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200 1976 REPORT' of THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., In THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., Inc. 110 East 59th Street, New York, N.Y. 10022

Organization and Policy The Council for Tobacco Research-U.S.A., Inc. is the sponsoring agency of a program of research into questions of tobacco use and health. It is the out- growth of an organization formed early in 1954 by tobacco manufacturers, growers and warehousemen. Research support has been mainly through a pro. gram of grants-in-aid supplemented by contracts for research with institutions and~ laboratories. The Council does not operate any research faeility: The Scientific Advisory Board to The Council meets regularly to evaluate applications for grants-in-aid and for contracts, judging them solely on the basis of scientific merit and relevance. The Council awards research grants to independent scientists who are as- sured complete scientific freedom in conducting their studies. Grantees alone are responsible for reporting or publishing their findings in the accepted scien- tific manner - through medical and scientific journals and societies. Through December 1976;, The Council approved research projects for 358' investigators in~ 239 medical schools, hospitals and research institutions. These awards totaled more than $40,000,000. This Report includes a brief summary of The Council's present program in the cardiovascular area as well as lists of current and previous research projects supported' by The Council'. Also ineluded are abstracts of 80 researcK papers, acknowledging Council support, that were published in scientific journals dur- ing 1976. Project recipients have so far published 1,420 such papers. ADDISON YEAMAN Chairman and President

® 202 SCIENTIFIC ADVISORY BOARD to The Council for Tobacco Research-U.S.A., Inc. as of~ December 31, 1976 SHELDON C. SOMMERS, M.D., Chairman Director o f Laboratories, Lenox Hill Hospital Clinical'Professor of Pathology College of Physicians & Surgeons of Columbia University New York, New York .' 3. RICHARD M. BING, M.D. Director of Cardiology and Intramural Medicine Huntington Memorial Hospital~ Pasadena, California ~~. Professor of Medicine University of Southern California School of Medicine Los Angeles, California - JOSEPH D. FELDMAN', M.D. Head, Department of Immunopathology Scripps Clinic and Research Foundation La Jolla, California ~ WILLIAM U. GARDNER, PH.D:. Scientific Director, The Council for Tobacco Research-U.S.A, Inc E. K. Hunt'Professor of Anatomy (emeritus)' ~ l i h ool of~ Medicine Ya e Un versity Sc New Haven, Connecticut ROBERT J. HUEBNER, M.D. ~ Chief, Laboratory of RNA Tumor Viruses National Cancer Institute Y Bethesda, Maryland LEON O. JACOBSON, M.D. Director, The Franklin McLean Memorial Research Institute ~ Regenstein Professor of Biological Sciences ~ University of Chicago "` Chicago, Illinois ~ AVERILL A. LIEBOW„M.D. A Professor of Pathology (emeritus) ~: University of' California School of Medicine San Diego, California 4' HENRY T. LYNCH, M.D. °~ Professor and Chairman Department of Preventive Medicine and Public Health ,i Creighton University School of! Medicine Omaha, Nebraska =4

nc 203 FANS MEIER, D.V.M., Dr. Med. Vec, M.R.S;H. Senior Stafl Scientist The Jackson Laboratory Bar Harbor,,Maine LEE W. WATTENBERG, M:D. Professor of'Pathology Department of', Laboratory Medicine and Pathology University ofMinnesot'a Medical SchoolMinneapolis; Minnesota JOHN P. WYATT, M.D. Director Tobacco and Health Research Institute University of Kentucky Lexington, Kentucky Scientific Staff of The Council WILLIAM U. GARDNER, PH.D. Scientific Director ROBERT C. HOCKETT, Px.D. Research Director JOHN H. KREISHER, PH.D. VINCENT F. LISANTI, D.M.D. Associate Research Director Associate Research Director DAVID STONE,Px.D. Associate Research Director I H I

204 . CONTENTS Studies Related to Cardiovascular Diseases and Function Abstracts of Reports , Cancer-Related Studies The Respiratory System Heart and Circulation Neuropharmacology and Physiology Immunology and Adaptive Mechanisms Epidemiology Active Frojects Completed Projects Index of Principal Investigators Index of Senior Authors P,' Counci monar, relevar. Ir of card Cardi A: from p the Un stroke;, 6721 failure. to preci 7$f I Ati Epide) 0c•  Nu posstble eases, TII The "ris relations it might Am elevated personali i predispo!'. factor, bi the other scientfists to what operativestrong, a personal but has t( observatic coherents prove vali 30-498 O V I I C

