It is a privilege and honor to be selected by the American Society of Pharmacology and Experimental Therapeutics for the Torald Sollmann Award. I wish to take this opportunity to comment on my experiences over the last 40 plus years as both a student and a faculty member. I was trained in pharmacology as well as medicine with the goal of entering an academic career that would permit me to engage in teaching and research. The subject of my presentation focuses on the events that helped to shape my career. Attention is given to those who made it possible for me to advance in my learning, teaching, and research. In addition to having been taught by excellent instructors, I have benefitted from having many outstanding undergraduate and graduate students, as well as postdoctoral fellows, without whom much of what I feel I have accomplished would not have been possible. Being surrounded by a supportive environment and accompanied by bright and eager young students gives me reason to look toward the future with enthusiasm. I chose to take the time and space allotted to me to present a brief overview of where I have been and how many individuals played important roles in helping to achieve my goals. In essence, this composition is a tribute to my family, professional associates, and current and former students.
My interest is in the futurebecause I am going to spend the rest of my life there Charles Kettering
It is an honor to be selected as the recipient of the 2001 Torald Sollmann Award and to have the opportunity to appear before the distinguished members and guests of the American Society of Pharmacology and Experimental Therapeutics. I thank the members of the selection committee for this recognition and the Wyeth-Ayerst Pharmaceutical Company for their generous support of the award.
Torald Sollmann had a dominant role in fostering pharmacology as a specialized medical discipline, as well as being one of the original organizers of ASPET and becoming its second president. He received his medical degree from Western Reserve and remained at that institution for fifty years. He served as Professor and Chairman of Pharmacology for 40 years and was concomitantly appointed as Dean for 16 years. Dr. Sollmann is noted for his exceptional textbook, Manual of Pharmacology, with the 8th and final edition published in 1958. In addition, he authored or coauthored over 500 publications, truly an outstanding record of scholarly achievement. His record of accomplishment was consistent with his belief that the future of pharmacology depended upon imaginative persons dedicated to hard work and self-development.
A Moment of Reflection
Occasions such as this cause one to pause and wonder, Why me? Why now? Am I deserving of this award? I would be remiss, therefore, if I did not take a moment to acknowledge the fact that many individuals, far too numerous to mention, had a profound influence in developing my career by providing opportunity and support during the past 50 years. First and foremost, I give recognition to my wife, Diana, whom I met in 1951 on the first day of college and finally convinced her to marry me in 1957. Shortly thereafter, she journeyed with me from New York City to start a new life in Ann Arbor, Michigan. She has been a source of strength and encouragement as well as a mother of five children, who developed into fine adults, married, and presented us with eight grandchildren.
Of course there are the many teachers who provided instruction, guidance, and support during those early years. Time does not permit me to give deserving recognition to all, but I am compelled to mention several. There is my first teacher of pharmacology, Alfred E. Livingstone, Ph.D. Dr. Livingstone, although retired from Temple University Medical School, was called from retirement when our regular instructor was inducted into the military during the Korean War. Dr. Livingston introduced me to the excitement associated with the study of drugs and the laboratory approach to research. He encouraged me to seek a graduate degree in pharmacology, but only after I had obtained a graduate degree in Physiology. I followed his advice, and after completing my Bachelor of Science degree, I enrolled as a graduate student at St. John's University where I studied under Dr. Daniel Lilly, Chairman of the Biology Department. I was a teaching assistant in the morning, a student in the afternoon, and a thesis researcher during the evening and early morning hours. I completed my thesis (Purine Metabolism in Stylonichia pustulata) in May 1957. Throughout the 6 years of attending St. John's University, I traveled each morning and night between the borough of Queens and the borough of Brooklyn by bus and subway; a round trip of more than 4 hours. During those commutes, I had the chance to read Goodman and Gilman's textbook three times from cover to cover.
Upon completion of my graduate degree in Physiology, I approached Dr. Livingston once again for advice regarding the choice of a graduate school. He suggested Georgetown University and the University of Pennsylvania. I asked his opinion on the University of Michigan, however, since he often made reference in class about the work on narcotic analgesics being done by Dr. Maurice Seevers. After a moment of hesitation, he implored me not to go to Michigan. When I asked for his reason, he replied, “Once you go to Ann Arbor, you will never leave”.
