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
1Pulmonary Critical Care Division, Department of
Medicine and 2Institute of Environmental Medicine,
4Department of Pharmacology, University of
Pennsylvania, Philadelphia 19104; and 3Department of
Pathology, Temple University Hospital, Philadelphia, Pennsylvania 19140
Submitted 21 January 2003
; accepted in final form 10 March 2003
 |
ABSTRACT
|
---|
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 PECAM-expressing 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 (anti-TM/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.
drug delivery; acute lung injury; platelet/endothelial cell adhesion molecule
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), caveoli-associated antigens, surface
adhesion molecules, and thrombomodulin (TM) deliver reporter cargo materials
to the pulmonary vasculature
(9,
3638,
4347,
5759).
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
dys-function 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
(21,24).
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.
 |
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 G418-sulfate 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 200400
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 susceptible 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 µg of anti-PECAM/catalase
or IgG/catalase were incubated with cells in 200 µl 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 51Cr-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 µg of anti-PECAM/125I-catalase or
IgG/125I-catalase in 100 µl 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-µm-thick frozen sections from optimum cutting temperature
compound-embedded tissues and 4-µm paraffin sections were prepared from
lungs harvested 30 min after intravenous injection of 200 µg 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 µg of anti-TM/GOX simultaneously with 100 µg of
anti-PECAM/catalase or IgG/catalase or 100 µg 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), 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/125I-catalase, but not
IgG/125I-catalase, whereas wild-type REN cells bound significantly
lesser amounts of 125I-catalase conjugated with either anti-PECAM
or IgG (Fig. 1A). The
delivered catalase was enzymatically active; REN/PECAM cells treated with
anti-PECAM/catalase degraded H2O2 markedly faster than
counterpart REN cells (*P <0.01, not shown). IgG/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).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1. Specific protection of endothelial cells against H2O2
by anti-platelet/endothelial cell adhesion molecule (PECAM)/catalase
conjugate. A: binding of anti-PECAM/125I-catalase (closed
bars) or IgG/125I-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.
|
|
Anti-PECAM/catalase accumulates in the pulmonary vasculature after
intravenous injection in mice. Anti-PECAM/catalase, but not the IgG
counterpart, accumulated in the lungs after intravenous injection in intact
mice. Pulmonary uptake of anti-PECAM/125I-catalase 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/125I-catalase was close
to 7, similar to this parameter obtained with anti-ACE carrier
(44,
45).

View larger version (15K):
[in this window]
[in a new window]
|
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.
|
|
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 23 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).

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 3. Visualization of anti-PECAM/catalase conjugate in mouse lungs. Catalase
immunostaining in lungs harvested 30 min after iv injection of 200 µg of
anti-PECAM/catalase conjugate (B and C) or vehicle (saline,
A) in intact mice. Magnification, x250. Note the positive
reaction in the pulmonary capillaries (B) and small venules or
arterioles but not in bronchioles or epithelium (C).
|
|
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, 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 µg 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.

View larger version (117K):
[in this window]
[in a new window]
|
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,
x500.
|
|
Oxidative stress induced by injection of 6075 µg 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).

View larger version (73K):
[in this window]
[in a new window]
|
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 µg of anti-TM/GOX (lane 1,
positive injury control) and lungs from mice injected with 75 µg of
anti-TM/GOX and 100 µg of anti-PECAM/catalase (lanes 24),
showing minimal injury with near normal appearance. Bottom:
representative lung tissue sections stained with hematoxylin and eosin
(x500 magnification) after injection of anti-TM/GOX alone (B),
polyethylene glycol (PEG)/catalase (C), or anti-PECAM/catalase
(D).
|
|
Anti-PECAM/catalase protects against
H2O2-induced
pulmonary edema and lethality. Injection of 30 µg 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
postanti-TM/GOX injection, P = 0.001 vs. anti-TM/GOX-injected group)
and reduced the lethality in this experiment to 20%
(Fig. 6A).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6. Anti-PECAM/catalase markedly attenuates anti-TM/GOX-induced lethality in
mice. Mice were injected with 30 (A), 65 (B), or 75 µg
(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).
|
|
Escalation of anti-TM/GOX dose to 65 µg 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 µg 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 µg), IgG/catalase
failed to prolong survival and reduce lethality, whereas anti-PECAM/catalase
markedly 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 µg) 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/GOX-injected 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/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 anti-PECAM/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 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
endothelium-specific 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 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,
5,
6). By all these parameters,
protection by anti-PECAM/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 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.
