Am J Physiol Lung Cell Mol Physiol 285: L283–L292, 2003;
10.1152/ajplung.00021.2003.
PECAM-directed delivery of catalase to endothelium protects
against pulmonary vascular oxidative stress
Melpo Christofidou-Solomidou,1 Arnaud Scherpereel,1 Rainer Wiewrodt,1 Kimmie Ng,1
Thomas Sweitzer,2 Evguenia Arguiri,1 Vladimir Shuvaev,2 Charalambos C. Solomides,3
Steven M. Albelda,1 and Vladimir R. Muzykantov2,4
1
Pulmonary Critical Care Division, Department of Medicine and 2Institute of Environmental
Medicine, 4Department of Pharmacology, University of Pennsylvania, Philadelphia 19104; and
3
Department of Pathology, Temple University Hospital, Philadelphia, Pennsylvania 19140
Submitted 21 January 2003; accepted in final form 10 March 2003
TARGETED DRUG DELIVERY to endothelium promises effective and specific means for therapies (1, 31, 38, 43). For
example, affinity carriers directed against normal and
pathological endothelial cells provide vascular targeting of reporter, imaging, and toxic agents to endothelium in vivo (38, 43, 47). The pulmonary vasculature is
a preferential vascular target, since lungs contain 30%
of the total endothelial surface in the body, collect
100% of cardiac blood output, and receive the first pass
of venous blood after intravenous injection. Antibodies
directed against surface endothelial determinants, including angiotensin-converting enzyme (ACE), caveoliassociated antigens, surface adhesion molecules, and
thrombomodulin (TM) deliver reporter cargo materials
to the pulmonary vasculature (9, 36–38, 43–47, 57–
59). However, potential therapeutic applications of
vascular immunotargeting for endothelial protection
have not been characterized. This study provides the
first in vivo evidence of the protective effect of vascular
immunotargeting of drugs to endothelium in the context of pulmonary vascular oxidative stress.
Reactive oxygen species (ROS; e.g., H2O2) cause endothelial injury leading to edema, thrombosis, and
inflammation, contributing to morbidity and mortality
in acute lung injury (ALI), ischemia-reperfusion (I/R),
and many other disease conditions (5, 19, 33, 52, 62,
65). In many cases, the pulmonary endothelium represents both the major target and the source of ROS
generated via diverse enzymatic mechanisms by leukocytes, alveolar macrophages, and endothelial cells
themselves (2, 11, 20, 34). ROS cause endothelial dysfunction manifested by increased permeability, leukocyte recruitment, adhesion and transmigration, thrombosis, and other pathways initiating and propagating
inflammation (20, 61). However, current means for
vascular protection have provided inconsistent results
in many animal and clinical studies, at least in part
due to suboptimal delivery of antioxidants to the endothelial cells.
For example, the antioxidant enzymes, including superoxide dismutases (SOD) and catalase (the latter
safely reduces H2O2 into water), theoretically can provide powerful therapeutic antioxidant modalities (21,
Address for reprint requests and other correspondence: V. R. Muzykantov, Institute of Environmental Medicine, Univ. of Pennsylvania Medical
Center, 1 John Morgan Bldg., 36th St. and Hamilton Walk, Philadelphia,
PA 19104-6068 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
drug delivery; acute lung injury; platelet/endothelial cell adhesion molecule
http://www.ajplung.org
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society
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Christofidou-Solomidou, Melpo, Arnaud Scherpereel, Rainer Wiewrodt, Kimmie Ng, Thomas Sweitzer,
Evguenia Arguiri, Vladimir Shuvaev, Charalambos C.
Solomides, Steven M. Albelda, and Vladimir R. Muzykantov. PECAM-directed delivery of catalase to endothelium
protects against pulmonary vascular oxidative stress. Am J
Physiol Lung Cell Mol Physiol 285: L283–L292, 2003; 10.1152/
ajplung.00021.2003.—Targeted delivery of drugs to vascular
endothelium promises more effective and specific therapies in
many disease conditions, including acute lung injury (ALI).