I made my decision and applied to the University of Michigan Horace H. Rackham School of Graduate Studies. Dr. Livingston's admonition still rang clear in my memory, and I accepted the opportunity to enroll at the University of Michigan. One thing is obvious, Dr. Livingston was correct. I can honestly say that I never entertained serious thoughts of leaving the University of Michigan and consider it an honor to have had the opportunity to be a student, as well as a faculty member, for the past 43 years.
The next person to impact my career development was Maurice Seevers, M.D., Ph.D., Professor and Chairman of Pharmacology. I recall my first meeting with Dr. Seevers as I prepared to begin my graduate studies at Michigan. He was an impressive figure seated behind the heavy wooden desk on which there was a skull of a rhesus monkey. I did not dare to ask the significance of that artifact. Dr. Seevers reviewed my academic record from St. John's University and noted that I had an advanced degree in Physiology, along with a strong background in Biochemistry. He outlined a course of instruction, which included medical school courses in Histology, Embryology, Neuroanatomy, Statistics, Pharmacology, and Pathology. I also was advised to audit the courses in Biochemistry and Physiology. It was customary in the late 1950s for graduate students in Pharmacology to take the same courses offered to medical students, with the exception of Gross Anatomy and Psychiatry, during their first two years. In addition, graduate students were required to take at least one graduate course each term outside of the Department of Pharmacology, as well as the graduate courses offered by the Pharmacology faculty members. Along with the course requirements, graduate students were rotated among the laboratories in the Department and participated in the teaching of medical students. In the third and fourth graduate years, teaching assignments included assisting in the medical student laboratories two times a week. I had the privilege of taking courses of instruction provided by persons known to many of us; names such as Lauren Woods, Edward Domino, Ted Brody, Donald Bennett, Lloyd Beck, Ted Carr, Henry Swain, and Edward Cafruny are but a few who influenced my development. In addition, I became acquainted with fellow graduate students who went on to launch their own distinguished careers. Classmates such as Duncan McCarthy, Michael Brody, Thomas Baum, Dean Calvert, Charles Ross, Glen Kiplinger, Ken Moore, JoAnn Moore, and Ben Zimmerman are just a few of the peer group who inspired me with their valuable insights.
Pharmacology and the Race into Space
Finally, I was assigned to work under the supervision of Harold F. Hardman, M.D., Ph.D., an association that had a lasting influence on my approach to research and learning. Working with Dr. Hardman was rewarding, although the classified nature of the project kept me from knowing exactly why we were studying a compound referred to only as EA1476. This oily, viscous substance was essentially insoluble in water, and it was my task to formulate the substance so that it could be administered by intravenous injection. My experience from pharmacy school in preparing emulsions helped me with this task. Once accomplished, we began dosing animals with EA1476 and observed a dramatic pharmacological event in which the animal (dog) essentially was put into a state of “suspended animation”. The task given to the investigative team was to find the antidote that would restore the animal to its normal state. The reason that the United States Government and the military wanted this information remained a mystery for many years until the project was later declassified.
On October 4, 1957, the Soviet Union stunned the world when Sputnik rode into orbit on a ballistic missile. The first artificial satellite had been launched. Its two radio transmitters sent “beeping” signals to the world as it orbited the earth. By American standards, Sputnik was huge; at 184.3 pounds, it was 20 times larger than the satellite the U.S. was attempting to launch. A month later, the Soviet Union compounded U.S. uneasiness by launching Sputnik II with a live dog named Laika as a passenger. America's failure to launch its small satellite in December 1957 further emphasized the magnitude of the Soviet accomplishment.
In the early days of the space race, the goal was to land on Mars. However, the United States lacked rockets of sufficient power to carry astronauts and the necessary life support equipment to great distances. Therefore, to reduce the necessary life-support payload, it was considered desirable to have a “drug” that would place the astronauts into a state of suspended animation that could be reversed by ground command upon the administration of an antidote. EA1476 was such an agent, if only an antidote could be found. This was the goal of our highly classified project.
Dr. Edward Domino and Dr. Harold Hardman discovered that the catatonic state induced by EA1476 was reversible by the intravenous administration of dextroamphetamine. Immediately, the animal could be restored to a fully functional state. The event made a lasting impression upon the new graduate students. My fascination with drug actions and mechanisms was reinforced. Subsequently, the project was declassified and it was made known to us that EA1476 was a potent derivative of delta-9-tetrahydrocannabinol. A detailed report of the study was the subject of a presentation at a New York Academy of Sciences Symposium (Domino, 1971) as well as in Pharmacological Reviews (Hardman et al., 1971).