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 anti-PECAM/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 µg),
exceeding those reported as protective in this paper (100 µg), do not cause
detectable pathological alterations in the lungs within 2 wk after intravenous
injection in animals (B. Kozower, M. Christofidou, and V. Muzykantov,
unpublished data). However, specific potential side effects of delivery of
antioxidants to endothelium must be rigorously addressed, especially in light
of the notion that H2O2 plays a physiological signaling
role in the vasculature (28).
It is possible that interception of normally produced ROS might lead to
untoward interventions in cellular signaling.
An additional area of development is upgrading the immunotargeting by use
of diverse affinity carriers and therapeutic cargoes. Carriers recognizing
inducible surface adhesion molecules, including ICAM-1, selectins,
angiotensin-converting enzyme, and caveolar antigens, facilitate drug delivery
to endothelium (9,
30,
38,
39,
41,
47,
54,
55). Delivery of diverse
antioxidant enzymes may permit more complete protection. For example,
targeting SOD or SOD mimetics may help to decompose superoxide anion and thus
prevent inactivation of nitric oxide and oxidative nitration in the tissues
(21,
64). A chimera construct
combining manganese SOD (that protects against intracellular
O2-) and heparin-binding domain of extracellular SOD
(that binds to charged components of endothelial glycocalix) showed protective
effect in animal models of transplantation and hepatic I/R injury
(4,
49); these recently reported
findings provide hope that this construct may accumulate and exert protective
effects in the pulmonary vasculature.
In summary, this study demonstrates that vascular immunotargeting of an
antioxidant enzyme (catalase) to an endothelial surface determinant (PECAM-1)
augments antioxidant defense and protects intact animals against otherwise
lethal ALI. From a general standpoint, to our knowledge, this is the first
documented proof of principle that vascular immunotargeting of drugs to
endothelium in intact animals provides a significant therapeutic, protective
effect. Specifically, this result supports a novel strategy for more specific
and effective means for treatment of ALI and other types of acute vascular
oxidative stress. Future translation of this strategy into the clinical domain
may have a significant impact in pulmonary and cardiovascular medicine.
 |
ACKNOWLEDGMENTS
|
---|
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. Christofidou-Solomidou), 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).
 |
FOOTNOTES
|
---|
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:
muzykant{at}mail.med.upenn.edu).
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.
 |
REFERENCES
|
---|
- Akerman ME,
Chan WC, Laakkonen P, Bhatia SN, and Ruoslahti E. Nanocrystal targeting in
vivo. Proc Natl Acad Sci USA
99: 1261712621,
2002.[Abstract/Free Full Text]
- Al-Mehdi A,
Zhao G, Dodia C, Tozawa K, Costa K, Muzykantov V, Ross C, Blecha F, Dinauer M,
and Fisher A. Endothelial NADPH oxidase as the source of oxidants with
lung ischemia or high K+. Circ Res
83: 730737,
1998.[Abstract/Free Full Text]
- Barnard M,
Baker P, and Matalon S. Mitigation of oxidant injury to lung
microvasculature by intratracheal instillation of antioxidant enzymes.
Am J Physiol Lung Cell Mol Physiol
265: L340L345,
1993.[Abstract/Free Full Text]
- Cerwinka WH,
Cooper D, Krieglstein CF, Ross CR, Mc-Cord JM, and Granger DN. Superoxide
mediates endotoxin-induced platelet-endothelial cell adhesion in intestinal
venules. Am J Physiol Heart Circ Physiol
284: H535H541,
2003.[Abstract/Free Full Text]
- Chabot F,
Mitchell JA, Gutteridge JMC, and Evans TW. Reactive oxygen species in
acute lung injury. Eur Respir J
11: 745757,
1998.[Abstract/Free Full Text]
- Christofidou-Solomidou M, Kennel S, Scherpereel A, Wiewrodt R, Solomides CC,
Pietra GG, Murciano JC, Shah SA, Ischiropoulos H, Albelda SM, and Muzykantov
VR. Vascular immunotargeting of glucose oxidase to the endothelial
antigens induces distinct forms of oxidant acute lung injury. Am J
Pathol 160:
11551169, 2002.[Abstract/Free Full Text]
- Christofidou-Solomidou M, Pietra GG, Solomides CC, Argiris E, Harshaw D,
FitzGerald G, Albelda SM, and Muzykantov VR. Immunotargeting of glucose
oxidase to endothelium in vivo causes oxidative vascular injury in the lungs.
