Download In Vivo Detection of Cell Death in the Area at Risk in Acute

In Vivo Detection of Cell Death
in the Area at
Risk in Acute Myocardial
Infarction
J Nucl Med. 2003 Mar; 44(3): 391-6
報告者: 實習醫師 唐之航
指導者: 住院醫師 謝宏仁
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Apoptosis / Programmed Cell
Death
Apoptosis, a Greek word that describes
the process of leaves falling from trees or
petals falling from flowers
Coined by Kerr and colleagues in 1972 to
describe a form of cell death exhibiting a
distinct set of morphological features
Cell death that is a normal part of the life
of a multicellular organism
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Characteristics of Apoptosis
Chromatin condensation
Fragmentation of DNA into
oligonucleosomal fragments
Membrane blebbing and “boiling” (zeiosis)
Loss of membrane asymmetry leading to
the exposure of phosphatidylserine (PS)
Induced by stress or death signals (Fas
ligand)
Activate caspase pathway
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Measurements of Apoptosis
Annexin V assay: externalization of
phosphatidylserine (PS)
Electron microscopy: apoptotic cells
Immunohistochemistry: detection of
caspase cleavage in tissue sections
TUNEL [terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nickend labeling] assay: detection of DNA
fragmentation
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PS Externalization and Annexin A5
Imaging
Anionic phospholipids such as PS and phosphatidyl
ethanolamine are actively recruited to the inner leaflet of
the lipid bilayer by “translocase”.
“Floppase” actively pumps cationic phospholipids such
as phosphatidyl choline and sphingomyelin to the outer
leafle.
During apoptosis, translocase and floppase enzymes are
deactivated and the asymmetric pattern of distribution is
lost.
The random bidirectional distribution is further
exaggerated by activation of “scramblase” enzyme.
PS is then expressed on the cell membraneand detected
by annexin A5.
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Externalization of PS During
Apoptosis
Anionic PS, P ethanolamine↓
↑Cationic PC, sphigomyelin
Blankenberg et al., Nucl Med Comm 21 (3), 241-50
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Van Engeland et al., Cytometry 31 (1), 1-9
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Apoptosis
Necrosis
Patterns of
death
Cell size
Single cells
Groups of
neighboring cells
Swelling
Plasma
membrane
Preserved continuity
Blebbed
Phosphatidylserine
on surface
Chromatin:
Clumps &
Fragmented
Smoothing
Early lysis
Contracted
"Apoptotic bodies“
Swelling
Disruption
Nuclei
Organelle
shape
Shrinkage
Fragmentation
Membrane disruption
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Apoptosis
Mitochondria Increased membrane
permeability
Contents released into
cytoplasm
Cytochrome c; Apaf1
Structure relatively
preserved
Necrosis
Swelling
Disordered
structure
DNA
degradation
Fragmented
Diffuse & Random
Internucleosomal cleavage
Free 3' ends
Laddering on
electrophoresis
DNA appears in cytoplasm
Cell
degradation
Phagocytosis
No inflammation
Inflammation
Macrophage
invasion
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Introduction
Previous study showed the presence of
programmed cell death (PCD) in the hearts of
animals and humans after acute myocardial
infarction (AMI) by detecting PS with annexin A5
on necropsy.
99mTc-4,5-bis(thioacetamido)pentanoyl-annexin
A5 (BTAP-anxA5) (Apomate, Theseus Imaging
Corp., Cambridge, MA) was used to monitor the
cell death in human hearts noninvasively and
reconstruction of oblique images as commonly
done in cardiac perfusion studies.
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Study Aim
To demonstrate the area at risk in the acute
phase of MI using 99mTc-sestamibi (MIBI)
To localize uptake of 99mTc-annexin A5 in vivo in
the area at risk using 99mTc-BTAP-anxA5
To study 99mTc-annexin A5 activities in the
subacute phase of AMI
To determined the size of 99mTc-MIBI defects in
acute and subacute phases of AMI
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Materials and Methods
Nine patients (6 male, 3 female; mean age, 61 y;
range, 42–77 y) with primary AMI who presented
within 6 h of the onset of symptoms were
included in this study.
All patients underwent percutaneous
transluminal coronary angioplasty (PTCA) of the
infarct-related vessels (primary PTCA: 4; rescue
PTCA: 5).
In 2 patients, the 99mTc-annexin A5 study was
repeated in the subacute phase.
12
Time Line of the Study Protocol
Thimister et al., J Nucl Med. 44 (3), 391-6
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Patient Characteristics
Thimister et al., J Nucl Med. 44 (3), 391-6
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Results-1
All patients showed decreased uptake of 99mTc-MIBI in
an area corresponding to the location of the infarct as
diagnosed on electrocardiography but an increased
uptake of 99mTc-annexin A5 was seen indicating the
presence of cardiomyocytes with externalized PS in the
infarct area.
Best images were obtained 15 h after the infusion,
showing 99mTc-annexin A5 activity at the infarct site and
little 99mTc-MIBI rest activity (“MIBI ghost”).
