The Evolution of the Discipline of Vascular Biology
The changes in vascular research over the past half century have been nothing short of extraordinary. From the evolution in technologies to the analytical process, advances that were not even imaginable 50 years ago have led to far-reaching insights into the biology of the blood vessel and the diseases that affect it.
This period has also witnessed two major shifts in the general experimental paradigm of vascular biology. At mid-century, vascular biologists used physiological methods to attempt to understand phenotype. Their primary focus was on vascular tone and its neurohumoral determinants in health and disease. Limited by available methods, they were essentially unable to apply biochemical or molecular analysis to the characterization of vascular function. That situation, however, changed approximately 25 years later by which time modern techniques of cell culture were applied to vascular cells, and microanalytical methods, as well as novel reagents, became available for more precise molecular characterization of vascular phenotype. Of note, both of these historical phases of research in vascular biology used the conventional analytical regimen of reductionism: an investigator typically identified a vascular reflex or endothelial product of primary interest, asked a scientific question about the role of this reflex or molecule on vascular phenotype, designed experiments and controls that focused on that question, then drew inferences from the observed experimental result—without regard for other factors in the vessel that might influence the observed phenomenon.
In the past decade, another major shift in the investigative paradigm has been developing as a result of access to large molecular data sets. The human genome project, together with expression array analysis, proteomics, and metabolomics, has begun to yield a rich treasure trove of data that offers the promise of great insight into understanding vascular biology and pathobiology. Yet, we are plagued by the limitations of conventional reductionist analysis applied to this complex data set.
Using a reductionist approach to the analysis of complex systems violates Einstein’s dictum that “things should be made as simple as possible, but not any simpler.” As gratifying and useful as reductionist biology is in identifying particular determinants of mechanism, this standard analytical paradigm by its very nature fails to take into account that biological systems are complex systems in which single parameters cannot conceivably change in isolation. The constancy of controlled conditions is an artificial experimental construct that, while essential for conventional scientific investigation, sorely limits one’s ability to appreciate the nuanced responses of a system in which a single perturbation begets a network response from a host of interacting elements.
Armed with genomic and proteomic technologies and using the methods and language of the evolving fields of bioinformatics, nonlinear dynamics, and mathematical modeling of complex systems, we can now actually consider developing a complete in silico model of the vasculature. The model will include not only a complete description of vascular phenotype in the steady state but also changes in the complete array of expressed genes, vascular proteins, and their metabolic products in response to an environmental or genetic perturbation. This kind of analysis, defined as systems biology, will afford us a method to link molecular biology to systems physiology, restoring our focus on physiological vascular phenotype, but now doing so by viewing the vasculature as a complex biological system governed by molecular mediators whose identity, regulation, and modulation can be precisely quantified.
Let me offer an example of this historical evolution that bears some personal connotations. We begin by reviewing the articles published in the first edition of Circulation Research. Among these are included several classical studies on cardiovascular hemodynamics, as well as cardiovascular neural reflexes. Most interestingly from my personal perspective is an article by Lanier and colleagues1 that examines the arterial vasodilator responses to several vasoactive agents, including methacholine. In this study, the authors showed that methacholine causes “marked vasodilation” in the isolated femoropopliteal vascular bed in dogs; yet, they, as many investigators thereafter, did not appreciate that another endogenous mediator was important for the response. The mechanism for this effect and the identity of that endogenous mediator did not become known for another 30 years, by which time endothelium-derived relaxing factor was identified as nitric oxide (NO) by Ignarro and colleagues.2 As Thornton Wilder pointed out, “[s]ome things are just too big to be seen,” and NO is, indeed, one huge biological effector whose footprints were scattered across the vascular landscape for many years, but whose identity eluded the investigative community.
This simple molecule has played a key role in my own scientific career, and it was not until I remembered an experience I had as a graduate student at the University of Pennsylvania that I realized how much it has crossed my path. At that time, the biochemistry department’s library was down the hall from the laboratory in which I worked, and I spent not an inconsiderable number of hours working at a desk in that quiet place throughout my student days. Scattered about the walls of the library was a collection of photomicrographs of crystals of hemoglobin taken by David Drabkin, a former professor at Penn who developed a biochemical reagent for measuring hemoglobin that bears his name. One of the forms of hemoglobin that he crystallized and photographed was nitrosylhemoglobin, whose crystals were striking for their brilliant hue. That particular photomicrograph hung just above the desk at which I preferred to sit. Little did I know at that time how much the simple molecule responsible for the striking color of the crystals would consume my interests and energies 10 years later.
