TABLE OF CONTENTS

• Background
• Introduction
• State of the Science
• Areas of Opportunity
• Summary of Recommendations
• References
• Roster
• Agenda

BACKGROUND

The National Heart, Lung, and Blood Institute relies upon the counsel of experts in academia and industry, soliciting their advice on a wide range of topics relevant to its mission. On March 29, 2000, an NHLBI Working Group Meeting on Atheroprotective Genes was held at the National Institutes of Health, Bethesda, Maryland.  At this one-day meeting, an expert panel considered the evidence in support of an atheroprotective gene hypothesis in the context of biomechanical paradigms of endothelial gene regulation and vascular homeostasis. The Working Group Participants (see attached Roster and Agenda) addressed the state of the science and provided a series of recommendations on research opportunities and challenges for the next decade. This document is a summary report of the Working Group's deliberations and recommendations.
 

INTRODUCTION

Atherosclerosis and its clinical complications, heart attack, stroke, and peripheral vascular insufficiency, remain a major cause of morbidity and mortality among women and men in this country. Multiple risk factors, both environmental and genetic, appear to be involved in the pathogenesis of this chronic inflammatory disease process of arteries. Various studies have pointed to the components of the blood vessel wall, in particular vascular endothelium, as the primary targets for certain biochemical factors. These components include oxidized lipoproteins associated with hypercholesterolemia, advanced glycation end products associated with diabetes and aging, elevated homocysteine levels associated with genetic metabolic abnormalities, as well as certain hemodynamic factors, such as the disturbed flows that are associated with lesion-prone arterial geometries. All are currently believed to play a central role in disease pathogenesis. However, given the multiplicity and chronicity of these various risk factors and the protracted time course and varying severity of disease progression observed in different individuals, the existence of "atheroprotective genes" whose expression might offset or ameliorate the pathogenesis of atherosclerosis has been suggested. This atheroprotective gene hypothesis states that the coordinated induction of a subset of endothelial genes (for example, anti-thrombotic, anti-inflammatory, and anti-oxidant genes) in response to certain stimuli such as biomechanical forces present in lesion-resistant arterial geometries, or gender-based differences in hormonal milieu, could be exerting a net vasoprotective effect, thus constituting a natural "anti-atherogenic" mechanism. Recent data obtained from high-throughput molecular genetic analyses support this hypothesis (Refs. 1-3).
 

STATE OF THE SCIENCE: Consideration of the Background, Supporting Data, and Implications of the Atheroprotective Gene Hypothesis

Blood vessels are highly adaptive biological entities that undergo remarkable structural and functional alterations such as changes in wall mass, cross-sectional area, and intrinsic reactivity in response to various physiological and pathological stimuli. Important examples include: developmental events such as the closure of the ductus arteriosus and the transition of the pulmonary vasculature from a low to high flow bed within minutes of birth; acute and chronic fluctuations in (patho)physiological demands, for example, aerobic exercise, hypertension, tissue ischemia, and zero-gravity; the surgical transposition of venous segments into a pulsatile, high pressure environment (saphenous vein-coronary artery bypass grafts); and the dramatic local deformation of major arteries, with lipid and scar tissue accumulation, that is the hallmark of atherosclerotic plaque formation.

As the lining of the cardiovascular system, vascular endothelium plays a pivotal role as a sensor, transducer, and primary integrator of both humoral and biomechanical input stimuli in various adaptive responses, and is itself the locus of early dysfunctional changes. Loss of endothelial-derived relaxation factor (EDRF) response and expression of pro-inflammatory and pro-thrombotic activities that contribute to disease initiation and progression illustrate the multiple roles of the vascular endothelium. Interactions with other cells of the blood vessel wall including smooth muscle, pericytes, and recruited leukocytes, as well as with circulating blood elements (lipoproteins, platelets, leukocytes) are integral to these adaptive and non-adaptive responses.

The current state of understanding of the cellular and molecular mechanisms of vascular adaptation can be conceptually schematized as a series of "(patho)physiological balances" (see below), comprised of multiple effector molecules. Often these are characterized as having mutually antagonistic actions. For example, pro-thrombotic activities (tissue factor elaboration and the generation of thrombin or the diminished production of inhibitors of platelet activation such as nitric oxide and prostacyclin) are balanced by the anti-thrombotic activities (thrombin neutralization via thrombomodulin, surface ADPase expression, or enhanced tPA production). The set-point of these various balances determines local vascular responsiveness for the parameter in question, and their disregulation can contribute to disease initiation and progression.

