nature biotechnology • VOLUME 21 • APRIL 2003 • www.nature.com/naturebiotechnology

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Immunotargeting of catalase to the pulmonaryendothelium alleviates oxidative stress andreduces acute lung transplantation injury

Benjamin D. Kozower1,5, Melpo Christofidou-Solomidou2,5, Thomas D. Sweitzer3, Silvia Muro3, Donald G. Buerk3,Charalambos C. Solomides4, Steven M. Albelda2, G. Alexander Patterson1, and Vladimir R. Muzykantov3*

Published online 24 March 2003; doi:10.1038/nbt806

Vascular immunotargeting may facilitate the rapid and specific delivery of therapeutic agents to endothelialcells. We investigated whether targeting of an antioxidant enzyme, catalase, to the pulmonary endotheliumalleviates oxidative stress in an in vivo model of lung transplantation. Intravenously injected enzymes, conju-gated with an antibody to platelet-endothelial cell adhesion molecule-1, accumulate in the pulmonary vascu-lature and retain their activity during prolonged cold storage and transplantation. Immunotargeting of catalaseto donor rats augments the antioxidant capacity of the pulmonary endothelium, reduces oxidative stress, ame-liorates ischemia-reperfusion injury, prolongs the acceptable cold ischemia period of lung grafts, and improvesthe function of transplanted lung grafts. These findings validate the therapeutic potential of vascular immuno-targeting as a drug delivery strategy to reduce endothelial injury. Potential applications of this strategy includeimproving the outcome of clinical lung transplantation and treating a wide variety of endothelial disorders.

The targeted delivery of drugs to endothelial cells has enormouspotential for the treatment of a variety of disorders, including cancer,cardiovascular, and pulmonary diseases1–4. Vascular immunotarget-ing using antibodies directed against surface endothelial determi-nants facilitates site-specific targeting5. For example, diverse cargocompounds conjugated with monoclonal antibodies to platelet-endothelial cell adhesion molecule-1 (anti-PECAM) accumulateintracellularly in the pulmonary endothelium after intravascularinjection in animals6–9. We evaluated the therapeutic potential ofPECAM-directed targeting of an antioxidant enzyme, catalase, toalleviate vascular oxidative stress and ischemia-reperfusion injuryassociated with lung transplantation.

Endothelial cells are a major source and target for reactive oxygenspecies during ischemia-reperfusion injury10. Endothelial oxidativestress causes thrombosis, inflammation, increased vascular perme-ability, and leukocyte recruitment11–14. Ischemia-reperfusion injuryis a substantial problem in 15–20% of lung transplant recipients andis the most common cause of primary graft failure15. Primary graftfailure is an acute lung injury manifesting as hypoxemia, pulmonaryedema, and prolonged ventilator dependence. It remains the princi-pal cause of perioperative morbidity and mortality16–18.

Antioxidant enzymes, including catalase, alleviate oxidative stress invarious models. However, their effects have been fairly modest andinconsistent in vivo, in part due to suboptimal endothelial bindingspecificity and intracellular delivery19. Our data demonstrate thatPECAM-directed vascular immunotargeting facilitates the delivery ofcatalase to the pulmonary endothelium in rats, reduces oxidative stress,and ameliorates acute lung-graft injury after prolonged cold storage.

ResultsPulmonary targeting and activity of anti-PECAM/β-galactosi-dase conjugate in rats. To evaluate PECAM-directed targeting ofactive cargo enzymes to the lung, we injected rats intravenouslywith β-galactosidase (β-Gal) conjugated with anti-PECAM orcontrol IgG. Anti-PECAM/β-Gal, but not IgG/β-Gal, markedlyaugmented β-Gal activity in the lungs within 7.5 minutes of injec-tion (P < 0.001) (Fig. 1A). β-Gal activity in the lungs was signifi-cantly higher than in liver, heart, and kidney (84.6 ± 6.0 versus47.6 ± 2.2, 11.2 ± 0.8, and 9.8 ± 1.1 mU/mg of protein, respective-ly; P ≤ 0.01). The activity of anti-PECAM/β-Gal in the lungsdeclined within 1 hour after intravenous injection (Fig. 1B).

