From the Division of Cardiology, Department of Medicine (F.S.V., M.L.P.);
Division of Cardiothoracic Surgery, Department of Surgery (R.J.J., W.R.W.);
and Department of Cell Biology and Physiology and Center for Biologic Imaging
(S.C.W., S.M.A.), University of Pittsburgh, Pittsburgh, Pa; and Mallinckrodt
Medical, Inc, St Louis, Mo (S.K., G.H.B.).
Correspondence to Flordeliza S. Villanueva, MD, University of Pittsburgh, 200 Lothrop St, S568 Scaife Hall, Pittsburgh, PA 15213. E-mail villanuevafs{at}msx.upmc.edu
Methods and Results—A perfluorobutane gas–filled lipid-derived
microsphere with monoclonal antibody to ICAM-1 covalently bound
to the bubble shell was synthesized. Bubbles with either nonspecific
IgG or no protein on the shell were synthesized as controls. Coverslips
of cultured human coronary artery ECs were placed in a
parallel-plate perfusion chamber and exposed to 1 of the 3 microbubble
species, followed by perfusion with culture medium. Experiments were
performed with either normal or interleukin-1ß–activated ECs
overexpressing ICAM-1, and bubble adherence was quantified with
epifluorescent videomicroscopy. There was limited adherence of
control bubbles to normal or activated ECs, whereas a 40-fold
increase in adhesion occurred when anti–ICAM-1–conjugated bubbles
were exposed to activated ECs compared with normal ECs
(8.1±3.5 versus 0.21±0.09 bubbles per cell, respectively,
P<0.001). Although diminished, this difference
persisted even after perfusion at higher wall shear rates.
Conclusions—A gas-filled microbubble with anti–ICAM-1 antibody
on its shell specifically binds to activated ECs overexpressing
ICAM-1. Diagnostic ultrasound in conjunction with targeted
contrast agents has the unique potential to characterize cell
phenotype in vivo.
Cell Culture
Microbubble Preparation
Experimental Protocol
In a second series of experiments, normal (n=2 coverslips) or
activated (n=3 coverslips) ECs were similarly exposed to
anti–ICAM-1 microbubbles. Normal ECs were thereafter perfused for 4
minutes at a wall shear rate of 25 s-1, followed
by a 2-minute perfusion at 1000 s-1.
Activated ECs were perfused for 2 minutes at 100
s-1, succeeded by another 2-minute perfusion at
1000 s-1.
After each perfusion, 20 randomly selected fields (at x1000) were
interrogated with epifluorescent videomicroscopy, and the
number of bubbles and ECs per field was counted. Selected coverslips
were fixed in paraformaldehyde and prepared for
multicolor fluorescent microscopy.
Multicolor Fluorescent Microscopy
Flow Cytometry
Statistical Analysis
Figure 2
In the 3 coverslips with activated ECs exposed to
anti–ICAM-1 microbubbles and perfused sequentially at 100 and 1000
s-1, the number of adherent microbubbles
decreased from 2.6±0.3 to 0.8±0.4, respectively (P=0.04).
There was minimal adherence of the targeted microbubbles to normal ECs
on the 2 coverslips perfused successively at 25
s-1 (0.2±0.0) and 1000
s-1 (0.1±0.0, P=0.25). Despite the
relatively small number of coverslips tested, binding of anti–ICAM-1
bubbles appeared to occur preferentially to activated ECs
(0.8±0.4) compared with unstimulated cells (0.1±0.0) even at wall
shear rates of 1000 s-1 (P=0.08).
Flow Cytometry
Targeted Binding of Microbubbles
Our data also suggest that adherence may decrease at the higher
wall shear rates (1000 s-1) present in the
microvasculature. Nonetheless, the number of targeted microbubbles
binding to activated ECs is still greater than that binding to
normal cells perfused even at this high shear rate. Thus, despite the
high wall shear rates present at the microvascular level,
differential adherence of targeted bubbles to activated ECs can
occur. Importantly, at the wall shear rate of 100
s-1, which characterizes larger arteries, a
substantial number of targeted bubbles continue to adhere to
activated ECs.
Comparison With Previous Studies
Study Limitations
Potential Future Clinical Applications
Received December 12, 1997;
revision received April 27, 1998;
accepted May 6, 1998.
2.
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expression of a mononuclear leukocyte adhesion molecule during
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© 1998 American Heart Association, Inc.
Brief Rapid Communications
Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Preclinical
atherosclerosis is associated with increased
endothelial cell (EC) expression of leukocyte adhesion
molecules (LAMs), which mediate monocyte adhesion during atherogenesis.
Identification of cell-surface LAMs may uniquely allow assessment of
endothelial function, but there are no in vivo methods
for detecting LAMs. We tested a new microbubble designed to bind to and
allow specific ultrasound detection of intercellular adhesion
molecule-1 (ICAM-1).