Am J Physiol Lung Cell Mol Physiol
278: L794L805,
2000.[Abstract/Free Full Text]
- Danel C,
Erzurum S, Prayssac P, Eissa M, Crystal R, Herve P, Baudet B, Mazmanian M, and
Lemarchand P. Gene therapy for oxidant injury-treated diseases:
adenovirus-mediated transfer of superoxide dismutase and catalase cDNAs
protects against hyperoxia but not against ischemia-reperfusion lung injury.
Hum Gene Ther 9:
14871496, 1998.[ISI][Medline]
- Danilov SM,
Gavrilyuk VD, Franke FE, Pauls K, Harshaw DW, McDonald TD, Miletich DJ, and
Muzykantov VR. Lung uptake of antibodies to endothelial antigens: key
determinants of vascular immunotargeting. Am J Physiol Lung Cell
Mol Physiol 280:
L1335L1347, 2001.[Abstract/Free Full Text]
- Davidson KG,
Bersten AD, Barr HA, Dowling KD, Nicholas TE, and Doyle IR. Lung function,
permeability, and surfactant composition in oleic acid-induced acute lung
injury in rats. Am J Physiol Lung Cell Mol Physiol
279: L1091L1102,
2000.[Abstract/Free Full Text]
- Eckenhoff RG,
Dodia C, Tan Z, and Fisher AB. Oxygen-dependent reperfusion injury in the
isolated rat lung. J Appl Physiol
72: 14541460,
1992.[Abstract/Free Full Text]
- Engelhardt JF. Redox-mediated gene therapies for
environmental injury: approaches and concepts. Antioxid Redox
Signal 1:
527, 1999.[Medline]
- Engelhardt JF,
Sen CK, and Oberley L. Redox-modulating gene therapies for human diseases.
Antioxid Redox Signal 3:
341346, 2001.[ISI][Medline]
- Epperly MW,
Travis EL, Sikora C, and Greenberger JS. Manganese [correction of
Magnesium] superoxide dismutase (MnSOD) plasmid/liposome pulmonary
radioprotective gene therapy: modulation of irradiation-induced mRNA for IL-1,
TNF-
, and TGF-
correlates with delay of organizing
alveolitis/fibrosis. Biol Blood Marrow Transplant
5: 204214,
1999.[Medline]
- Eppihimer MJ,
Russell J, Langley R, Vallien G, Anderson DC, and Granger DN. Differential
expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) in
murine tissues. Microcirculation
5: 179188,
1998.[Medline]
- Eppinger MJ,
Deeb GM, Bolling SF, and Ward PA. Mediators of ischemia-reperfusion injury
of rat lung. Am J Pathol 150:
17731784, 1997.[Abstract]
- Esmon CT and
Owen WG. Identification of an endothelial cell cofactor for
thrombin-catalyzed activation of protein C. Proc Natl Acad Sci
USA 78:
22492252, 1981.[Abstract]
- Folz RJ,
Abushamaa AM, and Suliman HB. Extracellular superoxide dismutase in the
airways of transgenic mice reduces inflammation and attenuates lung toxicity
following hyperoxia. J Clin Invest
103: 10551066,
1999.[Abstract/Free Full Text]
- Fox RB, Hoidal
JR, Brown DM, and Repine JE. Pulmonary inflammation due to oxygen
toxicity: involvement of chemotactic factors and polymorphonuclear leukocytes.
Am Rev Respir Dis 123:
521523, 1981.[ISI][Medline]
- Freeman BA and
Crapo JD. Biology of disease: free radicals and tissue injury.
Lab Invest 47:
412426, 1982.[ISI][Medline]
- Fridovich I. Superoxide radical and superoxide dismutases.
Annu Rev Biochem 64:
97112, 1995.[ISI][Medline]
- Gole MD, Souza
JM, Choi I, Hertkorn C, Malcolm S, Foust RF III, Finkel B, Lanken PN, and
Ischiropoulos H. Plasma proteins modified by tyrosine nitration in acute
respiratory distress syndrome. Am J Physiol Lung Cell Mol
Physiol 278:
L961L967, 2000.[Abstract/Free Full Text]
- Gow A, Branco
F, Cristofidou-Solomidou M, Schultz L, Albelda S, and Muzykantov V.