For PECAM-directed targeting to be useful for protecting trans-planted lung grafts against ischemia-reperfusion injury, conju-gates should remain active during cold ischemia, transplantation,and reperfusion. We injected rats with anti-PECAM/β-Gal imme-diately before lung-graft harvest and stored grafts at either 37 °Cor 4 °C. β-Gal activity was stable for 18 hours at 4 °C (Fig. 1C).β-Gal activity in lung grafts stored ex vivo at 37 °C declined tobackground after 12 hours, but at a slower rate than in vivo (com-pare with Fig. 1B). Furthermore, lung grafts transplanted after 6 hours of cold storage at 4 °C (conditions similar to clinical lungtransplantation) retained 60% of their peak β-Gal activity at thetime of reperfusion (Fig. 1D).

Effects of anti-PECAM/catalase conjugate in cultured cells andperfused rat lungs. Anti-PECAM/catalase, but not IgG/catalase,accelerated the rate of H2O2 decay (**, P < 0.01), thus augmentingthe antioxidant capacity of human umbilical vein endothelial cells

1Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, MO 63110. 2Department of Medicine and 3Institute for EnvironmentalMedicine, University of Pennsylvania Medical School, Philadelphia, PA 19104. 4Department of Pathology, Temple University Hospital, Philadelphia, PA 19140.

5The first two authors contributed equally to this study. *Corresponding author (muzykant@mail.med.upenn.edu).

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(HUVECs) (Fig. 2A). Anti-PECAM/catalase-mediated accelerationof H2O2 decay and protection against H2O2 toxicity (as determinedby 51Cr release) were PECAM specific and were not seen in controlREN cells lacking PECAM expression (Fig. 2B, C).

HUVECs internalized anti-PECAM/catalase (conjugate diameter100–250 nm), as well as polymer nanoparticles coated with anti-PECAM and catalase, in a temperature-dependent fashion (Fig.2D). Three hours after uptake, anti-PECAM/catalase provided min-imal protection from H2O2, concordant with the kinetics of lysoso-mal trafficking (Fig. 2E). Analysis of 51Cr release confirmed that theprotective effect of anti-PECAM/catalase disappears 5 hours afterinternalization (data not shown).

To extend this study from a static cell culture model to a flow-adapted, whole-organ setting, we used a perfused rat lung model.When added to the perfusate buffer, anti-PECAM/125I-labeledcatalase, but not control IgG/125I-labeled catalase conjugate, accu-mulated in the lungs (*, P < 0.05) (Fig. 3A). Anti-PECAM/catalaseaccelerated the degradation of H2O2 in the perfusate (Fig. 3B),thus protecting the pulmonary endothelium from circulatingH2O2. In lungs treated with anti-PECAM/catalase, bothangiotensin-converting enzyme activity in the perfusate (a markerof endothelial injury) and the ratio of wet to dry weight of thelung tissue (an index of pulmonary edema) were close to those incontrols (Fig. 3C, D).

Anti-PECAM/catalase conjugate ameliorates lung transplantischemia-reperfusion injury. To investigate anti-PECAM/catalaseprotection in a clinically relevant setting of acute lung injury, weused a rodent model of lung transplantation. Immediately beforelung-graft harvest, we injected donor rats with anti-PECAM/cata-lase, a mixture of nonconjugated anti-PECAM and catalase, orsaline and stored grafts for 18 hours at 4 °C before transplantation.

We also injected a separate recipient group with anti-PECAM/catalase at the time of post-transplantationreperfusion and included a sham thoracotomy group,without transplantation, as a negative injury control.Lung grafts were transplanted and their function wasassessed 4 hours after reperfusion.

Anti-PECAM/catalase treatment of donor ratsmarkedly reduced lung-graft edema, alveolar exudate,and inflammatory infiltrate as compared to the salinegroup. In fact, the lungs of the anti-PECAM/catalasegroup resembled those of sham-operated rats (Fig.4A–C). Morphometric analysis showed that anti-PECAM/catalase treatment also reduced the numberof intra-capillary/interstitial and intra-alveolar neu-trophil leukocytes, which approached those of thesham control lungs (P < 0.01 versus saline control)(Fig. 4D). Myeloperoxidase levels in lung homogenatesfrom the anti-PECAM/catalase-treated group weresignificantly lower than those for the group treatedwith a mixture of nonconjugated anti-PECAM andcatalase (0.24 ± 0.08 arbitrary units (AU) versus 0.54 ±0.19; P < 0.05). The mean myeloperoxidase level insham-operated rats was 0.09 ± 0.03 AU.