The Introduction of Dichloroisoproterenol
The pharmacology of the first β-adrenoceptor blocking agent was published in 1958 (Powell and Slater, 1958), followed shortly thereafter by the classic publication describing the cardiovascular effects associated with β-receptor blockade (Moran and Perkins, 1958). These events provided an opportunity to engage in a new and rapidly developing area of cardiovascular pharmacology.
At the time, I was working in Dr. Hardman's laboratory on a project involving the effects of ionization on drug actions. Although interesting, my attention was being drawn to the study of β-adrenergic receptor blocking agents.
I expressed my interests to Dr. Hardman. He was understanding and agreed I could pursue my own interests in the evening while I assisted with the studies on drug ionization in the daytime. He allotted a budget of $800 to support my studies. I became totally absorbed in my research and was able to demonstrate that dichloroisoproterenol (DCI) possessed antiarrhythmic properties independent of its ability to block the cardiac β-adrenergic receptor. My desire to continue the studies with DCI required that I obtain an additional research supply of the drug. Regrettably, the Eli Lilly Company did not share my enthusiasm, and the decision was made not to continue research on β-adrenoceptor blocking agents.
Fortunately, Imperial Chemical Industries had undertaken efforts to develop β-adrenoceptor blocking agents. It was fortunate that Dr. James Black (now Sir James Black) presented a seminar in the Department of Pharmacology in which he described the basic pharmacological properties of pronethalol and propranolol. I had the opportunity to show Dr. Black my data on DCI. To demonstrate that the antiarrhythmic action of this class of compounds was independent of receptor blockade, I requested and received a research supply of the respective dextro- and levo-optical isomers. The experimental data supported the hypothesis that both drugs possessed antiarrhythmic properties separate from their ability to inhibit cardiac β-adrenoceptors (Whitsitt and Lucchesi, 1967). Submission of our findings to the American Heart Journal was received with a total lack of enthusiasm. The Editor commented, “The drug you describe (propranolol) is of little interest to clinicians and your paper would be better suited for a specialty journal”. TheJournal of Pharmacology and Experimental Therapeuticsaccepted an earlier manuscript despite one of the reviewers recommending rejection (Lucchesi and Hardman, 1961). The journal's Editor, Dr. Neil Moran (Chairman, Department of Pharmacology, Emory University), instructed me to disregard the major objections since he had conducted similar studies and confirmed my results. This served as an important lesson and one that had a lasting influence on my behavior as an editor.
Propranolol was the first β-adrenergic receptor blocking agent to be approved by the U.S. Food and Drug Administration with the primary indication being that of an antiarrhythmic agent. My studies with this class of compounds formed the basis of my doctoral dissertation, which was completed in December of 1960, followed by my graduation in May of 1961.
My study of β-adrenoceptor blocking agents would not have been possible had it not been for Dr. Hardman, who allowed me to pursue my dream. I learned early in my career that when one comes upon a new finding, the excitement of discovery is a driving force. To this day, I try to maintain Dr. Hardman's approach in training graduate students and postdoctoral fellows. The pursuit of one's own findings is the most effective way of encouraging enthusiasm and a determination to seek new knowledge.
In the fall of 1959, Dr. Hardman left the University of Michigan. After his departure, I was asked to meet with Dr. Seevers, who offered me a half-time position as an Instructor in the Department of Pharmacology. This appointment came with the understanding that I would apply to medical school, a totally unanticipated sequence of events, but an opportunity I could not resist. So it was that I began my medical school studies in September of 1960. However, having taken essentially the first two years of medical school courses as part of the Ph.D. curriculum, I was given credit for those courses and only was required to take Gross Anatomy and Psychiatry over the next two years.
During this time, I was able to complete my dissertation and submit my first NIH grant, entitled “Analysis of Antiarrhythmic Agents”. Through grant support from the NIH-HLBI, the American Heart Association, and many pharmaceutical companies, cardiovascular research and training of graduate students and postdoctoral fellows in my laboratory has been on-going continuously for the past 38 years. Ten years of NIH support was through a Merit Award, the importance of which should not be underestimated. The ability to conduct research with the guarantee of a period of uninterrupted funding provided an opportunity to “follow the science”, as opposed to following the latest trends and fads in basic research. Long-term NIH-HLBI funding provided an opportunity to develop a number of experimental models embraced by many investigators in cardiovascular pharmacology. These are programs that we all must actively protect.
