National Heart, Lung, and Blood Institute
Working Group on Atheroprotective Genes
National Institutes of Health
Center Bethesda, MD
March 29, 2000
TABLE OF CONTENTS
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.
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".
- 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.
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;
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;
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;
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
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