Studies Related to Cardiovascular Diseases and Function Previous annual reports have presented the general plan and rationale of Council-sponsored' studies of carcinogenesis and the etiology of chronic pul- monary disorders. Their purpose was to provide a framework in whichi the relevance of individual contributions wouldi be more easily apparent. In the present issue, we describe similarly some of our approaches to study of cardiovascular diseases and function. Cardiovascular Diseases Among the many disorders of the cardiovascular system, those deriving from progressive atherosclerosis rank first as causes of disability and death in~ the UnitediStates. These include heart' attacks (myocardial infarction), angina,, stroke, arterial blockages irn the limbs, and some cases of congestive heart failure. Hypertension is believed not only to accelerate atherosclerosis, but also to precipitate acute events in damaged circulatory systems.. Atherosclerosis is, therefore, a principal focus of the present discussion. Epidemiology o f Atherosclerosis-Related Diseases Numerous epidemiological studies during the last 20 years have sought' possible "causes" (primary or contributory) of the atheroscleresis-related dis- eases. These studies often summarized their findings im terms of "risk factors." The "risk factor" is essentially a stati'sticali concept based upon mathematical relationships without necessarily any known or established mechanism, by which it might contribute to etiology. Among the many such "risk factors" frequently reported are hypertension, elevated serum cholesterol, diabetes or a prediabetic diathesis, cigarette smoking, personality type, inadeqpate physical' activity, and emotional stress. Genetic predisposition (e.g., hyperlipoproteinemia, homocysteinemia) is another such factor, both in its own right and as a contributor to most or possibly all of the others: Biochemists, physiologists, pharmacologists, psychologists, and other scientists have thus been challenged to fill the gaps and learni whether, how, to what extent and' in what kinds of persons each factor might actually be operative. The stimulus to conjecture, speculationi and hypothesis has been strong, and many scientists have apparently seized the occasion to test their personal hunches in the hope of a lucky strike. This was entirely legitimate„ but has tended to produce a welter of' fragmentary, confusing and inconclusivee observations subjected to speculative projections. Clarification may come as coherent, integrative theories emerge that can accommodate all the findings that prove valid. 5 30+498 0 ~- 78 - 14

Cigarette Smoking as a "Risk Factor" ~ The identification of a"risk factor" can be no more reliable than the statistics on which it is based. Smokers select themselves from among the .~; general population on a basis or bases that are little understood. Is it legitimate ~ to compare them with nonsmoker controls as animal experimenters eompare' ~ their test animals with genetically identical litter-mates? Tobacco use has many variations, both qualitative and' quantitative. To ~ what extent does voluntary adoption of any particular behavioral pattern select out a sub-population with innate characteristics or life-styles that' are'` linked to cardiovascular disease susceptibility? There are many evidences from studies of man that genetic predisposition'.~ plays an important part in determining which individ'uals are potentiali victims of atherosclerosis-related diseases. ln several types of rather extreme suscepti-,; bility, the patterns of inheritance have been well established. Review of the "risk factors" listed above indicates that most of them, are ; already known to be influencedi if not determined, by heredity. Studies of' spontaneous alcoholi consumption by inbred mice have shown ' substantiall strain differences in appetite, in behavioral responses and in, meta-` bolism. Analogous mouse studies of spontaneous nicotine and tobacco smoke ' intake have now been inaugurated by a Council grant to learn whether similarc genetic variations occur. a Twin Studies tary influences (even among twins reared apart), in determining initiation and ;: Identical twins provide the human~ counterpart of animals of inbred straini ~ Studies of all like-sexed twins in Sweden and! Finlan& are being assiste& by ~ The CounciP in an effort to determine broadly the relative influences of heredity ~ versus environment upon the incidence of atherosclerosis-based cardiovascular ~ diseases. A key strategy is to compare the incidence of such~ disDrders in smoking and nonsmoking identical twins, including symptomatology during life and, ~ eventually, age at death, the primary cause of decease and post-mortem evalua- tion of vascular pathology. Non-identical, like-sexed twin siblings serve as " controls. :. ;~ While the incidence of discordance in smoking practices is low among ' identical twins, an observation that in itself evidences a strong role of heredi- z~. maintenance of smoking; the numbers of discordants are great enough to accrue to increasingly significant levels in substantial twin populations followed over a sufficient period of time. These studies have now been extended beyond identical twins to other relatives of various defined degrees of consanguinity, such as half-sibs and the offspring of twins. These time-consuming, 'investigations will' require a considerable induction perio& before comprehensive new reports emerge. They may eventually provide more direct evidence whether cigarette smoking per se is a truly significant "risk factor" in these and perhaps some other diseases. They cannot, however, be expectedi to add greatly to elucidation of pathogenic processes or to ~ provide 6 V1 M M 0 e