Long Days and Short Nights—The Right Place and the Right Time
In June of 1962, I began my third year of Medical School. The next two years were the most taxing in terms of workload. In addition, my wife had given birth to our first child, Tom (November, 1959), our daughter, Mary (September, 1961), and was expecting our third child, Richard, (born 12:45 AM, January 1, 1963). The clinical years, along with the responsibility of conducting my research and tending to my teaching duties, were demanding. However, the excitement of the laboratory and the care of patients and clinical use of pharmacological interventions kept me infused with a desire to continue.
During my third year in medical school, I had the good fortune to be under the guidance of Dr. Richard Judge and Dr. Thomas Preston, cardiologists with degrees in electrical engineering. It was a time when there was a growing interest in developing implantable devices for pacing the human heart. Having a research laboratory at my disposal provided an opportunity to work with my instructors in developing the first pacemaker produced by the General Electric Company, along with an external device used to interrogate the implanted unit. This was an example of translational research in which basic animal laboratory studies evolved to the point of applying the knowledge to patients. My first clinical research experience produced personal satisfaction in knowing that patients benefited from our efforts. An added outcome of the study was the observation that certain pharmacologic factors are capable of causing measurable changes in the threshold energy requirements of the human heart. These studies were made possible by the development of an external device for making repetitive measurements of myocardial threshold and interelectrode impedance in patients with implanted pacemakers (Preston et al., 1967). Also important is that student and teacher, along with industry, worked together with each contributing their respective talents toward achieving a common goal.
My Full-Time Appointment
I completed my medical school training in June 1964 and began full-time employment as an Instructor in the Department of Pharmacology. It was about this time when the Surgeon General of the United States was expressing concern over the health hazards associated with the use of tobacco and tobacco products. Dr. Seevers was appointed to chair the committee on Tobacco and Health. The American Medical Association-Education Research Foundation (AMA-ERF) agreed to fund studies to examine the potential adverse effects of tobacco on health.
Dr. Seevers, himself a smoker (cigars), immediately issued orders that smoking would not be permitted in the Department. Furthermore, he was instrumental in removing cigarette vending machines from the student study areas, as well as prohibiting the sale of tobacco products in the University Hospital. Dr. Seevers led a one-man crusade against what is now recognized as a major public health hazard.
As a result of his long time interest in drug-related physical dependence, Dr. Seevers suggested I prepare a grant proposal to the AMA-ERF. The primary focus was to examine the hypothesis that nicotine was addictive or could lead to the development of physical dependence. The study involved the recruitment of healthy male and female volunteers over 18 years of age. I came to realize that the only way the study could be done was to conduct the investigation in a double-blind manner and to engage the subjects in an activity unrelated to smoking. It is important to note that the study was done before the establishment of current guidelines requiring informed consent.
During the screening interview, we observed whether or not the applicant possessed a packet of cigarettes or engaged in smoking during the interview. Once it was observed that the person was a tobacco user, he/she was sent forward for a complete medical examination and, if found to be in good health, was offered an opportunity to participate in the study. At no time were the subjects made aware of the nature or intent of the study or the substance to be administered. However, they were informed that they had been exposed to the “drug” on previous occasions.
Research subjects were asked to come to the laboratory at 7:00 AM. They were to have abstained from food, caffeine, alcohol, and tobacco from 7:00 PM of the preceding day. All personal belongings were removed, and the subjects were dressed in surgical scrub suits and placed in a soundproof chamber with a one-way window allowing observation from the other side. An intravenous line was placed in a hand vein, electrocardiographic leads were attached, and a blood pressure cuff was placed on the subject's forearm. After obtaining preliminary recordings of vital signs, subjects were given a standard breakfast, reading material of their choice and, upon their request, were permitted to smoke if they so desired. However, we provided the brand of cigarettes.
The door to the soundproof chamber was closed, and for the next 8 hours the subjects were engaged in a battery of psychological tests. At the appropriate time in the protocol, the subjects received either a placebo intravenous infusion of 0.9% sodium chloride solution for injection or an intravenous infusion of nicotine in a dose equal to that obtained by smoking one cigarette. The intravenous infusions were administered in a pulsed fashion to mimic the manner in which nicotine would be absorbed during smoking. The placebo or nicotine infusions were repeated every 30 min with each administration lasting 15 min. The question to be answered was simple: Would the exogenous administration of nicotine reduce the smoking frequency in a totally naı̈ve subject who otherwise had no intent in discontinuing the use of tobacco? The result of the study was surprising. We were able to demonstrate for the first time that nicotine administered systemically would reduce the frequency of smoking, thereby providing evidence to support the belief that the alkaloid produced a state of physical dependence. The study was published in the Journal of Clinical Pharmacology and Therapeutics (Lucchesi et al., 1967) and formed the basis for the subsequent use of nicotine patches and chewing gum for people who wanted to stop smoking.
