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EDITORIAL FOCUS
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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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drug delivery; acute lung injury; platelet/endothelial cell adhesion molecule
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.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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).
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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).
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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, 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.
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Oxidative stress induced by injection of 60–75 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).
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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).
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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.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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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.
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