This study evaluates the therapeutic effect of drug targeting to
PECAM (platelet/endothelial cell adhesion molecule-1) in vivo
in the context of pulmonary oxidative stress. Endothelial injury
by reactive oxygen species (e.g., H2O2) is involved in many
disease conditions, including ALI/acute respiratory distress
syndrome and ischemia-reperfusion. To optimize delivery of
antioxidant therapeutics, we conjugated catalase with PECAM
antibodies and tested properties of anti-PECAM/catalase conjugates in cell culture and mice. Anti-PECAM/catalase, but not
an IgG/catalase counterpart, bound specifically to PECAMexpressing cells, augmented their H2O2-degrading capacity,
and protected them against H2O2 toxicity. Anti-PECAM/catalase, but not IgG/catalase, rapidly accumulated in the lungs
after intravenous injection in mice, where it was confined to the
pulmonary endothelium. To test its protective effect, we employed a murine model of oxidative lung injury induced by
glucose oxidase coupled with thrombomodulin antibody (antiTM/GOX). After intravenous injection in mice, anti-TM/GOX
binds to pulmonary endothelium and produces H2O2, which
causes lung injury and 100% lethality within 7 h. Coinjection of
anti-PECAM/catalase protected against anti-TM/GOX-induced
pulmonary oxidative stress, injury, and lethality, whereas polyethylene glycol catalase or IgG/catalase conjugates afforded
only marginal protective effects. This result validates vascular
immunotargeting as a prospective strategy for therapeutic interventions aimed at immediate protective effects, e.g., for augmentation of antioxidant defense in the pulmonary endothelium and treatment of ALI.
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MATERIALS AND METHODS
The following reagents were used in the study: biotinylated glucose oxidase, catalase, PEG-catalase, components of
buffer solutions from Sigma (St. Louis, MO), fatty acid-free
BSA from Boehringer-Mannheim-Roche (Indianapolis, IN),
streptavidin and 6-biotinylaminocaproic acid N-hydroxysuccinimide ester from Calbiochem (San Diego, CA), Bradford
Bio-Rad protein microassay kit (Hercules, CA), and G418sulfate from Life Technologies (Rockville, MD). A rat MAb to
human creatine kinase was used as control isotype-matched
IgG2a (CKMM 14.15; American Type Culture Collection hybridoma, Manassas, VA). MAb 390 is a rat MAb to muPECAM-1
(7, 59), and MAb 34 is a rat MAb to murine TM (6, 29).
Conjugation of GOX and catalase with carrier antibodies.
Catalase was labeled with 125I using Iodogen-coated tubes.
Catalase and GOX were conjugated with antibodies using
streptavidin-biotin cross-linker without loss of enzymatic activity as described previously (44, 45). Dynamic light scattering (Brookhaven Instruments) showed that size of the conjugates indicated as anti-PECAM/catalase, IgG/catalase, and
anti-TM/GOX was within 200–400 nm, permitting optimal
intracellular targeting (59, 67).
Cell culture experiments. Human mesothelioma REN cell
line and REN cells stably transfected with murine PECAM
(REN/PECAM cells) were produced and maintained as described (25). Endothelial and REN cells are similarly suscepAJP-Lung Cell Mol Physiol • VOL
tible to oxidative stress induced by H2O2 (23). In confluent
REN/PECAM cells, PECAM localizes predominantly in the
intercellular borders, similarly to that in endothelial cells
(25). The cells were washed with serum-free medium without
phenol red, and 10 ␮g of anti-PECAM/catalase or IgG/catalase were incubated with cells in 200 ␮l of culture medium in
24-well culture dishes for 1 h at 37°C. After washing to
remove the unbound reagents, cell associated-125I-catalase,
H2O2 degradation, and H2O2 cytotoxicity were assayed as
described previously (23). Briefly, 5 mM H2O2 in 1 ml of
RPMI 1640 medium without phenol red was added to the
cells, and remaining H2O2 was measured in the aliquot
supernatant medium by peroxidase-catalyzed color reaction
determined by absorbance at 490 nm in a Bio-Rad 3550
Microtiter Plate Reader. H2O2 toxicity was determined in
51
Cr-labeled cells. Five hours after H2O2 exposure, radioactivity in supernatant medium and cell lysates was measured
in a Wallac 1470 Wizard gamma counter (Wallac-LKB), and
cellular death was expressed as the percent 51Cr release,
reflecting irreversible plasma membrane damage.