Key Words: cells • microspheres • cell adhesion molecules • endothelium
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The earliest
atherosclerotic lesions involve monocyte adhesion to
endothelial cells, which is mediated by abnormal
endothelial expression of specific
LAMs.1 2 Upregulated LAMs on coronary ECs
are a specific indicator of incipient endothelial
disease, yet there are no methods to identify LAMs on EC surfaces of
intact, living organisms. We recently showed that interactions between
endothelial surfaces and albumin microbubbles
used in the imaging technique of myocardial contrast
echocardiography3 may yield
insight into endothelial
phenotype.4 The purpose of this
investigation was to develop a method that uses ultrasound to study
specific cellular features of coronary
endothelium in real time. In the present study, a
new ultrasound microbubble contrast agent conjugated to monoclonal
antibody to human ICAM-1 is shown to bind specifically to
activated human coronary artery ECs overexpressing
ICAM-1 in a perfused cell culture model.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Perfusion Chamber
A previously described parallel-plate perfusion
chamber4 was used to expose ECs to small volumes
of microbubbles within a closed system. Coverslips with confluent EC
monolayers were mounted in a 200-µm-high perfusion chamber. The
chamber entry port was used to introduce microbubbles onto the
endothelial surface. The exit port was connected via
Silastic tubing to a syringe pump (Harvard Apparatus) set
in the withdrawal mode to perfuse the chamber. The chamber was mounted
on an inverted epifluorescent microscope (Axiovert 35, Zeiss)
connected to a videocamera.
Human coronary artery ECs (Clonetics Corp) were
subcultured at 37°C on glass coverslips and grown to confluence over
4 days in 5% serum culture medium composed of
endothelial basal media (Clonetics). To create
inflammatory endothelium, 100 U/mL IL-1ß (Sigma) was
added to the cultures 4.5 hours before the experiment.
Perfluorocarbon gas–filled microbubbles were prepared
with monoclonal antibody on the shell as the ligand for EC binding.
Perfluorobutane (PCR Inc) was dispersed by sonication (XL2020
sonicator, Misonix) in aqueous medium containing phosphatidylcholine
(Avanti Polar Lipids), a surfactant, a phospholipid derivative
containing a carboxyl group, and a fluorescein derivative
of phosphatidylethanolamine (Molecular Probes) in a molar ratio of
75:15:7:1. The perfluorobutane was encapsulated during sonication by
a lipid shell carrying the fluorescent label. The carboxylic
groups were exposed to the aqueous environment and used for covalent
attachment of antibodies to the microbubbles as follows. First, unbound
lipid dispersed in the aqueous phase was separated from the gas-filled
microbubbles by flotation. Second, carboxylic groups on the microbubble
shell were activated with 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (Sigma), and antibody was then covalently attached via its
primary amino groups with the formation of amide
bonds.5 Unbound antibody was thereafter removed
by flotation. Three different microbubble species were produced,
containing either anti–human ICAM-1 IgG monoclonal antibody (Endogen),
purified nonspecific mouse IgG (Sigma), or nothing (plain), on the
shell. Microbubble diameter ranged from 1 to 10 µm, and
concentration was 108 to
109 bubbles/mL.
The EC coverslips were briefly exposed to
10-5 mol/L quinacrine dihydrochloride (Sigma) as
a fluorescent label and mounted in the perfusion chamber, and
the system was primed with culture medium. A volume of 0.2 mL of
microbubbles, equivalent to the volume of the chamber, was injected
into the system. Because of buoyancy typical of most gas-filled
microbubbles, the chamber was inverted, with the ECs forming the roof
of the chamber, to maximize exposure of cells to bubbles. After a
3-minute static exposure time, the chamber was perfused for 3 minutes
with bubble-free culture medium (25 s-1 wall
shear rate) to wash away unbound bubbles. Experiments were performed
separately with either normal or IL-1ß–activated
endothelium. Each coverslip was exposed to 1 of the 3
preparations: microbubbles with anti–human ICAM-1 antibody, or control
microbubbles with either nonspecific IgG or no added protein on the
shell (plain).
Coverslips were fixed in 2% paraformaldehyde,
washed in PBS, and permeabilized with 0.1% Triton
X-100 (Sigma) in 2% paraformaldehyde for 5 minutes. To
stain for F-actin, the coverslips were incubated with
rhodamine-conjugated phalloidin (Molecular Probes) for 30 minutes and
washed with PBS. To stain DNA, coverslips were incubated with Hoechst
dye (Sigma) for 30 seconds and washed with PBS. Phalloidin binds to
F-actin and was used to qualitatively indicate EC activation and define
cell boundaries. Hoechst dye was used to identify the nuclei. The
slides were mounted in Gelvatol (Monsanto) and placed onto a coverslip
for light microscopy (Nikon FXA microscope).