My studies of the vascular effects of NO began as a cardiology fellow working in a hematology laboratory. I had developed an interest in thrombosis at a time when most cardiologists had little interest in the area. The seminal article on the role of thrombosis in acute myocardial infarction by DeWood and colleagues3 was not published until 1980, and it took a couple of years for the cardiology community to accept the importance of this pathobiology in coronary heart disease. I realized only later that this new-found focus on the role of thrombosis in atherosclerotic disease represented reacceptance of what earlier cardiologists clearly knew. A review of the 1949 edition of Friedberg’s Diseases of the Heart, the first major cardiovascular text published in the United States, showed that acute myocardial infarction was defined as coronary thrombosis.4 In fact, some of the very earliest clinical trials in cardiovascular disease performed in the 1950s involved the use of anticoagulants for the treatment of this acute coronary thrombosis. Owing to inadequate control of the degree of anticoagulation achieved at that time, bleeding complications were excessive, leading to a general reluctance to apply this therapy broadly. In the 1960s and 1970s, β-blockers and calcium channel blockers became available for the treatment of patients with ischemic heart disease, and focus switched from thrombosis to vasospasm as a key pathophysiological determinant of acute coronary syndromes. The angiographic demonstration of fresh coronary thrombus in patients with acute coronary syndromes by DeWood and colleagues,3 coupled with the subsequent development of recombinant tissue plasminogen activator for use in acute myocardial infarction,5 led to widespread acceptance of the importance of thrombosis in the pathobiology of coronary heart disease, and fueled the rapid development of the antithrombotic therapies central to contemporary treatment of acute coronary syndromes.
As a young investigator who had just finished clinical training in cardiology, I was struggling with how best to justify my existence as someone interested in thrombosis within a cardiovascular division in this transitional era. For this reason, I began to examine the role of nitrates on platelet function, and, finding no real effect at pharmacologically relevant concentrations, reviewed earlier work by Needleman who showed that the vasorelaxing effects of nitrates could be potentiated by thiols.6 Taking a cue from this earlier work, I showed that thiols potentiated the antiplatelet effects of nitrates and went on to demonstrate that this effect was, in part, a consequence of the metabolism of organic nitrates to S-nitrosothiols.7 This novel class of NO donors served as a major focus of our laboratory for a number of years. When endothelium-derived relaxing factor was identified as NO,2 we went on to demonstrate that S-nitrosothiols are important endogenously synthesized NO derivatives that stabilize NO and facilitate its transport and transfer within and between vascular cells.8,9 In addition and importantly, we demonstrated that S-nitrosation represents a form of posttranslational modification of proteins, altering protein function and cell phenotype.10,11
NO is a reasonably stable free radical that can react with a host of other molecules normally found within the vascular compartment, including molecular oxygen and superoxide anion. These reactive oxygen species served as another important focus of our explorations into the vascular actions and metabolism of NO. Molecular and genetic models of vascular oxidant stress were used to demonstrate its importance in regulating the bioavailability of NO in the vasculature and to determine the benefits of specific endogenous and exogenous antioxidant mechanisms in maintaining NO’s bioactivity. Deficiencies of cystathionine-β-synthase, causing hyperhomocysteinemia12; glucose-6-phosphate dehydrogenase13; and glutathione peroxidase-314 have all been shown to promote vascular oxidant stress and, thereby, limit the bioactivity of NO. In a series of studies over several years, we have shown that a deficiency of one of these antioxidant enzymes, glutathione peroxidase-3, leads to platelet-dependent arterial thrombosis.14,15 This work is notable in that it demonstrates for the first time in a convincing experiment of nature that oxidant stress in the vasculature limits bioavailable NO and can, thereby, lead to a thrombotic disorder. Thus, we have come full circle, from illustrating the antiplatelet effect of organic nitrates to demonstrating that endogenous NO has important antiplatelet effects in vivo. Equally importantly, we determined the molecular basis of an important pathobiological phenotype, ie, a state of vascular NO insufficiency caused by a genetically determined deficiency of a key extracellular antioxidant enzyme.
As gratifying as this work has been, it suffers from its reductionist underpinnings by failing to take into account the consequences of other key reactions of NO in the vasculature on the antiplatelet effects of NO. To show that endogenous NO is less bioavailable when an extracellular enzyme that reduces lipid peroxides and hydrogen peroxide is deficient tells us nothing about the precise biochemical mechanism for this impairment. What are the sources of reactive oxygen species, and which are most important? Why does the intracellular isoform, glutathione peroxidase-1, not limit the transcellular flux of reactive oxygen species? And how does the complex series of other key reactions in which NO can participate change in response to this limiting extracellular antioxidant enzyme? The answers to these questions can only come from a rigorous quantitative analysis of the entire reaction network, which requires a knowledge of the principal enzyme and nonenzyme sources of reactive oxygen species, their steady-state concentrations and kinetic constants, and the regulation of the genes that code for expression of these other enzymatic determinants. There are now a growing number of mathematical approaches for analyzing complex nonlinear systems, and these approaches will afford us the opportunity to characterize the vasculature as an NO generator quite precisely. Importantly, many of these approaches do not require precise knowledge of every kinetic constant or every reactant’s concentration. Rather, the system can be perturbed in a precisely defined way and changes in those network components whose concentrations can be measured then monitored16; by this perturbation analysis, the network can be defined with an incomplete set of initial conditions.
This systems biology approach will undoubtedly serve as the basis for future investigations in vascular biology and will lead to specific questions that can then be asked more rationally with conventional reductionist approaches. As astonishing as it may seem, we are on the verge of the very real possibility of understanding the vasculature as a biological system in its entirety, which promises to offer unique insights into mechanisms of disease and therapeutic strategies. The next 50 years should prove to be at least as exciting as the last 50 years for those of us working in this rapidly evolving discipline.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
The author wishes to thank Stephanie Tribuna for expert technical assistance.
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