Evaluation of pulmonary targeting of the conjugates in
intact mice. Animal experiments were performed in accordance with protocol no. 388100, approved by the institutional
animal care and use committee of the University of Pennsylvania. Normal BALB/c mice (Charles River) were injected
with 3 ␮g of anti-PECAM/125I-catalase or IgG/125I-catalase in
100 ␮l of saline via tail vein. One hour later, animals were
killed, and the internal organs were dissected, washed with
saline, blotted dry, and weighed. Radioactivity in organs was
determined in a gamma counter and used to calculate the
percent of injected dose per gram of tissue (%ID/g) and
lung-to-blood ratio. To visualize anti-PECAM/catalase in the
lung, 6-␮m-thick frozen sections from optimum cutting temperature compound-embedded tissues and 4-␮m paraffin sections were prepared from lungs harvested 30 min after intravenous injection of 200 ␮g of anti-PECAM/catalase. The
sections were stained using anti-catalase MAb (Sigma) and a
labeled secondary antibody using Vectastain kit (Vector
Labs).
Injection of catalase conjugates in the anti-TM/GOX injury
model. To inflict an acute H2O2-mediated endothelial injury
in the pulmonary vasculature in mice, we used intravenous
injection of anti-TM/GOX as described in detail previously
(6). Mice were injected with 30, 60, or 75 ␮g of anti-TM/GOX
simultaneously with 100 ␮g of anti-PECAM/catalase or IgG/
catalase or 100 ␮g of PEG-conjugated catalase. The lungs
were harvested from animals immediately postmortem or at
termination of the experiment (12 h), inspected, photographed, and processed for wet-to-dry weight ratio determination, cryosectioning, histological studies, and immunostaining. For histological studies, the lungs were instilled,
before removal from the animal, with 0.75 ml of buffered
formalin through a 20-gauge angiocatheter placed in the
trachea, immersed in buffered formalin overnight, and processed for conventional paraffin histology. Sections were
stained with hematoxylin and eosin and examined by light
microscopy. Pulmonary oxidative stress was detected by immunostaining of tissue sections counterstained with Neutral
Red (Sigma) using a rabbit polyclonal antibody directed
against iPF2␣-III isoprostane, a marker of lipid peroxidation,
formerly known as 8-epi or 8-isoPGF2␣ (53), and a rabbit
polyclonal antibody to nitrotyrosine, a marker of oxidative
protein nitration (22). Immunostaining was visualized by the
use of an alkaline phosphatase kit (Vector Labs).
Statistical analysis. Statistical differences among groups
were determined using one-way analysis of variance. When
statistically significant differences were found (P ⬍ 0.05),
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24). Encapsulation in liposomes, coupling of polyethylene glycol (PEG), lecithin, or albumin permit more
effective cellular uptake and prolonged circulation of
antioxidant enzymes, improving their therapeutic applicability (26, 27, 63). Furthermore, intratracheal administration of PEG-conjugated and liposome-loaded
antioxidant enzymes alleviates hyperoxia-induced pulmonary oxidative stress (3, 18). However, lack of specific affinity to endothelium limits protective effects of
intravascular administration of catalase and its derivatives in conditions dominated by endothelial oxidative
stress, such as ALI and I/R (2, 34, 47). Gene therapies
promise improved delivery of antioxidant enzymes (8,
12, 14, 35, 68) but cannot be used in acute situations
when protective intervention is required immediately.