207 strain. ed by nedity pscular Inoking e and, valua- rve as ,,. among heredi- n and accrue pd over beyond uinity, duction brovide pificant ~wever, `rovide I ~. methods usefuli in reducing risks due to biochemical aberrations imparted by the genes from ancestors. For this, other types of research are required. Cholesterol Metabolism Another prevalent "risk factor" in epidemiological studies has been "ele- vated serum cholesterol."' Contradictory findings over the years suggest that this concept; was an oversimplification, especially since many victims of heart attacks had no history of high cholesterol levels. That cholesterol is implicated in atherosclerosis has been assumed because mature arterial plaques contain the steroid both free and in combinations. Nevertheless, long-ter~m adminis- tration of drugs that lower serum cholesterol has not been as generally effective in arresting or reversing the process in man as had been hoped. Accumulating information about the states of combination of cholesteroli in blood is directing attention to the several cholesterol-containing combina- tions, classified as chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). The LDLs are the major carriers of' blood cholesterol and there are a number of experi- mental as well as epidemiological findings that an elevate& serum level of VLDL is correlated with progression of atherosclerosis, whereas elevated HDL is a favorable indication. Thus certain "lipoprotein profiles" are now considered by many to be better indicators of susceptibility to atherosclerosis than elevated totali ch& esterol. Certain~ of these profiles, associated; with very high susceptibility to disease, have been shown to be genetically determined. Barring some unforeseeable empirical discovery (which does occur im medical research and is a perennial hope), the rational route toward effective control is through systematic study of the pathogenesis of atherosclerosis, at biochemical' and physiological levels. This obviously must include a better understanding of the regulat'ion of synthesis, transport, function, and elimin• ation of cholesterol and of the aberrations in disease in the hope of altering these by targeted'treatment. The Council is presently reviewing the most promising concepts and leads that now exist in this complex field with the intent of increasing its support of basic study of athererosclerosis. The intent is not only to assist these im- portant developments, but also to seek more directly relevant assay systems for assessing the possible effects of cigarette smoke inhalation. The task is additionally difficult because the atherosclerotic disorders appear to be peculiarly human ones with few counterparts among animals under natural conditions. Highly contrived manipulations to produce animal "counterparts" tend to diminish probability of relevance to human experience. Human studies have been impeded by the lack of non-intervention tech- niques for assessing initiation and progression of the atherogenesis processes in the vasculature of man. New experimental techniques for visualizing the condition of the arterial walls, including scintillation photography, appear to show promise for the future. 7 1