The identification of these (patho)physiological balances implies the existence of counter-regulatory mechanisms that are invoked by various potentially injurious or physiologically disruptive stimuli and serve to mediate return to a normal range of structure and function. The evolution of these fundamental mechanisms of vascular homeostasis most certainly antedates atherosclerosis as an arterial disease process, in that the latter is limited largely to humans and subhuman primates, and does not exert much selective pressure during the reproductive age of a given individual. Thus, the basic process is perhaps better termed "vasoprotection", and the putative candidate genes, "athero/vaso-protective genes".

Perhaps the most striking evidence to date for the existence of "athero/vaso-protective genes", derives from the study of the geometric pattern of the lesions of atherosclerosis in the arterial tree of humans and various experimental animals. Regardless of species, gender, risk factor profile, dietary or molecular genetic manipulation, the earliest lesions of atherosclerosis are limited to certain arterial geometries - branch points, bifurcations, and areas of major curvature, which have been termed "lesion-prone" areas. Conversely, other geometries in the same individual - unbranched, uniformly tubular portions of arteries, often directly adjacent, remain relatively disease-free for prolonged periods despite exposure to the same systemic risk factor milieu. Recent molecular genetic strategies of analysis coupled with in vitro modeling of the local fluid mechanical environments of these distinct geometries, have suggested a potential explanation. Certain candidate "athero/vaso-protective genes" are stably over-expressed by the endothelium in response to the steady laminar shear stresses that are characteristic of lesion-protected geometries. These genes encode known effector molecules whose net biological functions are anti-thrombotic, anti-oxidant stress, anti-inflammatory, and pro-cell-survival. In contrast, these athero/vaso-protective genes are relatively under-expressed in lesion-prone geometries which do not experience steady laminar flow stimulation. High-throughput molecular strategies have also identified other novel candidate athero/vaso-protective genes, some of which encode cell surface receptors, intracellular signaling molecules, and transcription factors, whose functional roles in atherogenesis are currently under investigation (Ref. 2).

While the "athero/vaso-protective gene" hypothesis is strongly exemplified by this biomechanical paradigm of endothelial gene regulation, the concept clearly need not be limited to this cell type or form of input stimulus. For example, certain growth factors/cytokines may exert broad-reaching "vascular protective" effects beyond their ascribable functions as growth regulators (Ref. 4). Importantly, gender-based or other genetically determined biological differences in atherosclerosis susceptibility, may provide fruitful areas in the search for candidate athero/vaso-protective genes (Refs. 5,6).

Critical categories of vascular homeostasis supported by athero/vaso-protective genes include but are not limited to, anti-thrombotic, anti-inflammatory, anti-proliferative, anti-oxidant-stress, and pro-survival (anti-apoptotic) mechanisms. In addition to individual candidate genes that encode specific effector molecules such as the endothelial isoform of nitric oxide synthase, cyclooxygenase-2, various anti-oxidant enzymes and co-factors, and intrinsic down-regulators of cytokine activation pathways such as Smad-6,-7, the existence of programs of genes whose coordinate expression is regulated by one or more transcription factor/co-factor networks (Egr-1/NFk-B) needs also to be taken into account (Refs. 7,8). The latter may represent important loci of genetic regulation that may have global, as well as specific downstream effects.