I am forever indebted to Dr. Seevers for having provided me with this opportunity. When I entered the study, I was unaware that this would be the first time anyone had administered intravenous nicotine over an extended period to human subjects. Had I known that nicotine had never been administered in this manner, I certainly would have given more consideration to withdrawing from the idea. However, Dr. Seevers mentored us along the way. He always remained in the background, allowing Bob Schuster and me to share the credit for having conducted the first study demonstrating that nicotine is addictive.
Study of Antiarrhythmic Agents—Prevention of Sudden Cardiac Death
In 1982, my laboratory developed a canine model of sudden cardiac death used to detect the antifibrillatory and profibrillatory actions of new pharmacological agents (Patterson et al., 1982). The canine model provided definitive evidence for the antifibrillatory properties of bretylium (Anderson et al., 1980a, 1982b; Holland et al., 1983;Gibson et al., 1983) and led to a clinical trial in collaboration with my former graduate student, Dr. Eugene Patterson, and Dr. Jeffrey Anderson from the Division of Cardiology (Anderson et al., 1980b, 1981,1982a). The experimental model also proved valuable in providing the first demonstration of the proarrhythmic potential of flecainide (Kou et al., 1987; Lynch et al., 1987), predating the now infamous Cardiac Arrhythmia Suppression Trial (CAST Investigators, 1989; Ruskin, 1989).
Experimental Coronary Artery Thrombosis and its Prevention
The second experimental model developed in my laboratory was that of spontaneous arterial thrombosis resulting from arterial wall injury, which serves as a clinically relevant model for the assessment of antithrombotic and fibrinolytic agents (Romson et al., 1980). The experimental model was instrumental in the preclinical development of abciximab (ReoPro) (Mickelson et al., 1989; Rote et al., 1994). The study provided opportunities to interact with colleagues in cardiology and to participate in the first clinical trial demonstrating the efficacy of a new class of antiplatelet agents now known as glycoprotein IIb/IIIa receptor antagonists. The utility of the experimental model attracted numerous visitors, as well as industrial contracts for the study of antiplatelet and fibrinolytic agents. In addition, many students and postdoctoral fellows benefited as a result of the research projects involving the study of a class of pharmacologic agents that had yet to become approved for clinical use. My involvement in the area of antithrombotic drugs proved an exhilarating experience. As an aside, my first attempt to obtain NIH support for development of the canine model of arterial thrombosis met with rejection. This serves as an example of persistence being rewarded.
Events from the Past—All Over Again
While conducting studies on the coronary circulation, we made an interesting observation in which reperfusion of the canine heart, after a period of regional ischemia, was accompanied by immediate tissue injury. The event brought to mind a day in 1964 when a patient was undergoing repair of an aortic valve. The procedure was done while the heart was in arrest and globally ischemic, thus requiring the surgeon to proceed with the utmost speed. All went well until restoration of the coronary blood supply to reperfuse the heart. To my astonishment, the heart went into contracture and did not recover its function. This event had been described in the literature as the “stone heart phenomenon”. The mechanism, however, went unexplored. Unexpectedly, we were able to replicate the incident in the canine heart.
Examination of the literature in 1964 provided no insight into understanding the relationship between myocardial ischemia and reperfusion. However, a colleague from the Department of Biological Chemistry suggested the event was most likely related to “peroxidation of lipid membranes”. Beyond that, my colleague was unable to offer further input, so I was forced to “go with the data”. Subsequently, a proposal was submitted to the NIH in which I hypothesized that the reintroduction of oxygenated blood to the previously ischemic heart would lead to injury and cell death. I referred to this phenomenon as “reperfusion injury” and attributed the tissue injury to formation of oxygen free radicals most likely derived from invading neutrophils and the associated inflammatory response (Romson et al., 1983). The NIH “pink sheet” summary began as follows: “This is a naı̈ve concept… ”. The rest of the review and the ultimate decision are self-evident. My rebuttal resulted in a site visit that failed to convince the reviewers that the concept of oxygen free radical-induced reperfusion injury deserved study. My own graduate students and some of my faculty colleagues voiced skepticism and refused to engage in or collaborate on the project.