Treatment of donor rats with anti-PECAM/catalaseconjugate, but not a mixture of the conjugate compo-nents, markedly reduced the lung-graft wet-to-dry-weight ratio (P = 0.001 versus saline control) (Fig.4E). Furthermore, anti-PECAM/catalase treatment ofdonor rats improved the oxygenation of the isolatedlung graft as compared with the saline control group(P < 0.001) to a level near that of the uninjured shamgroup (Fig. 4F).

Anti-PECAM/catalase conjugate alleviates ischemia-reperfusion injury oxidative stress. To evaluate the extent of oxida-tive injury and identify products of tissue oxidation, we stainedtransplanted lungs with antibodies directed against nitrotyrosine (amarker of oxidative protein nitration) and iPF2α-III (an F2 iso-prostane reflecting lipid peroxidation)20. Importantly, oxidativestress was evident in the control lung grafts after 18 hours of coldischemia, before transplantation and reperfusion (Fig. 5A, B).Injection of donor rats with anti-PECAM/catalase markedlyreduced both nitrotyrosine staining (Fig. 5C–E) and iPF2α-III stain-ing (data not shown) in the transplanted lung grafts, indicating asignificant reduction in oxidative stress.

Safety profile of PECAM-directed immunotargeting. To exam-ine potential side effects of PECAM-directed immunotargeting, weassessed the effects of anti-PECAM/catalase injections in mice.Postmortem examination of hematoxylin and eosin (H&E)-stainedlung tissue from mice killed 1, 7, and 14 days after intravenousinjection with 300 µg of conjugate (12 mg/kg, >10 times higher perbody weight than the 0.9 mg/kg dose used in the rat transplantationexperiments) did not show any pathological alterations (Fig.6A–C). In addition, electron microscopy revealed no signs ofendothelial, interstitial, or alveolar injury in the pulmonary tissue ofthe mice (Fig. 6D, E).

To investigate the feasibility of transferring anti-PECAMimmunotargeting into the clinical domain, we evaluated the pul-monary targeting of anti-PECAM Fab fragments in intact anes-thetized rats. One hour after intravenous injection, 125I-labeledstreptavidin conjugated with biotinylated anti-PECAM Fab frag-ments showed preferential pulmonary targeting similar to thatfound with 125I-labeled streptavidin conjugated with whole biotiny-lated anti-PECAM (Fig. 6F, G). In addition, the amount of nonspe-cific hepatic and splenic uptake was lower for Fab-based conjugates.

Figure 1. Immunotargeting of anti-PECAM/β-Gal to the pulmonary endothelium in rats. Thedelivery and stability of β-Gal activity was examined after intravenous injection of anti-PECAM/β-Gal conjugates in rats. (A) Rats were injected with anti-PECAM/β-Gal, IgG/β-Gal, or saline. Lungs were harvested 7.5 min after injection and β-Gal activity wasmeasured in lung homogenates (*, P < 0.001 versus IgG control). (B) Activity of delivered β-Gal in the lung (�) and kidney (�) as a function of time after injection. Activity in the lungsis significant for 1 h as compared to the relatively constant, low β-Gal activity in the kidney(P = 0.05). (C) After anti-PECAM/β-Gal injection, lungs were flushed with saline, harvested,and stored at 37 °C (�) or 4 °C (�), and β-Gal activity in the lungs was measured atindicated time. (D) Lungs harvested and stored at 4 °C for 6 h were transplanted andsubjected to reperfusion in recipient rats. Rats were immediately killed and β-Gal activityremaining in the lungs was measured (*, P < 0.001 versus saline control).

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DiscussionTo improve the delivery of drugs and genetic materials to endothe-lial cells, vascular immunotargeting uses monoclonal antibodiesthat recognize endothelial surface antigens, including adhesionmolecules, angiotensin-converting enzyme, caveolae-localized antigens, VEGF receptor, and other mole-cules5–7,21–26. Notably, antibodies against pan-endothelialdeterminants, such as anti-PECAM, accumulate prefer-entially in the pulmonary vasculature. The pulmonarycirculation is the first major capillary bed encounteredafter intravenous injection, is exposed to the entire car-diac output, and contains 30% of the total endothelialsurface area27.

