Download From Better Understood Pathogenesis to Superior Molecular

JACC: CARDIOVASCULAR IMAGING
VOL. 1, NO. 3, 2008
© 2008 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION
PUBLISHED BY ELSEVIER INC.
ISSN 1936-878X/08/$34.00
DOI:10.1016/j.jcmg.2008.02.002
EDITOR’S PAGEiVIEW
From Better Understood Pathogenesis to Superior
Molecular Imaging, and Back. . .
Jagat Narula MD, PHD, FACC*
Editor-in-Chief, JACC: Cardiovascular Imaging
Vasken Dilsizian MD, FACC†
Associate Editor, JACC: Cardiovascular Imaging
olecular imaging is a unique research
discipline with the potential to detect
disease in pre-clinical and early stages.
It integrates fields of clinical medicine, cell biology, and signal transduction for appropriate selection of target to be imaged, and
synthetic chemistry and imaging sciences for
characterization of the pathology. In contrast to
traditional diagnostic techniques, molecular imaging employs noninvasive, quantitative, and repetitively feasible strategy of imaging targeted biological processes at both cellular and subcellular
levels within a living organism (1).
Due to recent advances in nanotechnology and
engineering innovations, molecular imaging has
undergone profound change during its rather
short development period as a scientific discipline.
By weaving medicine and computer technology
together, molecular imaging has uncovered simplicity behind the complexity of diseases. It has
offered quantitative assessment of pathophysiology, altered metabolic states, subcellular processes,
receptor modifications, and message or gene expression in various disorders. To effectively develop the concept of molecular imaging, it has become essential that we demonstrate the feasibility
of imaging in small transgenic animal models.
This has stimulated the development of advanced
small animal imaging technology including microscale positron emission tomography, singlephoton emission computed tomography, com-
M
*From the University of California Irvine, Irvine, California; and the
†University of Maryland School of Medicine, Baltimore, Maryland.
puted tomography, magnetic resonance imaging,
high-frequency ultrasound, and optical fluorochrome or bioluminescence imaging. It is expected that fusion technology involving computed
tomography, magnetic resonance imaging, or ultrasound imaging combined with nuclear or optical imaging would not only provide both morphological and functional information, but also better
localization of hitherto difficult foci of tracer
uptake.
It is exciting that the National Cancer Institute
has identified molecular imaging as 1 of the 6
most rewarding opportunities for development
(2), and a major thrust has been placed on molecular imaging in the newly formed National Institute of Biomedical Imaging and Biomedical Engineering. Although the National Heart, Lung, and
Blood Institute has yet to fully embrace this initiative, the field of cardiovascular molecular imaging has slowly but surely progressed. In addition
to addressing a diagnostic query, we strongly believe that targeting of key steps in cellular or subcellular biologic processes further clarifies critical
pathogenetic pathways. The pathophysiology of a
disease process and development of targeted imaging are interdependent and complementary. Review of the historical evolution of any molecular
imaging strategy should substantiate our claim.
Let’s consider the imaging of apoptosis in myocardium. It becomes amply clear that targeting
of a cell membrane phospholipid, phosphatidyl
serine (PS, a hallmark of apoptosis), not only allowed development of a diagnostic tool, but has
also led to better understanding of reversibility of
JACC: CARDIOVASCULAR IMAGING, VOL. 1, NO. 3, 2008
MAY 2008:406 –9
apoptotic process, in turn expanding diagnostic
applications and proposing novel intervention
strategies.