AREAS OF OPPORTUNITY AND STRATEGIES OF APPROACH

The current working hypothesis of athero/vaso-protective genes has its origins in studies of the patterns of atherosclerotic disease expression and the present understanding of pathogenic mechanisms at the cellular and molecular level. Its further exploration will be greatly aided by the powerful tools afforded by modern molecular genetics, including genome-wide analyses of phenotypic expression and individual genetic variability, and the predictive power of population-based studies (Ref. 9). Thus, one can envision a multi-level, multi-disciplinary, integrative approach to candidate gene identification that would employ state-of-the-art transcriptional profiling technologies applied to both in vitro and in vivo model systems, as well as actual human tissue specimens obtained from various stages of disease, for example, dissected by laser-capture microscopy. Correlation of allelic (SNP) variation in candidate genes utilizing banked tissue samples from human subjects with defined disease outcomes, could lead to prospective studies, employing newly developed biomarkers of endothelial dysfunction in human subject populations at risk. Proof-of-mechanism studies would involve molecular manipulations (loss of function/over-expression) in appropriate murine or other animal models of atherogenesis. The final stages of hypothesis testing would entail appropriately designed clinical studies, again taking advantage of newly developed biomarkers, as sensitive indices of disease progression/regression. Thus, a full spectrum of basic to clinical and translational research activities is envisioned. The overall goal would be to gain fundamental insight into the natural mechanisms by which the vascular system maintains its integrity in health, and thus allow a more rational approach to vascular disease diagnosis, prognosis, treatment and ultimately prevention. Promoting the expression of athero/vaso-protective genes in individuals at risk for vascular disease would bring new meaning to the old adage--"An ounce of prevention is worth a pound of cure".

RECOMMENDATIONS

• Incorporate the ather/vaso-protective gene concept into existing and future programmatic efforts, especially the proposed NHLBI Programs for Genomic Applications, to harness genomics databases and technologies to specific studies of vascular disease pathogenesis.

• Encourage the linkage of basic, clinical and translational research efforts via the identification of biomarkers of vascular dysfunction as quantifiable, preferably non-invasive, indices of the failure of athero/vaso-protective mechanisms, that can be exploited for diagnostic, prognostic and therapeutic purposes. In particular, explore the usefulness of allelic variations in athero/vaso-protective candidate genes for risk stratification of individual subjects.

• Encourage the development of academic platforms for bioinformatics and database mining to enhance the accessibility to, and value-added feature of, these disease-targeted functional genomic efforts.

• Design and implement innovative approaches to cross-disciplinary training in this disease-targeted area. For example, a competitive cross-training opportunity at an established multidisciplinary center for a senior scholar or advanced postdoctoral fellow, who would in turn bring their new expertise to another institution.

• Convene a workshop with broad representation from the cardiovascular community to explore the concept of atheroprotective genes from various viewpoints, including basic mechanistic studies, as well as in vivo validation in animal models and human populations.

SELECTED REFERENCES

1. Topper JN, Cai J, Falb D, Gimbrone MA Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: Cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc. Natl. Acad. Sci. 1996; 93:10417-10422.

2. Gimbrone MA Jr. Vascular Endothelium, Hemodynamic Forces and Atherogenesis. (Commentary). Amer. J of Path. 1999; 155(1):1-5.

3. Davies PF, Polacek DC, Handen JS, Helmke BP, DePaola N. A spatial approach to transcriptional profiling: Mechanotransduction and the focal origin of atherosclerosis. TIBTECH 1999; 17:347-351.

4. Zachary I, Mathur A, Yla-Herttuala S, Martin J. Vascular Protection: A novel non-angiogenic cardiovascular role for vascular endothelial growth factor. Arterioscler Thromb Vasc Biol. 2000; 20: 1512-1520.

5. Libby P, Egan D, Skarlatos S. Roles of infectious agents in atherosclerosis and restenosis: An assessment of the evidence and need for future research. Circulation 1997; 96:4095-4103.

6. Shi W, Haberland M, Jien M-L, Shih, D, Lusis A. Endothelial Responses to Oxidzed Lipoprotiens Determine Genetic Susceptibility to Atherosclerosis in Mice. Circulation 2000;75-87.

7. Marx N, Sukhova GK, Collins T, Libby P, Plutzky J. PPAR activators inhibit cyotkine-induced vascular cell adhesion molecule-1 expression in human endothelial cells. Circulation 1999; 99:3125-3131.

8. McCaffrey TA, Fu C, Du B, Eksinar S, Kent KC, Bush H Jr, Kreiger K, Rosengart T, Cybulsky MI, Silverman ES, Collins T. High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. J of Clin. Invest. 2000; 105:653-662.

9. Lenfant C. NHLBI genomics initiatives. Looking beyond the human genome project. (Editorial). Circulation 2000; 101:468-469.

• Roster

• Agenda

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