PECAM is an attractive target antigen for treatingclinical lung disease because the high PECAM expres-sion on the endothelial surface is not suppressed dur-ing pathologic conditions28. Although platelets andleukocytes express PECAM-1, the extent of expressionis orders of magnitude smaller than in endothelialcells28. Therefore, PECAM immunotargeting in vivo isdirected predominantly to endothelial cells7–9.Furthermore, the conjugation of proteins with anti-PECAM antibodies, providing conjugates with a diam-eter of 100–300 nm, facilitates both pulmonaryendothelial targeting and the intracellular uptake ofcargo compounds in intact mice and pigs6,8,9,29,30.However, the therapeutic value of PECAM-directedvascular immunotargeting has not been established.

Our study demonstrated the therapeutic potential ofPECAM-directed vascular immunotargeting in a clini-cally relevant model of lung transplantation. Pulmonaryvascular oxidative stress contributes to endothelial dys-function and acute lung injury during lung transplanta-tion and a variety of diseases11–18,31. Reactive oxygenspecies secreted by leukocytes diffuse into endothelialcells10,32, and endothelial cells synthesize reactive oxygenspecies during ischemia-reperfusion injury by diverseenzymatic pathways33. Furthermore, the endothelium isparticularly vulnerable to intracellular oxidative stressbecause intracellular reactive oxygen species are inacces-sible to circulating antioxidant enzymes34.

Antioxidant enzymes, such as catalase and superox-ide dismutase, have enormous potential for alleviatingoxidative stress but they have extremely short half-lives after intravenous injection. Modification withpolyethylene glycol, encapsulation in liposomes, andother methods are designed to improve the bioavail-ability and intracellular uptake of antioxidantenzymes35–37. However, even these means do notafford consistent protective effects in vivo, likelybecause of suboptimal specific affinity for and intra-cellular uptake by endothelium19.

In this study, immunotargeting of catalase protected endothelialcells in culture, perfused rat lungs, and an in vivo model of lungtransplantation. The extent of protection was sufficient to approxi-

Figure 2. Anti-PECAM/catalase immunotargeting, internalization, lysosomal trafficking, andprotection against H2O2 in endothelial cell culture. (A) Rate of H2O2 decay in the supernatantmedium of HUVECs pretreated with anti-PECAM/catalase (filled square) or IgG/catalase(filled circle) conjugates before challenge with H2O2. (B, C) HUVECs or REN cells (notexpressing PECAM) were treated in parallel with anti-PECAM/catalase and IgG/catalaseconjugates before challenge with H2O2. Cells were then assayed for (B) H2O2 decompositionand (C) H2O2-induced cytotoxicity (*, P < 0.05, **, P < 0.01 versus IgG/catalase controls).(D) HUVECs internalize anti-PECAM/catalase (upper panels) and nanoparticles coated with anti-PECAM and catalase (lower panels, images merged from phase-contrast andfluorescence micrographs). Internalized conjugates are green and conjugates on the cellsurface are yellow. Bar, 10 µm. (E) Lysosomal trafficking and inactivation of anti-PECAM/catalase nanoparticles. HUVECs incubated for 1, 2, and 3 h at 37 °C after internalization ofthe nanoparticles were inspected by fluorescence microscopy (upper panels; bar, 10 µm) orexposed to H2O2 and inspected by phase-contrast microscopy 15 min later (lower panels;bar, 50 µm). Upper panels: conjugates (green) and lysosomes (red) colocalize 3 h afteruptake (yellow). Insets show higher magnification of the areas indicated by arrows. Lowerpanels: the antioxidant protection by anti-PECAM/catalase nanoparticles was tested byincubation of HUVECs with 5 mM H2O2. Phase-contrast micrographs show protectedHUVEC monolayers 1 and 2 h, but not 3 h, after nanoparticles internalization.