Apoptosis, a programmed cell death process,
constitutes an integral part of the pathology associated with ischemic or inflammatory state of the
myocardium (3); it is believed to precede the necrotic change. It has been proposed that noninvasive imaging of apoptosis may allow early identification of myocardial damage and since damage
follows an ordained cascade, it may be amenable
to recovery. The apoptosis-inducing stimuli usually converge to release various mitochondrial proteins into a cytoplasmic compartment, which initiate a chain reaction to activate highly specific
proteolytic enzymes, referred to as terminal
caspases, such as caspase-3 (4). Activation of
caspase-3 is linked to the loss of asymmetry of
phospholipid distribution between the inner and
outer cell membrane layers, and has been exploited as one method of apoptosis imaging. PS,
which is normally restricted to the inner membrane tablet is exteriorized to the outer cell membrane layer, and has been targeted by appropriately labeled annexin A5 (AA5) for in vitro and
in vivo imaging; AA5 is a normally circulating
protein and has a nanomolar affinity for PS. Elegant cell culture experiments had paved the way
for initial imaging of apoptotic cells, and a radiolabeled AA5 has been used clinically for molecular imaging of apoptosis in myocardial infarction
and transplant rejection (5–7). As expected, the
AA5 uptake was confined to the coronary distribution territory in the infracted myocardium,
which corresponded to the perfusion deficit. On
the other hand, a diffuse or multifocal uptake was
observed in the inflammatory state not limited to
a coronary distribution. As postulated by the basic
science tenets, use of broad caspase inhibitor,
ZVAD-fmk, not only restricted the extent of
myocardial injury verified by annexin uptake, it
also delayed the onset and slowed the rate of occurrence of myocardial damage in experimentallyinduced murine myocardial infarction (8 –11).
These preclinical studies have demonstrated a direct correlation between the extent of apoptosis
defined by AA5 uptake and the tell-tale histologic
characteristics, thus establishing grounds for potential clinical trials (11).
Narula and Dilsizian
Editor’s Page
When these studies were underway, it was becoming clear that apoptosis precedes onset of necrosis and, therefore, pre-necrotic ischemic state
should already be associated with PS externalization. As such, AA5 imaging was attempted in the
brief ischemic episodes with the premise that
AA5 imaging would permit positive imaging of
inducible ischemia in a myocardial perfusion negative zone (12). Experimental evidence confirmed
AA5 uptake and biochemical activation of
caspase-3, but was not verified by histologic evidence of either apoptosis or necrosis. Ultracentrifugal separation of myocardial homogenates
surprisingly recovered radiolabeled AA5 from the
cytoplasmic compartment suggesting the internalization of PS-bound AA5. Subsequently, a series
of experiments revealed that PS exteriorization
occur within 5 min of ischemic insult, which is
preventable by preadministration of caspase inhibitors (13). In the absence of caspase inhibitors, PS
remains expressed on the outer cell membrane up
to at least 6 h after resolution of brief ischemia,
potentially facilitating after-the-fact imaging of
ischemia. The PS externalization was found to be
a continuous process until caspase-3 activation
spontaneously resolved. The PS-bound AA5 trimerized between 10 to 20 min and invaginated a
membrane patch into the cytoplasm as a vesicle
and removed the PS from surface; persistent expression of PS would have constituted an “eatme-signal” for phagocytosis of the doomed cell. It
becomes much clearer from the aforementioned
description that whereas bench-side cues are important for development of molecular imaging,
the observations made by molecular imaging
equally and substantially contribute to a better
understanding of the disease process.