Vascular immunotargeting may offer an alternative
strategy to optimize endothelial delivery of antioxidant
enzymes (44, 46). For example, monoclonal antibodies
(MAb) directed against platelet/endothelial cell adhesion molecule-1 (anti-PECAM) deliver diverse reporter
molecules and genetic materials to the pulmonary endothelium (7, 36, 45, 59). In the present work, we
studied endothelial targeting of and antioxidant protection by catalase conjugated with a rat MAb against
murine PECAM (muPECAM). To characterize the protective effects of anti-PECAM/catalase conjugate in an
animal model of H2O2-induced severe oxidative lung
injury, we utilized glucose oxidase conjugated with a
thrombomodulin antibody (anti-TM/GOX). After intravenous injection in mice, anti-TM/GOX accumulates in
murine lungs and generates H2O2, which causes edematous oxidative lung injury (6). The data shown in the
paper indicate that PECAM-directed vascular immunotargeting facilitating delivery of catalase to the pulmonary endothelium protects against acute lung oxidative stress.
AUGMENTATION OF PULMONARY ANTIOXIDANT DEFENSE BY CATALASE IMMUNOTARGETING
catalase did not significantly accelerate H2O2 degradation by either cell type. Augmentation of antioxidant
capacity by anti-PECAM/catalase resulted in a marked
protection against exposure to a toxic dose of H2O2. In
control cultures, 5 mM H2O2 caused high lethality of
both REN and REN/PECAM cells (80% release of 51Cr).
IgG/catalase did not protect either cell type against
H2O2. In contrast, anti-PECAM/catalase protected
REN/PECAM, but not REN cells, against H2O2 toxicity
(Fig. 1B).
Anti-PECAM/catalase accumulates in the pulmonary
vasculature after intravenous injection in mice. AntiPECAM/catalase, but not the IgG counterpart, accumulated in the lungs after intravenous injection in
intact mice. Pulmonary uptake of anti-PECAM/125Icatalase achieved 30% ID/g 1 h postinjection and was
10 times higher than that of IgG/125I-catalase, whereas
blood level of either conjugate was close to 4.5% ID/g
(Fig. 2). Thus the lung-to-blood ratio of anti-PECAM/
125
I-catalase was close to 7, similar to this parameter
obtained with anti-ACE carrier (44, 45).
We also characterized the kinetics of radiolabeled
anti-PECAM/catalase in mice. Blood levels of the conjugate declined very rapidly with the half-life ⬍30 min
(data not shown). The inset in Fig. 2 shows that level of
anti-PECAM/catalase reaches peak value rapidly after
intravenous injection, whereas the intraperitoneal
route provides a delayed and relatively modest pulmonary accumulation of anti-PECAM/catalase. After intravenous injection, the anti-PECAM/catalase level in
the lungs declined after a 30-min steady level, and 2–3
h postinjection, it was reduced to 50% of the initial
peak level. This result corroborates our previously published data on the half-life in mice of reporter enzymes
conjugated with anti-PECAM (59).
By indirect immunostaining, anti-PECAM/catalase
was detected in the alveolar capillaries, on the luminal
surface of pulmonary venules and arterioles, but not in
the airways and interstitium (Fig. 3).
individual comparisons were made using the Bonferroni/
Dunn test (Statview 4.0).
RESULTS
Anti-PECAM/catalase binds to and protects against
H2O2 cells expressing muPECAM. First, we characterized the targeting, enzymatic activity, and protective effect of anti-PECAM/catalase in culture of human mesothelioma cell line (REN) transfected with muPECAM,
i.e., REN/muPECAM cells, which represent a useful
model to study the PECAM-directed targeting (23, 45).
REN/PECAM cells specifically bound anti-PECAM/
125
I-catalase, but not IgG/125I-catalase, whereas wildtype REN cells bound significantly lesser amounts of
125
I-catalase conjugated with either anti-PECAM or
IgG (Fig. 1A). The delivered catalase was enzymatically active; REN/PECAM cells treated with antiPECAM/catalase degraded H2O2 markedly faster than
counterpart REN cells (*P ⬍0.01, not shown). IgG/
AJP-Lung Cell Mol Physiol • VOL
Fig. 2. Pulmonary targeting of 125I-anti-PECAM/catalase in mice.
The level of 125I in blood and lung 1 h after intravenous (iv) injection
of IgG/125I-catalase (open bars) or anti-PECAM/125I-catalase (closed
bars) in intact mice. Data are means ⫾ SE (n ⫽ 4). Inset: kinetics of
pulmonary accumulation of anti-PECAM/125I-catalase after iv (top
curve) vs. intraperitoneal (ip) (bottom curve) administration of the
conjugate. %ID/g, percent of injected dose per gram of tissue.