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Figure 3. Anti-PECAM/catalase targeting protects perfusedlungs from oxidative stress. (A) Accumulation of anti-PECAM/125I-labeled catalase and IgG/125I-labeled catalase inthe lungs were measured 1 h after recirculating perfusion withconjugates in an isolated perfused whole rat lung model.(B) Isolated perfused rat lungs were treated as described in (A).After washout of excess conjugate, the lungs were challengedwith H2O2 in a nonrecirculating perfusion. H2O2 in the effluentwas measured with an electrochemical sensor and expressedin terms of current (I). (C, D) After challenge with H2O2 asdescribed in (B), both angiotensin-converting enzyme activity inthe perfusate (C) and lung wet-to-dry weight ratio after 1 h ofisolated lung perfusion (D) were determined.The dotted lineindicates the baseline value of normal lungs (*, P < 0.05).

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mate uninjured lungs in regard to several parameters: angiotensin-converting enzyme release in the perfusate, lung wet-to-dry-weightratio after transplantation, isolated lung-graft oxygenation, histolog-ical appearance, and results of immunostaining for tissue oxidation(Figs. 3, 4, and 5). These results validate the therapeutic value of vas-cular immunotargeting and identify PECAM as a potentially usefultarget. Furthermore, the protective effect of anti-PECAM/catalaseidentifies H2O2 as one of the principal pathological mediators oftransplantation-induced endothelial injury.

Interestingly, anti-PECAM/catalase did not reduce acute lunginjury when administered to recipient rats after transplantation (Fig.4, recipient group). Fisher et al. demonstrated that abrupt flowdeprivation of flow-adapted endothelial cells, in cell culture and inperfused lungs, produces an almost immediate activation of NADPHoxidase and generates reactive oxygen species, including H2O2, dur-ing the ischemic period10,33. In our transplantation model, nitrotyro-sine immunostaining showed that lung grafts suffer significantoxidative stress during cold ischemia, and others have also noticedthat oxidative stress occurs during lung-graft harvest and cold stor-age18 (Fig. 5). These data help to explain why anti-PECAM/catalasetreatment is more effective when administered to donor rats beforeorgan harvest than when administered after transplantation (Fig. 4,conjugate group).

The injection of nonconjugated anti-PECAM mixed with cata-lase did not reduce acute lung injury (Fig. 4, mixture group). Thisimportant control indicates that the inhibition of PECAM alone isnot responsible for the therapeutic benefit of anti-PECAM/cata-lase targeting. However, this result does not exclude the possibilitythat PECAM antibodies may provide a secondary benefit in othersettings by inhibiting the transmigration of leukocytes duringinflammation38,39.

Vascular immunotargeting has a number of characteristics ideal-ly suited for improving clinical transplantation. Anti-PECAM con-jugates attain their peak endothelial uptake within 10 minutes ofintravenous delivery (Fig. 1). This rapid targeting is crucial, as

organ donors could be treated at the time oforgan harvest. In addition, it would be rela-tively easy to inject therapeutic conjugatesdirectly into the pulmonary circulation orother afferent vessels, such as recentlydescribed in coronary arteries9. Endothelialischemia-reperfusion injury has an impor-tant role in transplantation injury in diverseorgans. Theoretically, the immunotargetingstrategy described may be useful in heart,liver, and kidney transplantation. It istempting to speculate that the targeting ofdiverse therapeutics (such as complementinhibitors or anti-thrombotic agents) willpermit more comprehensive managementof transplantation-associated pathology,including thrombosis40.

Further optimization of regimens of con-jugates administration is an important tech-nical challenge. Repetitive administration ofthe conjugates might produce immune reac-tions. Nevertheless, the use of immunotar-geting in the transplant setting may help tolimit immune reactions against antibody-enzyme conjugates. If the donor organ istreated, transplant recipients will only receiveconjugate that is already bound to the pul-monary endothelium (much of which isinternalized and will not be released into the

circulation), markedly reducing systemic exposure. Potential prob-lems in immunotargeting certain endothelial antigens are the inhibi-tion of their function or enhanced shedding or internalization. Forexample, immunotargeting of drugs to thrombomodulin appears topredispose lungs to vascular thrombosis20. However, the rapid kinet-ics of internalization, lysosomal trafficking, and conjugate inactiva-tion (Fig. 2) imply that potential side effects of anti-PECAM/catalasewill be transient and tolerable (and presumably will be confined tothe graft).