Immunotargeting of glucose oxidase: intracellular production of
H2O2 and endothelial oxidative stress. Am J
Physiol Lung Cell Mol Physiol 277:
L271L281, 1999.[Abstract/Free Full Text]
- Greenwald RA. Superoxide dismutase and catalase as
therapeutic agents for human diseases. Free Radic Biol
Med 8:
201209, 1990.[ISI][Medline]
- Gurubhagavatula I, Amrani Y, Pratico D, Ruberg FL, Albelda SM, and
Panettieri RA Jr. Engagement of human PECAM-1 (CD31) on human endothelial
cells increases intracellular calcium ion concentration and stimulates
prostacyclin release. J Clin Invest
101: 212222,
1998.[Abstract/Free Full Text]
- Igarashi R,
Hoshino J, Ochia A, Morizawa Y, and Mizushima Y. Lecithinized superoxide
dismutase enhances its pharmacologic potency by increasing its cell membrane
affinity. J Pharmacol Exp Ther
271: 16721677,
1994.[Abstract]
- Inoue M, Ebashi
I, Watanabe N, and Morino Y. Synthesis of a superoxide dismutase
derivative that circulates bound to albumin and accumulates in tissues whose
pH is decreased. Biochemistry
28: 66196624,
1989.[ISI][Medline]
- Jankov RP,
Negus A, and Tanswell AK. Antioxidants as therapy in the newborn: some
words of caution. Pediatr Res
50: 681687,
2001.[Abstract/Free Full Text]
- Kennel SJ,
Falcioni R, and Wesley JW. Microdistribution of specific rat monoclonal
antibodies to mouse tissues and human tumor xenografts. Cancer
Res 51:
15291536, 1991.[Abstract]
- Kiely JM,
Cybulsky MI, Luscinskas FW, and Gimbrone MA Jr. Immunoselective targeting
of an anti-thrombin agent to the surface of cytokine-activated vascular
endothelial cells. Arterioscler Thromb Vasc Biol
15: 12111218,
1995.[Abstract/Free Full Text]
- Langer R.
Drug delivery. Drugs on target. Science
293: 5859,
2001.[Free Full Text]
- Lee YM,
Hybertson BM, Cho HG, and Repine JE. Platelet-activating factor induces
lung inflammation and leak in rats: hydrogen peroxide production along
neutrophil-lung endothelial cell interfaces. J Lab Clin
Med 140:
312319, 2002.[ISI][Medline]
- Leff J, Parsons
P, Day C, Taniguchi N, Jochum M, Fritz H, Moore F, Moore E, McCord J, and
Repine J. Serum antioxidants as predictors of adult respiratory distress
syndrome in patients with sepsis. Lancet
341: 777780,
1993.[ISI][Medline]
- Li C and
Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation
injury. Am J Physiol Cell Physiol
282: C227C241,
2002.[Abstract/Free Full Text]
- Li L, Crockett
E, Wang DH, Galligan JJ, Fink GD, and Chen AF. Gene transfer of
endothelial NO synthase and manganese superoxide dismutase on arterial
vascular cell adhesion molecule-1 expression and superoxide production in
deoxycorticosterone acetate-salt hypertension. Arterioscler Thromb
Vasc Biol 22:
249255, 2002.[Abstract/Free Full Text]
- Li S, Tan Y,
Viroonchatapan E, Pitt BR, and Huang L. Targeted gene delivery to
pulmonary endothelium by anti-PECAM antibody. Am J Physiol Lung
Cell Mol Physiol 278:
L504L511, 2000.[Abstract/Free Full Text]
- Maruyama K,
Kennel SJ, and Huang L. Lipid composition is important for highly
efficient target binding and retention of immunoliposomes. Proc
Natl Acad Sci USA 87:
57445748, 1990.[Abstract]
- McIntosh DP,
Tan XY, Oh P, and Schnitzer JE. Targeting endothelium and its dynamic
caveolae for tissue-specific transcytosis in vivo: a pathway to overcome cell
barriers to drug and gene delivery. Proc Natl Acad Sci
USA 99:
19962001, 2002.[Abstract/Free Full Text]
- Minshall RD,
Pavcnik D, Halushka PV, and Hermsmeyer K. Progesterone regulation of
vascular thromboxane A2 receptors in rhesus monkeys. Am
J Physiol Heart Circ Physiol 281:
H1498H1507, 2001.[Abstract/Free Full Text]
- Mulligan MS,
Till GO, Smith CW, Anderson DC, Miyasaka M, Tamatani T, Todd RF III, Issekutz
TB, and Ward PA. Role of leukocyte adhesion molecules in lung and dermal
vascular injury after thermal trauma of skin. Am J
Pathol 144:
10081015, 1994.[Abstract]
- Murciano JC,
Muro S, Koniaris L, Christofidou-Solomidou M, Harshaw D, Albelda S, Granger D,
Cines D, and Muzykantov VR. ICAM-directed vascular immunotargeting of
plasminogen activators to the endothelial luminal surface.