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Fig. 1. Specific protection of endothelial cells against H2O2 by antiplatelet/endothelial cell adhesion molecule (PECAM)/catalase conjugate. A: binding of anti-PECAM/125I-catalase (closed bars) or IgG/
125
I-catalase (open bars) to REN and REN/murine (mu) PECAM
cells. B: cytotoxic effect of H2O2 determined by 51Cr release in REN
cells (closed bars) and REN/muPECAM cells (open bars). Either cell
type was pretreated with either PBS vehicle (control and H2O2 bars),
anti-PECAM/catalase, or IgG/catalase.
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Anti-PECAM/catalase ameliorates H2O2-induced oxidative stress in murine lungs. To test whether catalase
immunotargeting protects pulmonary vasculature
against H2O2-induced oxidative stress, we utilized an
original model of ALI induced by TM-directed immunotargeting of H2O2-producing enzyme GOX to pulmonary endothelium. As described in detail previously,
AJP-Lung Cell Mol Physiol • VOL
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Fig. 3. Visualization of anti-PECAM/catalase conjugate in mouse
lungs. Catalase immunostaining in lungs harvested 30 min after iv
injection of 200 ␮g of anti-PECAM/catalase conjugate (B and C) or
vehicle (saline, A) in intact mice. Magnification, ⫻250. Note the
positive reaction in the pulmonary capillaries (B) and small venules
or arterioles but not in bronchioles or epithelium (C).
anti-TM/GOX accumulates in murine lungs after intravenous injection and induces an acute, edematous pulmonary injury (6).
To evaluate qualitatively the extent of oxidative
stress induced in the murine lungs by H2O2 generated
in the pulmonary vasculature by anti-TM/GOX, the
lung tissue sections were stained with antibodies directed against nitrotyrosine, a marker of protein oxidative nitration, and iPF2␣-III, an F2 isoprostane, a
marker of lipid peroxidation. Positive immunostaining
for nitrotyrosine and iPF2␣-III was detected in the
lungs harvested 6 h after injection of 30 ␮g of anti-TM/
GOX (Fig. 4). Coinjection of IgG/catalase or PEG-catalase with anti-TM/GOX reduced the nitrotyrosine
staining to some extent. At this dose, anti-TM/GOX
inflicted lung injury that could be partially alleviated
by these “nontargeted” conjugates (see below). However, anti-PECAM/catalase provided a more marked
reduction of both the nitrotyrosine and iPF2␣-III staining in the lungs harvested after anti-TM/GOX injection
(Fig. 4). Therefore, endothelium-targeted anti-PECAM/
catalase more effectively detoxifies H2O2 produced in
the pulmonary vasculature than its nontargeted counterparts.
Oxidative stress induced by injection of 60–75 ␮g of
anti-TM/GOX led to severe lung injury, manifested by
a brownish hemorrhagic appearance, on a gross morphology examination (Fig. 5A), and vascular congestion, accumulation of leukocytes, and alveolar edema
on histological examination (Fig. 5B). Anti-PECAM/
catalase, but not PEG-catalase or IgG/catalase, markedly attenuated pulmonary injury induced by anti-TM/
GOX both at levels of gross morphology and lung histology (compare Fig. 5, C and D).
Anti-PECAM/catalase protects against H2O2-induced pulmonary edema and lethality. Injection of 30
␮g of anti-TM/GOX caused 100% lethality within 8 h
(Fig. 6A). This dose caused acute pulmonary edema
(wet-to-dry ratio 7.6 ⫾ 0.2 at postmortem vs. 5.2 ⫾ 0.2
in control mice killed 12 h postinjection of saline).
PEG-catalase injected with anti-TM/GOX slightly attenuated edema (6.9 ⫾ 0.2), delayed death, and reduced the lethality to 80% (Fig. 6A). Anti-PECAM/
catalase injected with anti-TM/GOX markedly attenuated edema (5.7 ⫾ 0.4, animals killed 12 h postantiTM/GOX injection, P ⫽ 0.001 vs. anti-TM/GOXinjected group) and reduced the lethality in this
experiment to 20% (Fig. 6A).