In support of this notion, light and electron microscopy showedno significant pathological changes in lungs after intravenous injec-tion of a relatively large dose of anti-PECAM/catalase (Fig. 6). Inaddition, our data demonstrate that anti-PECAM Fab fragments arean excellent targeting alternative to intact antibody (Fig. 6). Thiseliminates potential side effects mediated by Fc fragments and great-ly enhances the feasibility of evaluating the targeting of therapeuticcargoes to PECAM.

In summary, we validated the therapeutic potential of vascularimmunotargeting by treating oxidative stress. Our findings showed thatPECAM-targeted enzymes accumulated in the pulmonary vasculatureafter intravenous injection and retained their activity for a prolongedperiod of cold storage, transplantation, and reperfusion. PECAM-directed targeting delivered a therapeutic antioxidant enzyme, catalase,to the pulmonary endothelium that augmented antioxidant defenses,reduced oxidative stress, reduced transplantation-associated acute lunginjury, and improved recipient lung-graft function. Potential clinicalapplications may follow. This immunotargeting approach may beextremely valuable in several respects. First, it could potentially reduce ischemia-reperfusion injury after clinical lung transplantation.Second, pretreatment of grafts with immunoconjugates may increasethe amount of time that lung grafts can be successfully stored (cold ischemia), thus increasing the pool of donor lungs for use in clin-ical transplantation. In addition, this strategy has the potential toimprove the treatment of a variety of clinical disorders associated withendothelial injury.

Figure 4. Anti-PECAM/catalase ameliorates lung transplant ischemia-reperfusion injury. Three donorgroups of rats were treated at the time of lung-graft harvest with either saline, a mixture ofunconjugated anti-PECAM antibody and catalase (mixture), or anti-PECAM/catalase conjugate(conjugate). A recipient group received anti-PECAM catalase at the time of reperfusion (recipient) anda sham thoracotomy group, without transplantation, was used as a negative control (sham). (A–C)H&E-stained sections from lungs of (A) saline-treated, (B) conjugate-treated, and (C) sham-treatedrats killed 4 h after reperfusion (500×). Bar, 50 µm. (D) Counts for neutrophils (PMN) were performedin 10 randomly selected high-power fields at a magnification of 1,000×. Alveolar PMN and PMNpresent in interstitium and capillaries were counted separately (*, P < 0.01 versus saline control). (E)As a measure of ischemia-reperfusion injury, we measured lung-graft wet-to-dry ratio at 4 h afterreperfusion (*, P < 0.001 versus saline control). (F) Blood oxygenation was measured in the aortaafter the right hilum was ligated for 5 min (reflecting isolated left lung-graft oxygenation) (*, P < 0.001versus saline control).

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Experimental protocolImmunoconjugate preparation. Biotinylated catalase (20,000 U/mg) andSA-β-galactosidase (β-Gal; Sigma, St. Louis, MO) were conjugated withbiotinylated anti-PECAM or biotinylated IgG without loss of enzymaticactivity as described8,21. Conjugates had a mean diameter of 100–300 nm asassessed by dynamic light scattering29. β-Gal activity was measured in con-jugate preparations and organ homogenates using the β-Gal EnzymeActivity Assay Kit (Promega, Madison, WI) as described8.

Conjugate kinetics and organ distribution in intact rats. Anesthetizedmale Fischer 344 (F344) rats (250–300g) were injected intravenously withanti-PECAM/β-Gal conjugate, control polyclonal IgG/β-Gal conjugate, orsaline via the dorsal penile vein. All β-Gal conjugate experiments were per-formed using a dose of 100 µg β-Gal enzyme per conjugate injected.Animals were killed 7.5–120 min after injection (n = 4/time point) and thepulmonary and systemic circulation were flushed with heparinized saline(60 U/ml) before harvesting the lungs, hearts, livers, and kidneys.

Orthotopic left lung transplantation. Orthotopic left lung transplantationwas done by the modified cuff technique41,42. Briefly, donor F344 rats wereanesthetized, heparinized, and mechanically ventilated. After laparoster-notomy, the main pulmonary artery was flushed with 20 ml of 4 °C low-potassium dextran glucose buffer (Gibco-BRL, Carlsbad, CA) at 20 cmH2O pressure. The heart-lung block was removed with the lungs inflated atend-tidal volume (2.5 ml). The left lung graft was prepared and stored inlow-potassium dextran glucose buffer at 4 °C until transplantation.