Flow cytometry was used to quantify cell-surface expression of
ICAM-1 as previously described.4 Briefly, EC
monolayers were treated with collagenase (0.2 mg/mL) and
2 mmol/L EDTA in PBS. The harvested cells were diluted with Medium
199 (Sigma), centrifuged at 250g for 6 minutes, and
the pellet was incubated with PE-conjugated anti–human ICAM-1 murine
monoclonal IgG (3.12 µg/mL, Immunotech) or nonspecific PE-conjugated
murine IgG (5 µg/mL, Becton Dickinson) for 20 minutes at
4°C. The cells were washed with PBS and fixed in 1%
paraformaldehyde in PBS. Samples were analyzed
for fluorescence (FacScan, Becton-Dickinson), and 5000 events
were captured for each sample (540 nm/L for PE).
Results are expressed as mean±SEM. Data were
analyzed with a 2-factor ANOVA with 2 levels of activation
(unactivated versus activated) in the first factor and
3 levels of bubble type (plain versus nonspecific IgG–labeled versus
anti–ICAM-1–labeled) in the second factor, with statistical
significance defined as P<0.05. When a significant
difference was found, comparisons were made by Student's t
test, with Bonferroni criteria applied for multiple comparisons and
statistical significance defined as P<0.01 (2-tailed).
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Data from 40 coverslips perfused at a wall shear rate of 25
s-1 are expressed in terms of total number of
adherent bubbles normalized to the number of cells observed per
coverslip and are summarized in Figure 1
.
Binding of all microbubble types to normal ECs was minimal. There was a
significant interaction between EC activation status and bubble type by
ANOVA [F(2,34) for interaction=3.65]. In post
hoc analysis it was found that, as predicted, a greater number
of anti–ICAM-1–labeled bubbles adhered to activated ECs
(8.0±3.5) compared with normal ECs (0.21±0.09, P<0.001).
In contrast, there was no difference in the number of "plain"
microbubbles adhering to normal (0.04±0.02) versus activated
(0.05±0.02) ECs (P=0.42). Similarly, bubbles containing
nonspecific IgG had no preference for activated (0.04±0.02)
versus normal (0.03±0.01) endothelium.
View larger version (29K):
[in a new window]
Figure 1. Adherence of 3 microbubble species to normal vs
activated ECs. Adherence increased when inflamed ECs were
exposed to microbubbles containing anti–ICAM-1. Adherence of other
types of microbubbles to normal cells was minimal and comparable to
that seen with activated cells. shows fluorescent
micrographs of ECs exposed to nonspecific IgG–labeled bubbles (2A and
2B) or anti–ICAM-1–containing bubbles (2C and 2D) under normal
conditions or after endothelial activation with
IL-1ß. EC nuclei appear blue, rhodamine-labeled F-actin filaments are
red, and microbubbles exhibit green fluorescence. Figure 2
shows no adherence of nonspecific IgG–conjugated bubbles to ECs under
basal (2A) or activated (2B) conditions. There is scant
adherence of anti–ICAM-1–containing microbubbles to ECs under basal
conditions (2C) and extensive adherence of these bubbles to
activated ECs (2D). There is dense rhodamine staining of
F-actin in the IL-1ß–stimulated cells, indicative of activation.
Figure 2E shows a single activated EC with multiple adherent
anti–ICAM-1 microbubbles.
View larger version (117K):
[in a new window]
Figure 2. Fluorescent micrographs of ECs after
exposure to either nonspecific IgG–conjugated bubbles (A and B) or
anti–ICAM-1–conjugated bubbles (C and D). Cytosolic F-actin is
stained with rhodamine-phalloidin (red), nuclei are stained with
Hoechst dye (blue), and microbubbles are labeled with
fluorescein (green). Microbubbles have lost their gas and
spherical shape during preparation for microscopy. A, Scale bar=10
µm. There was no adherence of nonspecific IgG–labeled bubbles to
normal (A) or activated (B) cells. Although small numbers of
anti–ICAM-1 bubbles adhered to normal ECs (C), numerous bubbles
attached to activated ECs (D). Note intense
rhodamine-phalloidin staining of F-actin indicative of activation (B
and D) in cells exposed to IL-1ß. E, Enlargement of a single
activated cell exposed to anti–ICAM-1 bubbles.
The relative distribution for ICAM-1 binding (number of
events versus fluorescent intensity) in normal and
IL-1ß–stimulated cells is shown in Figure 3, which indicates a shift in the extent
of ICAM-1 expression after IL-1ß stimulation. The percentage of ECs
expressing ICAM-1 above background (nonspecific IgG binding) was
increased (P<0.0001) after IL-1ß exposure (83±5%, n=4
coverslips) compared with baseline (39±10%, n=5 coverslips).