208 In Vitro Studies of Arteries and Veins Meanwhile, in vitro techniques provide one method of studying synthests :~ and uptake of lipids an& cholesterol by human arteries an& veins, both _-` " " and atherosclerotie inct comparison with those of animals , dire. -, normal Though in vitro conditions cannot be made to duplicate the in vivo situation ~ perfectly, they may provide useful guides to in vivo research. Such studies have already been made with CTR support as.deseribed in the Council's Annual Reports for 1972-1975. It has been reported (and is reiterated in a current review) that human atherosclerotic and normal coronary arteries as welli as saphenous veins, perfuse& under pulsatile arterial pressures with human plasma containing labeled cholesterol an& acetate, do not synthe= size cholesterol from acetate and produce only small amounts of' cholesterol esters. Cholesterol is taken up in identical amounts by normal and diseased vessels and this uptake is increase& at higher pulse pressures, which seems consistant with the reputed effects of hypertension. Labeled acetate is incor- porate& into various lipids. Both normal and diseased coronary arteries and saphenous veins synthesize free fatty acids, triglycerides and phospholipids.' Addition of nicotine to the perfusion fluid did not influence cholesterol uptake. Analogous experiments with dog arteries appeared to shown an in- fluence of nicotine upon cholesterol uptake. If this is confirmed, it suggests caution in extrapolations from this species to man (1972' report). Carbon monoxide in the perfused plasma was reported' to enhance chol; esterol uptake by all arteries in this in virro system. Several' new studies report a dramatic inhibition of cholesterol uptake by `.' both~ human and animal arteries, under these in vitro conditions, by 7-keto-q~ cholesterol. : Living rabbits also showed am inhibition of cholesterol uptake, but to a much smaller extent. The low solubility of the 7-keto compound; imposed t technical problems. Improved techniques may increase the efficiency of the; ' effect. Meanwhile, the disparity, between the in vitro an& whole animal re-T sponses is another reminder that extrapolations must be cautious. ~A ° Other current studies deal with elucidation of possible mechanisms bf_` the inhibition and with the metabolic fate of the injected 7-keto compound iA; rabbits. Role o f Smooth Muscle Cells in Atherosclerotic Plaques The heretofore prevalent theory of atherosclerotic plaque formation posti-l-lated that the infiltration of fatty substances from the bloodstream into tbd ` arterial wall gives rise to cholesterol deposits that act as an irritant, causinr inflammation and proliferation of cells by processes akin to ordinary healiny This "insudation"' concept was consistent with the associations of atherosclet F, osis with elevated blood cholesterol, high blood pressure and high fat diets.. Animals fed large amounts of saturated fats and cholesteroli sometimes s* plemented with~ hormonal or other treatments, developed lesions superficiMresembling those observed in man at autopsy: These lesions often regressod in animals when the diets were altered, an observation, that stimulated hum22 dietary studies involving restriction of! cholesteroli and saturatedl fat consump'. 6 cc VE m ia ht is Ce sn to vc m m 01 tl rf 0 si st b, ai li1 ce th w cc in e% m E th hZ c r 0 s

ibed and' ron ressurt synthe~ leste ise see in ies xolipids: :)lesterd an ta~ ugge n postu- into the causing healing. TF, eroscler- '* ~t diets.~ es sup- _ ~~erficiallyregressed ` 4 human "` ~- ~onsump- _` 209 tion. Complicated by difficulties of control, the results in man have been equivocal. Recent electron microscopic studies of human arterial' plaques have re- vealed that the major component' of authentic plaques is proliferating smooth muscle cells like those normally composing the center (medial) layer of arter- ial walls rather than fibroblast cells such as proliferate to heal a wound. Early human arterial lesions (streaks) contain little lipid, suggesting that insudation is not the prime factor in typical atherosclerosis. Cholesterot deposits and cellular debris appear later. Present debate centers on what incites normali smooth muscle cells to migrate from the mediali layer and what causes them to proliferate. Presumably, damage to the inner surface cell' layer of the vessels is implicated and a number of theories as to how this endothelial layer may become damaged have been advancedi Many of the plaques generated in animal arteries by non-physiological manipulations are reported to be radically different in composition from those of genuine human atherosclerosis and their relevance to the human diseases is therefore questionable. Recognition of the difference has, however, led to reports that certain animals can develop human-type atherosclerosis under other more appropriate conditions. Smooth muscle cells from primate arteries are now being maintained successfully in culture media. Low-density lipoproteins added to the media stimulate them to multiply. Further, it has been reported that the ever-present blood platelets, normally involved in the clotting process in response to injury an& im other, functions, also secrete a substance that strongly stimulates pro- liferation of these smooth muscle cells. Damage to endothellal and' intimal cell layers of an artery, which ordinarily separate the blood from contact with the smooth muscle cells of the medial layer, can bring these cells into contact with~ platelets and presumably incite multiplication in the artery wall. Another concept holds that the smooth muscle cells multiply as the consequence of a mutatiom akin to that which transforms other normal cells into malignant ones. This "monoclonaV' theory„ which is supporte& by striking evidence but is nevertheless in some dispute, would suggest quite different mechanisms of action by external agents than those described heretofore. Endothelial Cells and Blood Platelets A single layer of endothelial cells composes the thin membrane lining the inner surfaces of arteries, performing, an important function in retaining the red corpuscles while allowing water and some other substances to pass through. As mentioned, damage to the endothelium is believed to contribute to all three of the mechanisms of atherosclerosis described. Many possible causes of endothelial damage have been suggested andl are being studied. It is possible that severali may be involved. The damaged endothelium can repair itself, rather slowly, but if damage is sustained or repeated too often, repair may not be achieved. Clots (thrombi) forming in an artery are thought to injure the endo- thelium by pressure and by impeding the access of oxygen. The blood platelets have long been• known to play a role in the complex events that produce