Escalation of anti-TM/GOX dose to 65 ␮g caused
even more severe injury: a wet-to-dry ratio of 8.1 ⫾ 0.2
and 100% lethality within 4 h (Fig. 6B). At this dose of
anti-TM/GOX, IgG/catalase partially attenuated
edema (wet-to-dry ratio 7.0 ⫾ 0.3), delayed death (50%
death time was 6 h), and reduced lethality to 60%,
whereas anti-PECAM/catalase-treated mice survived
12 h after injection of 65 ␮g of anti-TM/GOX (Fig. 6B)
and had a practically normal wet-to-dry ratio after
death (4.9 ⫾ 0.1).
At the highest anti-TM/GOX used in the study (75
␮g), IgG/catalase failed to prolong survival and reduce
lethality, whereas anti-PECAM/catalase markedly
AUGMENTATION OF PULMONARY ANTIOXIDANT DEFENSE BY CATALASE IMMUNOTARGETING
L287
prolonged the survival and reduced lethality from
100% to 30% (Fig. 6C). Thus at all anti-TM/GOX doses
used in the study, anti-PECAM/catalase conjugate affords markedly more effective protection than nontargeted catalase counterparts.
In a separate experiment, we injected a moderate
dose (50 ␮g) of anti-TM/GOX in mice 2 min after
injection of either anti-PECAM/catalase or anti-PECAM/
streptavidin conjugates of a similar size (290- and
300-nm diameter). Mice were killed 4 h after injection,
and protein levels in the bronchoalveolar lavage fluid,
which are sensitive and reproducible parameters of
alveolar edema, were determined. In anti-TM/GOXinjected mice, bronchoalveolar lavage fluid protein was
elevated to 0.54 ⫾ 0.05 mg/ml vs. control level of 0.14 ⫾
0.07 (means ⫾ SE, n ⫽ 4, P ⬍ 0.01). Anti-PECAM/
AJP-Lung Cell Mol Physiol • VOL
catalase conjugate markedly suppressed protein elevation (0.27 ⫾ 0.04 mg/ml, P ⬍ 0.01 vs. anti-TM/GOX
group), whereas anti-PECAM/streptavidin conjugate
was not protective at all (0.69 ⫾ 0.08 mg/ml). This
result indicates that the protective effect of antiPECAM/catalase is due to specific delivery of the enzyme and not to PECAM-1 blocking or steric inhibition
of anti-TM/GOX binding.
DISCUSSION
Endothelial cells are vulnerable to oxidative stress
and represent an important therapeutic target. Most
therapeutic agents, however, have no specific affinity
to endothelium, and suboptimal delivery limits the
therapeutic efficacy. Our hypothesis was that vascular
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Fig. 4. Anti-PECAM/catalase attenuates oxidative
stress in the lungs. Anesthetized mice were injected
with saline (control, A and B), glucose oxidase coupled with thrombomodulin antibody (anti-TM/GOX)
alone (C and D), or in combination with IgG/catalase
(E and F) or with anti-PECAM/catalase (G and H).
The lung tissue sections were stained with antibody
directed against nitrotyrosine, a marker of protein
oxidative nitration (left column), or 8-epi iPF2a-III
F2 isoprostane, a marker of lipid peroxidation (right
column). The positive immunostaining was revealed
by secondary antibody conjugated with alkaline
phosphatase (blue color). Neutral Red counterstaining was used to mark individual cells. Magnification,
⫻500.
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AUGMENTATION OF PULMONARY ANTIOXIDANT DEFENSE BY CATALASE IMMUNOTARGETING
immunotargeting could improve delivery and enhance
therapeutic interventions. We reasoned that PECAM/
catalase conjugate is a good candidate for proof of the
concept in the context of endothelial protection against
oxidative stress. High and stable endothelial expression of PECAM under normal and pathological conditions permits robust drug delivery (7, 45, 59). PECAM-1
plays an important role in leukocyte transmigration
through endothelium, and PECAM antibodies suppress this function (15, 48, 51). Therefore, inhibition of
PECAM by the conjugates could possibly provide a
secondary anti-inflammatory benefit.