Blood. In press.
- Muro S,
Wiewrodt R, Koniaris L, Albelda S, Muzykantov V, and Koval M. Antibody
conjugates targeted to endothelial cell adhesion molecules are internalized
via a protein kinase C-dependent pathway that resembles macropinocytosis.
J Cell Sci 116:
15991609, 2003.[Abstract/Free Full Text]
- Muzykantov V. Immunotargeting of drugs to the pulmonary
vascular endothelium as a therapeutic strategy.
Pathophysiology 5:
1533, 1998.
- Muzykantov V,
Atochina E, Ischyropoulos H, Danilov S, and Fisher A. Immunotargeting of
anti-oxidant enzymes to the pulmonary endothelium. Proc Natl Acad
Sci USA 93:
52135218, 1996.[Abstract/Free Full Text]
- Muzykantov V,
Christofidou-Solomidou M, Balyasnikova I, Harshaw D, Schultz Fisher AB, and
Albelda SM. Streptavidin facilitates internalization and pulmonary
targeting of an anti-endothelial cell antibody (PECAM-1): a novel strategy for
intraendothelial drug delivery. Proc Natl Acad Sci USA
96: 23792384,
1999.[Abstract/Free Full Text]
- Muzykantov V,
Martynov A, Puchnina E, and Danilov S. In vivo administration of glucose
oxidase conjugated with monoclonal antibody to ACE: targeting into the lung.
Am Rev Respir Dis 136:
14641473, 1989.
- Muzykantov VR. Targeting of superoxide dismutase and
catalase to vascular endothelium. J Control Release
71: 121,
2001.[ISI][Medline]
- Nakada MT, Amin
K, Christofidou-Solomidou M, O'Brien CD, Sun J, Gurubhagavatula I, Heavner GA,
Taylor AH, Paddock C, Sun QH, Zehnder JL, Newman PJ, Albelda SM, and DeLisser
HM. Antibodies against the first Ig-like domain of human platelet
endothelial cell adhesion molecule-1 (PECAM-1) that inhibit PECAM-1-dependent
homophilic adhesion block in vivo neutrophil recruitment. J
Immunol 164:
452462, 2000.[Abstract/Free Full Text]
- Nelson SK, Gao
B, Bose S, Rizeq M, and McCord JM. A novel heparin-binding, human
chimeric, superoxide dismutase improves myocardial preservation and protects
from ischemia-reperfusion injury. J Heart Lung
Transplant 12:
12961303, 2002.
- Nemzek JA, Call
DR, Ebong SJ, Newcomb DE, Bolgos GL, and Remick DG. Immunopathology of a
two-hit murine model of acid aspiration lung injury. Am J Physiol
Lung Cell Mol Physiol 278:
L512L520, 2000.[Abstract/Free Full Text]
- Newman PJ.