Recipient F344 rats were anesthetized, intubated, and mechanically venti-lated. We performed a left thoracotomy, dissected the pulmonary hilum,and anastomosed the left lung graft using the modified cuff technique43.

Cell culture studies. HUVECs and REN cells grown in 24-well culturedishes29 were labeled with 51Cr (ref. 42), washed with serum-free medium,and incubated with the conjugates at 37 °C for 1 h. After washing to remove the unbound reagents, we added H2O2 to the cells and assayedfor H2O2 degradation and cytotoxicity (determined by both 51Cr releaseand morphological abnormalities of the endothelial monolayer)44.Internalization in HUVECs was visualized in a fluorescence microscope asdescribed previously29. HUVECs were incubated for 1 h at either 4 °C or 37 °Cwith standard anti-PECAM/catalase conjugates or with FITC-labelednanospheres (200 nm diameter) coated with anti-PECAM and catalase.After washing, the cells were fixed and stained with a Texas Red–conjugatedgoat anti-mouse IgG to reveal the conjugates or nanospheres on the cellsurface (double labeling, yellow color). The cells treated with anti-PECAM/catalase were subsequently permeabilized and stained with aFITC-goat anti-mouse IgG. Thus, green color (single labeling) revealedinternalized conjugates or nanospheres. In a separate series, HUVECsloaded with red lysosomal marker (Texas Red dextran, 2 mg/ml) wereincubated at 4 °C with FITC-labeled anti-PECAM/catalase nanospheres,warmed, and incubated for a further 1–3 h at 37 °C to detect nanospheresin lysosomes, visible as double-labeled yellow particles.

Perfused rat lungs. Lungs were isolated from anesthetized Sprague-Dawleyrats (170–200 g), ventilated in a thermostat-controlled chamber using anSAR-830 rodent ventilator (CWE, Ardmore, Pennsylvania), and perfusedwith recirculating Krebs-Ringer buffer (pH 7.4) as previously described45.Unless otherwise indicated, we perfused 1 µg of anti-PECAM/125I-labeledcatalase or 100 µg of unlabeled anti-PECAM/catalase for 1 h followed by 5 min of a nonrecirculating buffer to eliminate unbound material. H2O2

was added to the perfusate (5 mM final concentration) and the lungs wereperfused for an additional hour. The angiotensin-converting enzyme activ-ity was determined in the perfusate using a fluorogenic substrate45. The

Figure 5. Anti-PECAM/catalase alleviates oxidative stress in transplantedlungs. Lung tissue sections were stained with antibody directed againstnitrotyrosine, a marker of protein oxidative nitration. Positive immuno-staining was revealed by secondary antibody conjugated with alkalinephosphatase (blue color). Neutral Red counterstaining was used to markindividual cells. (A) Lungs stained immediately after harvest. (B) Lungsstored at 4 °C for 18 h before staining. (C) Lungs harvested from saline-injected rats transplanted after 18 h at 4 °C and reperfused for 4 h.(D) Lungs harvested from anti-PECAM/catalase-injected rats transplantedafter 18 h at 4 °C and reperfused for 4 h. (E) Lungs from sham-operatedlungs. Bar, 50 µm.

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EFigure 6. Safety of anti-PECAM/catalase immunotargeting. (A–E) Light(A–C) and electron (D, E) microscopy evaluation of murine lungs fixed 1 d(A, D), 1 week (B), and 2 weeks (C, E) after intravenous injection of anti-PECAM/catalase conjugate. Lu, vascular lumen; RBC, red blood cells;EC, endothelial cells; type II, type II epithelial cells in alveoli (alv). Bars:A–C, 50 µm; D, E, 2 µm; inset in E, 5 µm. (F, G) Tissue distribution of 125I-labeled SA/anti-PECAM conjugates (F) and 125I-labeled SA/anti-PECAM Fab conjugates (G) 1 h after injection in rats (mean ± s.e.m., n = 4). Closed bars show control IgG conjugates.