To obtain a decisive proof of principle, we tested
anti-PECAM/catalase in an original in vivo model of
oxidative pulmonary stress induced by H2O2 generated
by GOX targeted to TM (6). TM is an endotheliumspecific surface glycoprotein enriched in the pulmonary
vascular lumen (17). After intravenous injection, anti-TM
accumulates in the murine lungs and delivers conjugated liposomes and other materials to the lungs (37).
Anti-TM/GOX accumulates in the lung after injection
in mice, produces H2O2 in the pulmonary vasculature,
and causes oxidative lung injury similar, by many
pathological features, to human ALI syndrome (6).
This contrasts with many currently available animal
models of ALI/acute respiratory distress syndrome
(ARDS), where oxidative stress in the pulmonary vasculature is initiated indirectly and represents a very
AJP-Lung Cell Mol Physiol • VOL
complex interplay between various oxidants and other
proinflammatory mediators generated in the lung by
vascular, alveolar, and blood cells (10, 40, 50, 56, 60,
66). Importantly, anti-TM/GOX-induced lung injury is
dose dependent and acute (Fig. 6).
Anti-PECAM/catalase conjugates, but not nontargeted catalase counterparts, accumulate in the pulmonary vasculature (Figs. 2 and 3). Anti-PECAM/catalase
intervention in anti-TM/GOX-injected mice reduced
oxidative stress, attenuated pulmonary edema and injury, delayed lethality, and increased survival (Figs.
4–6). By all these parameters, protection by antiPECAM/catalase markedly exceeded the modest protection afforded by nontargeted catalase preparations
(PEG-catalase and IgG/catalase).
Vascular oxidative stress initiated and/or propagated by ROS plays a central role in pulmonary and
cardiovascular disease conditions such as ALI, hyperoxia, sepsis, I/R, myocardial infarction and stroke, hypertension, and diabetes (16, 19, 20, 32, 65). Some of
these disease conditions may be amenable antioxidant
therapies, pending adequate delivery and timing of the
therapeutic intervention. The exact onset of oxidative
stress is known in certain settings, including oxygen
ventilation therapies, radiation injury, and lung transplantation, which are ideal for initial testing of the
immunotargeting of antioxidant enzymes administered exactly at or immediately before the time of
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Fig. 5. Anti-PECAM/catalase protects against pulmonary injury inflicted by anti-TM/GOX. A: typical gross
morphology appearance of lungs harvested from a mouse injected with 75 ␮g of anti-TM/GOX (lane 1, positive
injury control) and lungs from mice injected with 75 ␮g of anti-TM/GOX and 100 ␮g of anti-PECAM/catalase (lanes
2–4), showing minimal injury with near normal appearance. Bottom: representative lung tissue sections stained
with hematoxylin and eosin (⫻500 magnification) after injection of anti-TM/GOX alone (B), polyethylene glycol
(PEG)/catalase (C), or anti-PECAM/catalase (D).
AUGMENTATION OF PULMONARY ANTIOXIDANT DEFENSE BY CATALASE IMMUNOTARGETING
insult. Results of pilot studies in a rat lung transplantation model indicate that anti-PECAM/catalase protects the lung graft against acute transplantation injury. This paper presents the decisive evidence that
catalase immunotargeting affords significant protective effect in animals.
The present results obtained with the anti-TM/GOX
model of pulmonary oxidative stress are encouraging
yet must be interpreted cautiously in terms of potential
translation into treatment of human pathologies, including ALI/ARDS. For example, damage to alveolar
epithelial cells is an important component of human
ALI/ARDS, whereas endothelial injury dominates
pathogenesis of anti-TM/GOX model. Further experiments in models that include epithelial injury (e.g.,
hyperoxia) will test whether anti-PECAM/catalase effect is limited to endothelium protection or provides
more generalized protection in the lung tissue.