At the time, Stanley Jolly, Ph.D., who completed his doctoral studies under the guidance of Dr. Harold Hardman in Milwaukee, was a research fellow in my laboratory. Stan was given the charge of testing the free radical hypothesis in reperfusion injury. His skillful techniques and brilliant experimental protocol proved the correctness of the hypothesis. The paper was published in Circulation Research(Jolly et al., 1984), and the observations were confirmed by independent studies from investigators at Johns Hopkins University (Ambrosio et al., 1986). Resubmission to the NIH for the grant entitled “Pharmacologic Protection of the Ischemic Myocardium” resulted in funding for 17 consecutive years.
The project on reperfusion injury was the one in which I found the most excitement and personal satisfaction. Initially, it was dealing with the concept about which many expressed doubt and for which initial funding was unavailable. Perhaps it was the challenge that forced me to continue. Unlike members of a Study Section, I had the advantage of having made the laboratory observations and the conviction that what my students and I observed departed from the current understanding of the problem. Joseph Romson, Ph.D., a graduate student in my laboratory, confirmed the earlier report (Jugdutt et al., 1980) that ibuprofen reduced myocardial reperfusion injury (Romson et al., 1982a). Romson and others went on to demonstrate that the cardioprotection by ibuprofen was accompanied by impaired neutrophil infiltration upon reperfusion of the ischemic heart (Romson et al., 1982b). These observations were followed by studies demonstrating that antibody-induced neutropenia resulted in a reduction of tissue injury secondary to reperfusion (Romson et al., 1983). The findings related to ibuprofen were confirmed by others (Flynn et al., 1984) who, in addition, reported that ibuprofen, but neither aspirin, indomethacin, nor dexamethasone, reduced myocardial infarct size. However, an additional significant observation by the latter group went largely unappreciated. Ibuprofen, in contrast to other nonsteroidal antiinflammatory drugs (aspirin, indomethacin), was noted to inhibit complement (C5a)-mediated activation of polymorphonuclear leukocytes.
The findings from a number of laboratories provided compelling evidence that the polymorphonuclear neutrophil contributed to the progression of tissue injury. Therefore, we initiated studies directed at inhibition of neutrophil adhesion to the injured vascular wall. A monoclonal antibody (904) that binds to a leukocyte cell adhesion-promoting, heterodimeric glycoprotein (Mac-1; CD11b/CD18) was administered to open-chest anesthetized dogs 45 min after the induction of regional myocardial ischemia. Ischemia was produced by occluding the left circumflex coronary artery for 90 min, then reperfusing for 6 h. The mean size of the myocardial infarct was reduced 46% with anti-Mo1 antibody. The study demonstrated that administration of the anti-Mo1 monoclonal antibody after the induction of regional myocardial ischemia resulted in reduced myocardial reperfusion injury as measured by ultimate infarct size (Simpson et al., 1988). Our initial observation with the Mac-1 antibody was confirmed by other investigators (Litt et al., 1989) using antibodies directed against either the CD11b or CD18 components of the heterodimeric neutrophil adhesion receptor. The extravascular distribution of the inflammatory cells during early reperfusion occurs primarily in the border zone of the myocardium, thereby jeopardizing myocytes supposedly viable before instituting reperfusion. The localization of inflammatory cells in regions of otherwise viable tissue promotes extension of tissue injury dependent upon reperfusion and is independent of the ischemic insult.
Despite the evidence supporting neutrophil involvement in reperfusion injury, there were few studies indicating how neutrophils were directed to transmigrate into the reperfused myocardium. The role of the complement system in myocardial infarction had been demonstrated earlier in the rat heart subjected to permanent coronary artery occlusion (Hill and Ward, 1971). In the intact, blood-perfused heart, it was noted that in the absence of reperfusion, the membrane attack complex, C5b-9, accumulation occurs as a late event when most of the jeopardized myocardium has become necrotic. In the presence of reperfusion, however, the complement system is activated rapidly, and this could play a role in the pathogenesis of reperfusion injury (Mathey et al., 1994). Subsequent studies indicated that the lytic C5b-9 membrane attack complex leads to reperfusion injury in the early phase (30 min) of reperfusion, resulting in a larger infarct. After 2 h of ischemia, complement activation enhances the no-reflow phenomenon but no longer affects infarct size (Ito et al., 1996).