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concentration of H2O2 in the outflow was continuously measured with anelectrochemical sensor46 (n = 4 per group). The H2O2 sensor was fabricatedfrom platinum wire, polarized at an optimum H2O2 oxidation potential of+650 mV relative to an Ag/AgCl reference electrode, and calibrated at 37 °Cby adding small aliquots of 30% H2O2 stock solution to a stirred reservoirusing a precision syringe as described46. Electrochemical oxidation cur-rents were amplified with a sensitive electrometer. The output was low-pass-filtered using an analog circuit with 5-Hz cutoff and digitized bycomputer with 12-bit resolution at 1 sample/sec.

Effects of anti-PECAM/catalase conjugate on lung transplant ischemia-reperfusion injury. F344 rats were divided into five groups (n = 6 trans-plants/group) and 300 µg of catalase in a total volume of 500 µl was used forall intravenous treatments. Three donor groups were treated during lung har-vest with anti-PECAM/catalase, a mixture of unconjugated anti-PECAM andcatalase, or saline. A recipient group was treated with anti-PECAM/catalaseimmediately after reperfusion. The final group was a sham control (left tho-racotomy without transplantation).

Donor lungs were harvested and stored for 18 h before left lung trans-plantation. After transplantation or sham operation, the thoracotomy wasclosed in layers, pleural air was evacuated, and the transplanted rat wasextubated. At 4 h after reperfusion (or recovery, for the sham group), we re-anesthetized recipient rats and assessed the function of the isolated lunggrafts as previously described41,42. Briefly, the right hilum was ligated andthe left lung graft ventilated for 5 min, after which an arterial blood gasanalysis was done. The pulmonary artery was flushed with normal saline,and then the left lung graft was removed and its wet-to-dry-weight ratioand myeloperoxidase activity were determined.

Histology and morphometry. Transplanted lungs were fixed intratracheallywith formalin and processed for H&E staining as described20. Counts for neu-trophils (PMNs) were performed in ten randomly selected high-power fieldsat ×1,000 magnification under oil. Alveolar PMNs and PMNs present in inter-stitium and capillaries were counted separately. Immunohistochemistry wasperformed to evaluate oxidative stress using rabbit polyclonal antibodies spe-cific for iPF2α-III and nitrotyrosine as described20. Visualization was achievedusing an Alkaline Phosphatase Kit (Vector Laboratories, Burlingame, CA).

Biodistribution of Fab fragment-based anti-PECAM/streptavidin conju-gates in rats. F(ab)2 fragments prepared from anti-PECAM or controlmouse IgG (provided by M. Nakada, Centocor) were biotinylated and con-jugated with 125I-labeled streptavidin as previously described6. One hourafter intravenous injection of anesthetized rats, the rats were killed and 125Ilevel in the organs was determined as described21,22.

Statistical analysis. Data are reported as the mean ± s.e.m. Data were ana-lyzed using one-way analysis of variance. Multiple comparisons were madeusing Fisher’s least-significant-difference test.

Policy on experiments on animals. Animal studies were done in compli-ance with laws of states Pennsylvania and Missouri and were approved bythe protocols nos. 388100 and 19990188 by the Institutional Animal Careand Use Committee of the University of Pennsylvania and WashingtonUniversity, respectively.

AcknowledgmentsThe authors thank M. Nakada (Centocor, Malvern, Pennsylvania) for a gen-erous gift of anti-PECAM mAb 62, R. Wiewrodt and V. Shuvaev (Universityof Pennsylvania) for their advice and valuable help in characterization of theconjugates size by Dynamic Light Scattering, A.P. Thomas for help in conju-gate preparation and experiments with cell cultures, and D.W. Harshaw forhelp in experiments with perfused rat lungs. The authors also acknowledgethe help of T. Tagawa and S. Kanaan (Washington University) for their support with the transplantation experiments and R. Schuessler and B.W. McKane for their statistical and laboratory assistance. S.M. is support-ed by a fellowship from the Fundacion Ramon Areces (Spain). The work was supported by US National Institutes of Health SCOR in Acute Lung Injury(NHLBI HL 60290, Project 4 to V.R.M. and S.M.A.), ALA Research Grant(no. RG-087-N to M.C.S.) and National Institutes of Health grant 1 R01HL41281 (to G.A.P.).

Competing interest statementThe authors declare that they have no competing financial interests.

Received 11 December 2002; accepted 19 January 2003

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