AJP-Lung Cell Mol Physiol • VOL
The strategy used here will be further optimized for
clinical applications. One area currently under optimization is control of the size of the conjugates. Recent
findings indicate that conjugates within 100- to
350-nm diameter permit optimal pulmonary targeting
in vivo and intracellular delivery of cargoes, including
active enzymes (6, 59, 67). Another area that needs
further optimization is timing of injections and duration of the protective effect. Our previous studies revealed that the duration of active reporter (␤-galactosidase) anti-PECAM conjugates in the pulmonary vasculature varies from 30 min to several hours after a
bolus injection in mice and pigs (58, 59). Ongoing
studies indicate that human endothelial cells internalize anti-PECAM conjugates via an unusual endocytotic
mechanism, mediated by neither clathrin-coated pits
nor caveoli (42). In cell culture, this pathway leads to a
relatively slow lysosomal trafficking and degradation
of the conjugates (3 h vs. 15 min for clathrin-mediated
endocytosis), although the kinetics of lysosomal degradation may be faster in vivo. However, the uptake,
trafficking, and lysosomal degradation are inhibited in
endothelial cells by auxiliary pharmacological agents,
including the clinically useful drugs amiloride and
chloroquine (S. Muro, V. Muzykantov, and M. Koval,
unpublished data). It is plausible that pharmacological
interventions with auxiliary drugs might help to prolong the effect of the conjugates in vivo. At the present
time, it is unclear whether anti-PECAM/catalase intervention at the onset of ALI affords effective protection.
Our pilot experiments show that injection of antiPECAM/catalase 30 min after anti-TM/GOX is still protective. Further studies will systematically characterize
optimal regimens and potential limitations of administration of the anti-PECAM/catalase conjugates.
Delivery of proteins has an advantage of an immediate therapeutic intervention. Most likely, individual
conjugates that differ in their cargoes, cross-linking
methods, and molecular composition will have different kinetics of metabolism. Slow infusions of the conjugates or loading them into controlled release devices
may help to extend both the prophylactic and therapeutic windows in acute situations. Gene therapy
strategies, including targeting of viral or nonviral genetic materials to endothelial cells, provide a more
stable and prolonged delivery of antioxidant enzymes
that may afford prophylaxis and protection in chronic
conditions (8, 13, 14, 57). However, gene therapy would
not be effective in acute situations when protective
intervention is required immediately. It is tempting to
speculate that combined immunotargeting of therapeutic proteins and genes encoding these proteins will
permit effective management of vascular oxidative
stress and, perhaps, other disease conditions, such as
thrombosis and inflammation.
“Humanization” of carrier antibodies and use of Fab
fragments, manufacturing, and quality control of conjugates will help to solve some general issues related to
safety of systemic administration of conjugates. Our
pilot data indicate that large doses of anti-PECAM/
catalase (300 ␮g), exceeding those reported as protec285 • AUGUST 2003 •
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Fig. 6. Anti-PECAM/catalase markedly attenuates anti-TM/GOXinduced lethality in mice. Mice were injected with 30 (A), 65 (B), or
75 ␮g (C) of anti-TM/GOX conjugate (n ⫽ 10/group) alone or in
combination with anti-PECAM targeted catalase or nontargeted
catalase conjugates (PEG/catalase in A, IgG/catalase in B and C).
L289
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AUGMENTATION OF PULMONARY ANTIOXIDANT DEFENSE BY CATALASE IMMUNOTARGETING
The authors thank Dr. S. Kennel (Oakridge National Laboratory)
for thrombomodulin monoclonal antibody used for GOX targeting,
Alyssa Bohen for technical support with the animal experiments and
tissue/sample processing, and Anu Thomas for technical support in
preparation of the anti-PECAM conjugates.
DISCLOSURES
This work was supported by the National American Heart Association (AHA 0030192N) (to M. Christofidou-Solomidou), the National American Lung Association (RG-087-N) (to M. ChristofidouSolomidou), and American Heart Association Established Investigator Grant and Project 4 in National Institutes of Health Specialized
Center of Research in acute lung injury (to V. R. Muzykantov).
AJP-Lung Cell Mol Physiol • VOL
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