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . • . . . . . . . • • . . • • . . • . .. 168 List of Sponsors ...........................•..•......•..••.••...•......•••.. 169 OPENING ADDRESS by G. Schettler This conference on Molecular Biology of the Arterial Wall has been organized by the Heidelberg Academy of Sciences and a group of truly outstanding international experts who are acting as chairpersons. It is intended to be the first in a series of scientific meetings organized by the Academy in the years to come focusing on special topics related to basic medical research. Backed by a prestigious tradition. the Academy seeks to create an open forum for discussions of wide-ranging develop ments in basic research and for interdisciplinary scientific cooperation among Inter national scientists of world renown. More and more frequently in the last decades. researchers guided by quite different basic concepts and using different methods have obtained results which can be synthesized in unexpected ways to produce interesting conclusions that require us to view scientific questions in a new light. After a long period in which researchers working on the biology and pathobiology of the arterial wall were somewhat isolated from the mainstream of cellular biology. it seems that as cellular biologists approach the level of single cells and single genes. we are also developing a more fundamental understanding of the pathomechanisms of diseases of the arterial wall. The situation in the past was often frustrating. Cellular interactions in the arterial wall were too complex to be studied more specifically. Furthermore. arterial lesions evolve very slowly. which seemed to hinder an experimental approach. Finally. there was the attractive and popular lipid hypothesis that plasma components can be a sufficient cause for atherosclerosis and its clinical syndromes. This hypothesis has encouraged the study of atherogenesis in terms of plasma lipoproteins and apolipoproteins. which are available in large quantities and can be screened for mutations .. However. dis eases and cellular changes in the arterial wall are not just a consequence of hyper lipidemia and an increased influx of lipids into the arterial wall. The atherosclerotic plaque is not a degenerative process. As already hypothesized by the Cerman pathologist Rudolf Virchow in 1856. specific injuries of the arterial wall cause a disturbance of the critical balance between cell proliferation and cell destruction. Following this assumption. one can postulate that specific lesions in the arterial wall are constantly forming. regressing. or progressing. As new cellular and biochemical techniques have become available in the last years. researchers have developed new tools to address their investigations to the cellular basis of both normal arterial homeostasis and the diseased state when lesions of the arterial wall are developing. Consequently. our knowledge of the biology of arterial wall cells and molecular interactions. e.g .• on the level of cellular receptors. is ex panding more and more rapidly. It is becoming evident that many of the new notions will contribute to generating a basis for an understanding of Virchow's hypothesis on a molecular level. Workshop I Lipoproteins and Lipoprotein Receptors LDL Receptor Mutations in Patients with Familial Hypercholesterolemia M.A. Lehrman, H.H. Hobbs, M.S. Brown, J.L. Goldstein, and D.W. Russell Department of Molecular Genetics, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, Texas 75235 USA INTRODUCTION The low-density lipoprotein (LDL) receptor plays a major role in the maintenance of normal LDL level in humans (1). Mutations in the LDL receptor gene result in Familial Hypercholesterolemia (FH), an autosomal dominant disorder charac terized by elevated levels of plasma LDL (1). The LDL receptor is normally synthesized in the endoplasmic reticulum (ER) as a precursor of 120,000 daltons that contains immature N- and O-linked oligosaccharides. During synthesis, an amino-terminal signal sequence is cleaved from receptor. Maturation of the oligosaccharides in the Golgi complex generates the mature form of the receptor with a molecular weight of 160,000. The receptors are then transported to the cell surface, where they cluster in coated pits and can bind LDL. By receptor mediated endocytosis, the bound LDL is internalized as the coated pits invaginate to form coated vesicles. The receptor and ligand are then trans ported to endosomes, where they dissociate. The LDL is eventually transported to lysosomes where it is degraded. In contrast, the receptor returns to the cell surface to repeat the cycle (1). The LDL receptor gene consists of eighteen exons that span at least 40 kb qf DNA. Exon 1 encodes a cleavable signal sequence. Exons 2 through 18 encode the mature receptor protein, which consists of 839 amino acids and is thought to be composed of five domains (1). Listed from the amino to the carboxy terminus, they are: 1) a putative ligand binding domain of 292 amino acids (exons 2-6 of the receptor gene); 2) a domain of 417 amino acids with homology to the epidermal growth factor (EGF) precursor (exons 7-14); 3) a domain of 58 amino acids rich in serine and threonine-linked oligosaccharides (exon 15); 4) a 22 amino acid membrane-spanning region (exons 16 and 17); and 5) a cyto plasmic domain of 50 amino acids (exons 17 and 18). In order to begin to understand the functional roles of these domains, we have characterized the mutations that occur in patients with FH by a combination of DNA and RNA blotting, SI nuclease mapping, molecular cloning, and DNA sequencing. These studies have also provided insight into the genetic basis for these mutations and the mechanisms by which the receptor gene can be rearranged. RESULTS AND DISCUSSION Two mutations have been identified that eliminate part of the putative ligand binding domain. The mutation in one patient, FH 626, which was characterized by cloning and sequencing, was caused by a 0.8 kb deletion that eliminated exon 5, and generates a receptor that is smaller than normal and does not bind LDL (2). Thus, this domain of the receptor must function in the binding of ligand. A second mutation, FH 563 (3) lost approximately 30 nucleotides (10 amino acids) in exon 4, as judged by SI nuclease mapping. This receptor gets transported to the cell surface at a very slow rate. Since assays that measure LDL binding require that the receptors be present on the cell surface (1), it is not known if this mutation in the binding domain affects the ability of the receptor to bind LDL.