Recalling an earlier study (Flynn et al., 1984) in which it was suggested that ibuprofen prevented C5a-induced neutrophil activation, we advanced the hypothesis that the complement system was a primary mediator of reperfusion injury and that the complement proteins necessary for formation of the membrane attack complex could be derived entirely from the myocardium. The hypothesis was based upon published observations, as well as by the chance finding that reperfusion injury in an isolated, crystalloid perfused heart could be prevented if the heart had been exposed to heparin in vivo. A postdoctoral fellow in my laboratory, Gregory Friedrichs, Ph.D., subjected 49 nonhuman primate hearts to global ischemia for 30 min followed by reperfusion. Before removal of the hearts, the animals received either saline, aspirin, abciximab, hirudin, or heparin. All but six of the hearts developed contracture during the ischemic interval or upon reperfusion. Upon unblinding the study, it was ascertained that the six hearts surviving the procedure were obtained from monkeys that had received heparin during the early course of the experiment. Subsequent studies in the rabbit isolated heart confirmed the observations obtained in the nonhuman primate heart experiments (Friedrichs et al., 1994). The experimental results were unanticipated. However, it was known that heparin and related glycosaminoglycans had the ability to inhibit the complement cascade. Therefore, one way to explain the findings in the monkey isolated heart was to suggest that pretreatment with heparin prevented activation of the tissue-derived complement system and formation of the terminal lytic membrane attack complex. If true, then complement components must be present in the myocardial tissue or synthesized at the time of ischemia and/or reperfusion.
Dr. Patrick McGeer from the University of British Columbia, a noted authority on the complement system, agreed with my hypothesis. Dr. McGeer had data to show the microglia in the brain are able to synthesize all the complement proteins necessary for formation of the membrane attack complex (Walker et al., 1998). In a collaborative study, we used the reverse-transcriptase polymerase chain reaction technique to establish that the mRNAs for complement proteins C3 and C9 are expressed in rabbit heart. Rabbit liver, brain, spleen, and kidney also were shown to express C3 and C9 mRNAs. Western blotting established that the mRNAs in heart are translated into the corresponding proteins. Furthermore, the rapid up-regulation of mRNAs occurred in Langendorff-perfused rabbit isolated hearts subjected to ischemia and reperfusion. C3 mRNA always was expressed at higher levels than C9 mRNA, but C9 mRNA showed greater up-regulation under stress. Compared with levels in control hearts subjected to 5 min of normoxic perfusion, hearts subjected to 30 min of ischemia followed by 1 h of reperfusion had a 4.72-fold increase in C3 mRNA and a 19.5-fold increase in C9 mRNA. By contrast, C3 mRNA in hearts subjected to 3.5 h of normoxic perfusion showed no change, and those subjected to 3.5 h of ischemia showed only a 1.72-fold increase, whereas C9 mRNA levels increased by 5.17-fold after 3.5 h of normoxic perfusion and 12.5-fold after 3.5 h of ischemia. The results of this study demonstrated for the first time that heart tissue is capable of expressing genes and proteins of the complement system, although it is not yet known which cell types are responsible. The data further demonstrate that ischemia and reperfusion of the heart promotes a rapid up-regulation of the mRNAs encoding the complement proteins C3 and C9 and that the abnormal levels considerably exceed those of normal liver. These observations are consistent with the hypothesis that local production of complement proteins may contribute significantly to the degree of ischemic injury to the myocardium and that complement expression is augmented by reperfusion. Our data were in full agreement with those of others who indicated rapid formation of the membrane attack complex upon reperfusion (Mathey et al., 1994). The major difference is that our observations dispelled the assumption that the liver is the primary source of complement components. The data provide an opportunity to develop pharmacological agents targeting inhibition of tissue-derived complement proteins.