SPECT reconstruction was difficult in the acquisitions 20
h after the injection because of advanced decay of 99mTcMIBI and 99mTc-annexin A5 activity.
15
Thimister et al., J Nucl Med. 44 (3), 391-6
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Results-2
99mTc-MIBI
imaging 5–19 d after onset of the MI
(n=7) still showed a defect, although much
smaller, corresponding to the area of increased
99mTc-annexin A5 uptake and no sign of
ischemia.
Quantification on 99mTc-MIBI SPECT pictures: all
patients on whom 99mTc-MIBI in both the acute
and the subacute phase was performed (n=6)
showed decreased 99mTc-MIBI defects on the
subacute phase images when compared with
the acute phase of 12% on average.
17
Thimister et al., J Nucl Med. 44 (3), 391-6
18
Results-3
99mTc-annexin
A5 uptake was reduced on the 4-d
images when compared with the initial images.
After 8 d, no 99mTc-annexin A5 activity was
present at the infarct site.
201Tl scintigraphy performed 1 mo after the acute
MI showed complete normalization of the
affected area (i.e., reversible lesions).
There was no 99mTc-annexin A5 activity present
in the myocardium of the patient studied 3 mo
after the MI onset.
19
Thimister et al., J Nucl Med. 44 (3), 391-6
20
Discussion-1
Apoptosis in AMI may cause cardiomyocyte dysfunction
and irreversible cell loss which can lead to the decline in
left ventricular contraction.
Better understanding of the cellular apoptotic pathway
may lead to the development of new therapeutic drugs
that might limit this cardiomyocyte loss.
Animal studies have shown that increased 99mTcannexin A5 uptake in transplanted hearts of rats
correlated well with the in vitro detection of apoptosis
using annexin A5 immunofluorescence microscopy.
Animal studies also indicated that ischemia of the heart,
followed by reperfusion, induces reperfusion injury with a
substantial loss of cardiomyocytes through apoptosis.
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Discussion-2
Combining 99mTc-MIBI and 99mTc-annexin A5 images
could well define the area at risk on the MIBI pictures.
Because of the storage of 99mTc-MIBI in the
mitochondrion of cardiomyocytes (and lack of
redistribution), imaging could be postponed until after
revascularization was achieved, which is advantageous
to the use of 201Tl-chloride for the detection of
cardiovascular perfusion defects.
A MIBI image of the heart could still be seen vaguely
(“99mTc-MIBI ghost-image”) in the unaffected myocardial
area on the 99mTc-annexin A5 scintigraphy.
This technique allowed better SPECT reconstruction and
much better localization of the 99mTc-annexin A5 activity
in the heart.
22
Discussion-3
Necrosis features were also reported in the
annexin A5–positive cardiomyocytes
More necrosis than apoptosis?
The decreased 99mTc-MIBI defect in the
subacute phase, when compared with the acute
phase of the MI, strongly suggests that at least
part of the myocardial 99mTc-annexin A5 activity
as present in the acute phase represent
potentially reversible myocardial cell damage
rather than necrosis.
Cardiac biopsy is more straightforward but not
ethically acceptable.
23
Narula et al., J Nucl Med. 44 (3), 397-9
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Discussion-4
Decreased 99mTc-annexin A5 activity in a patient
who was repeatedly studied 3 and 8 d after the
MI onset.
Because no activity remained after 8 d,
regeneration of myocardial cells might have
taken place, in which case PS expression is no
longer present.
This would imply either that the ischemic part of
the MI has been restored to viable tissue or that
cells have been removed as necrotic material.
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Discussion-5
The main clinical utility of this in vivo detection of
cell death with 99mTc-annexin A5 scintigraphy
would be the evaluation of new therapeutic
strategies that intervene in myocardial cell death,
namely, cell death inhibitors.
The application of this methodology may be
extended to any situation of progressive
myocardial dysfunction.
Performing 99mTc-annexin A5 scintigraphy will
soon be possible to provide more information
about the kinetics of the cell death process in
different clinical forms of myocardial disease.
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Conclusion
Apoptosis can be detected in vivo at the
infarct area.
The area at risk can be well defined with
this protocol.
The decrease in 99mTc-MIBI defect size in
the subacute phase of the MI further
suggests that in parts of the area at risk,
reversible myocardial damage rather than
necrosis is present in cardiomyocytes.
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Future Studies
Can ischemia be subsumed within the spectrum
of apoptosis?
Could annexin-positive myocytes constitute an
indication for intervention?
What is the temporal window for treating PSexpressing ischemic cells?
Is it possible to develop a model of annexin
uptake vis-a`-vis the extent of troponin leakage
as an index of reversibility?
Can inhibitors of apoptosis abrogate ischemic
injury beyond reperfusion?
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Prospects
If annexin positivity identifies a significant
fraction of salvageable myocytes, we can
expect a major shift in the management of
acute coronary syndromes, somewhat
similar to the one that began 2 decades
ago with the advent of thrombolytic
therapy.
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Thank You for Your Attention!
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