View larger version (25K):
[in a new window]
Figure 3. Flow cytometry indicating relative distribution of
ICAM-1 binding (number of events vs fluorescent intensity) in
(b) unstimulated and (c) IL-1ß–stimulated ECs. Background
nonspecific IgG binding is shown in (a).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
This study demonstrates that a microbubble conjugated to a ligand
for a specific molecular epitope selectively adheres to a biological
surface expressing this epitope. We show that a novel perfluorocarbon
gas–filled microbubble incorporating monoclonal antibody to human
ICAM-1 on its outer shell preferentially binds to activated
human coronary artery ECs overexpressing ICAM-1. To the best of
our knowledge, this is the first demonstration of targeted microbubble
attachment to cells expressing a surface protein seen in early
atherosclerosis. These data raise the possibility of in
vivo ultrasound imaging to identify cell markers that are pathognomonic
of disease states and that are not otherwise identifiable
antemortem.
A 40-fold increase in the extent of bubble adhesion occurred
when activated ECs were exposed to anti–ICAM-1–conjugated
microbubbles. The lack of adherence of plain microbubbles to normal or
activated endothelium indicates that the
components of the lipid shell have no avidity for ECs. This observation
and the fact that nonspecific IgG microbubbles also do not adhere
indicate that anti–ICAM-1–conjugated bubble adhesion to
activated cells is due to a specific interaction with ICAM-1.
This interaction is further supported by flow cytometry data confirming
upregulation of ICAM-1 in IL-1ß–exposed ECs. Interestingly, there is
limited binding of anti–ICAM-1–conjugated microbubbles to ECs under
basal conditions, presumably because of low-level constitutive
expression of ICAM-1 by normal ECs.6
Published data on site-specific ultrasound contrast agents
are limited. Lanza et al7 enhanced the
echogenicity of thrombi with a biotinylated particle designed to bind
to "pretargeted" thrombus precoated with avidin-biotinylated
antibody complexes. This strategy differs from ours, in that we
conferred binding specificity by manipulating the ligand on the bubble,
rather than the characteristics of the target itself. McCreery et
al8 recently developed a microbubble that binds
directly to thrombus. Another study described solid liposomes
conjugated to antibody to fibrinogen binding to fibrin on glass and
atherosclerotic plaque.9 Bloemen et
al10 described a non–gas-filled liposome
conjugated to antibody to ICAM-1 that bound to detached, suspended
umbilical vein endothelial or bronchial epithelial
cells. Flow cytometry was used to quantify liposomal attachment, but
this approach could not distinguish between surface binding versus
uptake of the liposome into the cells.10 A major
difference between our study and those of others is that we were able
to visualize the specific adherence of a gas-filled, targeted bubble to
the surface of activated coronary ECs directly, in
situ.
The goal of this study was to establish proof of principle,
and the experiment was thus designed to optimize conditions most
favoring bubble adherence. Cultured coronary artery ECs were
therefore used as an approximation of the in vivo
situation,4 and cells were exposed to bubbles
under static conditions and washed with nonpulsatile flow in the
absence of a leukocyte-bearing environment. It remains to be determined
whether similar binding would occur in vivo and be detected with
ultrasound. In this regard, we recently reported that these bubbles
(without attached antibody) produce a strong backscatter signal in the
left ventricle when injected intravenously in
dogs.11 We have also shown that similar,
biotinylated bubbles attached to avidin-coated glass surfaces at
surface densities of 3% and bubbles contiguous to the wall of
saline-filled plastic bags in surface densities <0.8% can be detected
by conventional ultrasound systems.11 Studies
using both fundamental and harmonic imaging will be necessary to extend
these findings to clinical settings.
The development of an ultrasound imaging agent specific to a
molecular marker of early endothelial disease could
have implications for the identification of incipient
atherosclerosis. Tissue-specific ultrasound contrast
imaging could have applications to other disease states: the
incorporation of different ligands on the bubble could allow ultrasound
localization or characterization of components of atherosclerotic
plaque, restenotic lesions, or transplant vasculopathy or could
have other noncardiac applications in fields such as oncology.
Selected Abbreviations and Acronyms
EC
=
endothelial cell
ICAM-1
=
intercellular adhesion molecule-1
IL-1ß
=
interleukin-1ß
LAM
=
leukocyte adhesion molecule
PE
=
phycoerythrin
Acknowledgments
Dr Villanueva is supported by a FIRST Award from the National
Institutes of Health (R29-HL-58865-01). Dr Wagner and Ron Jankowski are
supported by the McGowan Center for Artificial Organs. Dr Sri
Muddassani, Dr David Ashai, and Arun Krishnan provided technical
assistance.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Libby P, Hansson GK. Involvement of the immune
system in human atherogenesis: current knowledge and unanswered
questions. Lab Investigation. 1991;64:5–15.[Medline]
[Order article via Infotrieve]
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