The story goes a bit further into apoptosis imaging of the myocardium. A slow and smoldering
apoptosis occurs in remodeling myocardium and
contributes to an inexorable decline in ventricular
function in heart failure (HF). Cytokine and/or
oxidative stress results in release of cytochrome c
from the mitchondria into cytoplasmic compartment, and consequent activation of pro-apoptotic
caspase-3 results in myofilament proteolysis and
DNA fragmentation (14). However, simultaneous
upregulation of antiapoptotic factors, such as
BCl2- and XIAP-like proteins, and loss of DNA
407
408
Narula and Dilsizian
Editor’s Page
JACC: CARDIOVASCULAR IMAGING, VOL. 1, NO. 3, 2008
MAY 2008:406 –9
Table 1. Evolution of Molecular Imaging of Apoptosis in Myocardium
Disease
Process
Pathogenetic Basis of Imaging
Imaging Results
Imaging Leading to Pathogenetic Clues
Myocardial
infarction
Apoptosis and necrosis in the
areas of myocardial damage
(1)
AA5 uptake observed in the areas
corresponding to perfusion deficit (5)
Caspase inhibitors resolve AA5 uptake (8,10), decrease both
apoptosis and necrosis (9), establish apoptosis-necrosis
continuum, extend window for intervention (8)
Myocardial
ischemia
Apoptosis precedes necrosis (1)
AA5 uptake observed in ischemic area not
associated with histoloically-verified
apoptosis or necrosis; AA5 recovered
from cytoplasm (11)
Transient PS expression and AA5 uptake, AA5 removes PS from
membrane by invagination as a protective phenomenon (13),
caspase inhibition hastens recovery
Heart
failure
Incomplete apoptosis contributes
to ventricular dysfunction
(3,4,15)
In worsening HF patients AA5 uptake is
associated with poor outcome (16)
Simultaneous upregulation of pro- and anti-apoptotic factors
establish the basis of incomplete apoptosis in heart failure
and suggest novel avenue for reverse remodeling (4,16)
fragmentation factors prevent completion of apoptotic process (apoptos is interruptus) (4,15). The
amount of activated caspase-3 is determined by
the balance of antiapoptotic and proapoptotic factors. Because caspase-3 is activated in HF, it was
proposed that the attendant PS externalization
should be amenable to AA5 imaging in HF (1).
The AA5 uptake was observed in up to 45% of
patients with worsening HF, who went on to
demonstrate a 10% decrease in their ejection fraction after 1-year follow up. On the other hand,
the remaining patients with negative scans improved LV function by 7% in the follow-up. The
results suggested that fewer the endogenous antiapoptotic factors, the greater the residual
caspase-3, the more PS externalization, the higher
likelihood of an AA5-positive scan, the poorer
the prognosis, and the greater the necessity for
supportive therapy. It is evident from the foregoing discussion that AA5 imaging has contributed
immensely to our understanding of the intricacies
of heart muscle cell damage in various cardiovascular diseases (Table 1).
The convergence of disciplines in medicine,
molecular and cellular biology, and computer
technology will likely translate into development
of better diagnostic and management strategies. It
is important that we strive to understand the
pathogenesis of various cardiovascular disorders.
Identification of the worthy targets which are
prominently overexpressed in the diseased tissue
would provide a high target-to-background contrast for hot-spot imaging. Alternatively, identification of moieties which are conspicuously absent
from the target tissue would allow a cold-spot
pattern of imaging. Molecular imaging is not limited by imaging modality. Once a target and targeting agent is identified, the imaging can be performed by conjugation of the imaging probe with
appropriate reporting tracer and the target should
become amenable to respective modality of imaging. In this issue of the JACC: Cardiovascular Imaging, one of the papers proposes the imaging of
angiotensin-II type 1 receptors, which is likely to
uncover susceptibility to the development of adverse myocardial remodeling and HF (17). The
paper (17) and the editorial comment (18) are
linked to CVN interviews that highlight the
role that molecular imaging will play in clinical
medicine. It is mandatory that an effort be
made to express the conceptual premises and
practical execution in a simplified manner so as
to stimulate close collaboration among the clinicians, biologists, and imagers alike. Molecular
imaging teaches us that targets are a plenty in
health and disease, each with a story to tell.
Imaging and imagers are the future story tellers, the Rosetta stone of pathophysiology and
advanced therapeutics.
Happy targeting, folks!
Address correspondence to:
Jagat Narula MD, PhD, FACC
Editor-in-Chief, JACC: Cardiovascular Imaging
3655 Nobel Drive, Suite 630
San Diego, California 92122
E-mail: [email protected]
JACC: CARDIOVASCULAR IMAGING, VOL. 1, NO. 3, 2008
MAY 2008:406 –9
REFERENCES
1. Wu J, Narula J. Molecular imaging and future of nuclear cardiology.
In: Braunwald E, Dilsizian V, Narula J. Atlas of Nuclear Cardiology.