I consider myself to be fortunate to have been both a student and a faculty member at the University of Michigan Medical School. During the course of obtaining my Ph.D. and M.D. degrees, I was instructed by outstanding teachers. I am indebted to them for their patience and guidance and for sharing their knowledge so that I might grow and serve as a productive member of the Medical School Faculty and the scientific community. I am indebted also to the many graduate students and postdoctoral fellows who labored long hours conducting the studies often credited to me, but truly belonging to them. I have had the honor of teaching the following graduate students who received their Ph.D. degree while working in my laboratory: Janice L. Stickney, Leighton S. Whitsitt, Richard R. Dean, W. Mark Vogel, Frank J. Kniffen, Donald A. Kroll, Larry R. Bush, Eugene Patterson, Joseph L. Romson, Stephanie E. Mitsos, Paul J. Simpson, Jonathon W. Homeister, Kenneth S. Kilgore, Liguo Chi, Michael R. Gralinski, James L. Park, and Elaine J. Tanhehco; and the following students who received their M.S. degree: Robert Shivak, Robert J. Hodgeman, Timothy E. Lomas, Yuk-Ping Li, Brian T. Eller, William E. Burmeister, and Paul T. Hoff. I also have been fortunate to mentor the following postdoctoral research fellows from the United States: David D. Ku, John K. Gibson, Gregory A. Kopia, Stanley R. Jolly, Gup P. Curtis, William A. Schumacher, Joseph J. Lynch, C. Van Jackson, Jan M. Kitzen, Mark R. Cunningham, William E. Rote, Gregory S. Friedrichs, Ruth A. Washington, and Sonya D. Coaxum; and from other countries: Marcelo Medina, Volker Fiedler, Masaru Minami, Wolfgang Söhngen, Yasuo Tamura, Andrew C. G. Uprichard, Paul S. Satoh, Shawn C. Black, S. Oluwole Fagbemi, Dun-Xue Mu, Yuji Sudo, J. Nelson Abreu Rivera, Sam S. Rebello, Katsuharu Saito, Jinbao Huang, Terrance D. Barrett, and James K. Hennan; and finally the clinical research fellows: Allen Peterson, Jeffrey Murray, James Stewart, Michael Shea, Philip Kirlin, Harry Colfer, David J. Wilbur, Steven W. Werns, Steven D. Nelson, Judith K. Mickelson, George C. Li, Daniel G. Walsh, David Muller, and Peter S. Fischbach.
Last, I thank the National Institutes of Health-Heart, Lung and Blood Institute for providing 38 years of uninterrupted support and numerous challenges through the years. Perhaps this is the part I enjoyed most. I owe a special thanks to the many pharmaceutical companies for their support of fellowships and research contracts, without which the training of many students and postdoctoral fellows would not have been possible.
The measure of one's success goes beyond the number of publications or dollar value in grant support. The measure of success in an academic setting should not exclude the human factor as determined by the training of future academicians and scientists in the discipline of pharmacology. More than ever, the training of young scientists will require careful nurturing and guidance in the formative years and perhaps beyond. It is essential that those in a position of leadership be willing to set aside personal pursuits of scientific achievement and devote part or all of their efforts toward developing the next generation of scholars. Being a leader (Departmental Chairman) should be viewed as a full-time endeavor in which the major goal is to ensure the success of the discipline and the appropriate training of those who will inherit the responsibility for the development of new therapeutic advances in medicine. This is a lesson I learned early in my career and one I find comforting as I try to devote my activities toward encouraging students to develop a broad interest in the biomedical sciences, and to explore the wonders of human physiology and basis for disease with an emphasis on developing therapeutic interventions. This is what the discipline of pharmacology is about.
Before closing, I wish to cite from a monograph written by Dr. Maurice Seevers, and published by the American Society for Pharmacology and Experimental Therapeutics (Seevers, 1969). The following excerpts are taken from Chapter 8 in a section wherein the author reflects on the future of pharmacology as a medical discipline. Dr. Seevers writes
“If pharmacology is submerged it will be in institutions where the pharmacologist, even though medically trained, identifies pharmacology only in laboratory terms. It is not likely to happen where pharmacology occupies an important position in the basic and clinical teaching of medical students throughout their educational program; where clinical pharmacology conducts training programs at the postdoctoral level and is recognized as a bridge between general pharmacology and clinical medicine; where the clinical pharmacologist is trained in both; where he/she is formally and physically associated with both; where he/she interprets laboratory findings in clinical terms and serves as a coordinator in all things of a clinical-pharmacological nature. In the long run, it may be that this type of cooperative activity will be a principal reason why general pharmacology as an independent discipline will survive in medical schools.”
Dr. Seevers continues by stating
“Unless pharmacology continues to assume the role of principal interpreter of the effects of chemicals and drugs in man, it will have abandoned its heritage, since this is its only function that is unique.”
Send reprint requests to: Dr. Benedict R. Lucchesi, Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, Ann Arbor, MI 48109-0632. E-mail:
- American Society of Pharmacology and Experimental Therapeutics
- National Institutes of Health-Heart, Lung and Blood Institute
- American Medical Association-Education Research Foundation
- The American Society for Pharmacology and Experimental Therapeutics