The biology of PECAM. J Clin Invest
99: 37,
1997.[Free Full Text]
- Pittet JF,
Mackersie RC, Martin TR, and Matthay MA. Biological markers of acute lung
injury: prognostic and pathogenetic significance. Am J Respir Crit
Care Med 155:
11871205, 1997.[ISI][Medline]
- Pratico D,
Iuliano L, Mauriello A, Spagnoli L, Lawson JA, Mclouf J, Violi F, and
FitzGerald GA. Localization of distinct F-2-isoprostanes in human
atherosclerotic lesions. J Clin Invest
100: 20282034,
1997.[Abstract/Free Full Text]
- Predescu D,
Predescu S, and Malik AB. Transport of nitrated albumin across continuous
vascular endothelium. Proc Natl Acad Sci USA
99: 1393213937,
2002.[Abstract/Free Full Text]
- Predescu SA,
Predescu DN, and Palade GE. Endothelial transcytotic machinery involves
supramolecular protein-lipid complexes. Mol Biol Cell
12: 10191033,
2001.[Abstract/Free Full Text]
- Punch J, Rees
R, Cashmere B, Oldham K, Wilkins E, and Smith DJ. Acute lung injury
following reperfusion after ischemia in the hind limbs of rats. J
Trauma 31:
760765, 1991.[ISI][Medline]
- Reynolds PN,
Nicklin SA, Kaliberova L, Boatman BG, Grizzle WE, Balyasnikova IV, Baker AH,
Danilov SM, and Curiel DT. Combined transductional and transcriptional
targeting improves the specificity of transgene expression in vivo.
Nat Biotechnol 19:
838843, 2001.[ISI][Medline]
- Scherpereel A,
Rome JJ, Wiewrodt R, Watkins SC, Harshaw DW, Alder S, Christofidou-Solomidou
M, Haut E, Murciano JC, Nakada M, Albelda SM, and Muzykantov VR.
Platelet-endothelial cell adhesion molecule-1-directed immunotargeting to
cardiopulmonary vasculature. J Pharmacol Exp Ther
300: 777786,
2002.[Abstract/Free Full Text]
- Scherpereel A,
Wiewrodt R, Christofidou-Solomidou M, Gervais R, Murciano JC, Albelda SM, and
Muzykantov VR. Cell-selective intracellular delivery of a foreign enzyme
to endothelium in vivo using vascular immunotargeting. FASEB
J 15:
416426, 2001.[Abstract/Free Full Text]
- Shenkar R,
Coulson WF, and Abraham E. Anti-transformation growth factor-
monoclonal antibodies prevent lung injury in hemorrhaged mice. Am J
Respir Cell Mol Biol 11:
351357, 1994.[Abstract]
- Siflinger-Birnboim A and Malik AB. Neutrophil adhesion to
endothelial cells impairs the effects of catalase and glutathione in
preventing endothelial injury. J Cell Physiol
155: 234239,
1993.[ISI][Medline]
- Sittipunt C,
Steinberg KP, Ruzinski JT, Myles C, Zhu S, Goodman RB, Hudson LD, Matalon S,
and Martin TR. Nitric oxide and nitrotyrosine in the lungs of patients
with acute respiratory distress syndrome. Am J Respir Crit Care
Med 163:
503510, 2001.[Abstract/Free Full Text]
- Turrens JF,
Crapo JD, and Freeman BA. Protection against oxygen toxicity by
intravenous injection of liposome-entrapped catalase and superoxide dismutase.
J Clin Invest 73:
8795, 1984.[ISI][Medline]
- Vujaskovic Z,
Batinic-Haberle I, Rabbani ZN, Feng QF, Kang SK, Spasojevic I, Samulski TV,
Fridovich I, Dewhirst MW, and Anscher MS. A small molecular weight
catalytic metalloporphyrin antioxidant with superoxide dismutase (SOD) mimetic
properties protects lungs from radiation-induced injury. Free Radic
Biol Med 33:
857863, 2002.[ISI][Medline]
- Ward P and
Hanninghake G. Lung inflammation and fibrosis. Am J Respir Crit
Care Med 157:
S123S129, 1998.[ISI][Medline]
- Warner RL,
Lewis CS, Beltran L, Younkin EM, Varani J, and Johnson KJ. The role of
metalloelastase in immune complex-induced acute lung injury. Am J
Pathol 158:
21392144, 2001.[Abstract/Free Full Text]
- Wiewrodt R,
Thomas AP, Cipelletti L, Christofidou-Solomidou M, Weitz DA, Feinstein SL,
Schaffer D, Albelda SM, Koval M, and Muzykantov V. Size-dependent
immunotargeting of cargo materials to endothelial cells via poorly
internalizable surface adhesion molecules. Blood
99: 912922,
2002.[Abstract/Free Full Text]
- Zwacka RM, Zhou
W, Zhang Y, Darby CJ, Dudus L, Halldorson J, Oberley L, and Engelhardt JF.
Redox gene therapy for ischemia/reperfusion injury of the liver reduces AP1
and NF-
B activation. Nat Med
4: 698704,
1998.[ISI][Medline]