2nd edition. Philadelphia, PA: Current Medicine, 2005;253–76.
2. National Cancer Institute: The Nation’s
Investment in Cancer Research for Fiscal
2004. Available at: http://plan.cancer.gov/
discovery/imaging.htm.
3. Narula J, Haider N, Virmani R, et al.
Apoptosis in cardiomyocytes in endstage heart failure. N Engl J Med
1996;335:1182–9.
4. Narula J, Haider N, Arbustini E,
Chandrashekhar Y. Apoptosis interruptus in heart failure and functional
reversibility. Nature Cardiovasc
Med (Clinical Practice) 2006;3:
681– 8.
5. Hofstra L, Liem IH, Dumont EA,
et al. Visualisation of cell death in
vivo in patients with acute myocardial infarction. Lancet 2000 Jul 15;
356:209 –12.
6. Narula J, Acio ER, Narula N, et al.
Annexin-V imaging for noninvasive
detection of cardiac allograft rejection.
Nat Med 2001;7:1347–52.
7. Blankenberg FG, Katsikis PD, Tait
JF, et al. In vivo detection and imag-
ing of phosphatidylserine expression
during programmed cell death. Proc
Natl Acad Sci U S A. 1998 May 26;
95:6349 –54.
8. Dumont EA, Reutelingsperger CP,
Smits JF, et al. Real-time imaging of
apoptotic cell-membrane changes at
the single-cell level in the beating
murine heart. Nat Med 2001;7:
1352–5.
9. Yaoita H, Ogawa K, Maehara K, Maruyama Y. Attenuation of ischemia/
reperfusion injury in rats by a caspase
inhibitor. Circulation 1998;97:276 – 81.
10. Chandrashekhar Y, Sen S, Anway R,
Shuros A, Anand I. Long-term
caspase inhibition ameliorates apoptosis, reduces myocardial troponin-I
cleavage, protects left ventricular function, and attenuates remodeling in rats
with myocardial infarction. J Am Coll
Cardiol 2004;43:295–301
11. Sarai M, Hartung D, Petrov AD, et
al. Broad and specific caspaseinhibitor induced acute repression of
apoptosis in atherosclerotic lesions
evaluated by radiolabeled annexin A5
imaging. J Am Coll Cardiol 2007;50:
2305–12.
12. Petrov A, Acio ER, Narula N, et al.
Sarcolemmal phosphatidyl serine expression in ischemic myocardial syndromes can be detected by 99mTc-
Narula and Dilsizian
Editor’s Page
annexin V imaging. Circulation 2000;
102:II544.
13. Kenis H, van Genderen H, Bennaghmouch A, et al. Cell surface-expressed
phosphatidylserine and annexin A5
open a novel portal of cell entry. J Biol
Chem 2004;279:52623–9.
14. Communal C, Narula J, Solaro J,
Hajjar RJ. Functional consequences of
caspase activation in cardiac myocytes.
Proc Natl Acad Sci USA 2002;99:
6252– 6.
15. Narula J, Pandey P, Arbustini E, et al.
Apoptosis in heart failure: release of
cytochrome c from mitochondria and
activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci
USA 1999;96:8144 –9.
16. Kietselaer BLJH, Reutelinsperger
CPM, Boersma H, et al. Noninvasive
detection of programmed cell loss
with 99mTc-labeled annexin A5 in
heart failure. J Nucl Med 2007;48:
562–7.
17. Verjans JWH, Lovhaug D, Narula N,
et al. Noninvasive imaging of angiotensin receptors after myocardial infarction. J Am Coll Cardiol Img 2008;
1:354 – 62.
18. Greenberg B. Noninvasive molecular
imaging of the remodeling heart: the
next step forward. J Am Coll Cardiol
Img 2008;1:363–5.
409