Original Contribution

PRETREATMENT WITH POLYNITROXYL ALBUMIN (PNA) INHIBITSISCHEMIA-REPERFUSION INDUCED LEUKOCYTE-ENDOTHELIAL

CELL ADHESION

JANICE RUSSELL,* NAOTSUKA OKAYAMA ,* J. STEVEN ALEXANDER,* D. NEIL GRANGER,* and

CARLETON J. C. HSIA†

*Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, LA, USA, and†SynZyme Technologies, Inc., Irvine, CA, USA

(Received10 April 1997;Revised12 January1998;Accepted13 January1998)

Abstract—Recently published evidence indicates that polynitroxylated albumin (PNA) protects tissues against isch-emia/reperfusion (I/R) injury, possibly by enhancing tissue redox activity. The objective of this study was to determineif PNA treatment alters the leukocyte-endothelial cell adhesion that is normally elicited by I/R. PNA, human serumalbumin (HSA) or saline were administered (i.v.) 5 min before reperfusion. Venular diameter, red blood cell velocity,wall shear rate, systemic hematocrit, systemic arterial pressure, as well as the number of adherent and emigratedleukocytes were monitored in rat mesenteric venules before and after 20 min of ischemia and 30 min of reperfusion. Insaline-treated rats, I/R elicited a 5.3-fold increase in leukocyte adhesion and a 1.8-fold increase in leukocyte emigration.HSA-treated animals exhibited 4.0 and 2.3-fold increases in leukocyte adherence and emigration, respectively. InPNA-treated rats, the number of adherent leukocytes increased only 2.1-fold increase in adherent leukocytes, whileleukocyte emigration was completely inhibited. The PNA-induced attenuation of leukocyte adherence/emigration couldnot be attributed to alterations in systemic or local hemodynamics (red blood cell velocity or wall shear rate). PNA wasalso shown to be a potent inhibitor of xanthine-xanthine oxidase mediated adhesion of human neutrophils to culturedhuman endothelial cells. These findings indicate that PNA may protect tissues against I/R injury by attenuatingleukocyte-endothelial cell adhesion. © 1998 Elsevier Science Inc.

Keywords—Superoxide dismutase, Inflammation, Free radical scavengers

INTRODUCTION

Reperfusion of ischemic tissues results in an acute in-flammatory response that is characterized by an in-creased fluid filtration and steric hinderance (plugging)of leukocytes in capillaries, with a concomitant recruit-ment of adherent and emigrating leukocyte in down-stream postcapiliiary venules.1–7The results of numerousstudies suggest that there is a cause-effect relationshipbetween the accumulation of inflammatory cells and themicrovascular (and parenchymal cell) dysfunction thatare elicited by ischemia and reperfusion (I/R). Supportfor this contention is provided by studies demonstrating

an attenuated I/R injury in mutant mice that are deficientin leukocyte (CD11/CD18) or endothelial cell (P-selectinor ICAM-1) adhesion molecules8 or in animals receivingmonoclonal antibodies directed against glycoproteinsthat mediated leukocyte-endothelial cell adhesion.9 Aconsequence of these observations is the growing effortto develop therapeutic agents for I/R injury that are basedon their efficacy in inhibiting leukocyte-endothelial celladhesion. These agents include oxygen radical scaveng-ing enzymes such as superoxide dismutase (SOD)8,12 orcatalase,10 and nitric oxide-releasing compounds.11

In exploring the therapeutic application of the SOD-mimetic activity of stable nitroxide free radicals, we haverecently shown that covalent attachment of multiple ni-troxides to albumin (PNA) enhanced redox activity of thenitroxide, when compared to albumin-free nitroxide(4-hydroxyl-2,2,6,6-tetramethyl-peridenyl-1-oxyl (TPL)).13

The PNA was also shown to exhibit remarkable protective

Address correspondence to: D. Neil Granger, PhD, Department ofPhysiology, LSU Medical Center, 1501 Kings Hwy, P.O. Box 33932,Shreveport, Louisiana 71130-3932; Tel: 318-675-6013; Fax: 318-675-6005; E-Mail: dgrang@lsumc.edu.

This work is supported in-part by grants from the National Institutesof Health, NS 36119 (CJCH) and HL26441 (DNG).

Free Radical Biology & Medicine, Vol. 25, No. 2, pp. 153–159, 1998Copyright © 1998 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/98 $19.001 .00

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153

actions in a model of transient focal ischemia of the brain.14

While the beneficial effects of PNA may be attributed to itsprevention of superoxide-mediated tissue injury, it is alsopossible that PNA may afford protection against I/R injuryby interfering with leukocyte-endothelial cell adhesion. Thelatter possibility is supported by published reports showinga potent anti-adhesive effect of superoxide dismutase.1,11Inthe present communication, we demonstrate that PNA isindeed capable of significantly reducing the recruitment ofadherent and emigrating leukocytes in postcapillary venulesexposed to I/R. In addition, PNA was shown to be a potentinhibitor of xanthine-xanthine oxidase mediated adhesionof human neutrophils to cultured human umbilical veinendothelial cells. The covalently attached nitroxide on PNAappears to be responsible for the observed anti-inflamma-tory action of the drug.

MATERIALS AND METHODS

Test solutions

PNA (lot number SZ-111695) was prepared essen-tially as described previously.8 Briefly, human serumalbumin (HSA) solution was reacted with 40 molarequivalents of 4-(2-bromoacetamido)-2,2,6,6-tetrameth-ylpiperidinyl-1-oxyl (BrAcTPO) at 60° for 4 hours withmixing. The resulting mixture was extensively washedby diafiltration with sterile saline solution (McGaw Lab-oratories, Irvine, California) to remove albumin-free ni-troxide. The protein concentration was adjusted to 23.5g/dl. The solution was sterile-filtered, packaged into 20ml vials, and stored at 4°C until use. The nitroxideconcentration, determined using electron paramagneticresonance (EPR) spectroscopy, was 110 mM. (4-hydrox-yl-2,2,6,6-tetramethyl-peridenyl-1-oxyl (TPL) (Lot#SY3799) was custom synthesized by Isotech (Ohio).The stock solution was prepared by dissolving 86 mg ofTPL in 5 ml of 0.9% saline followed by 0.22mm filtra-tion before use.

In vivo assessment of leukocyte-endothelial celladhesion

Surgical procedure. Male Sprague–Dawley rats (200–380 g) were maintained on a purified laboratory diet andfasted for 18–24 h before each experiment. The animalswere anesthetized with thiobutabarbital (120 mg/kg,i.p.). A tracheotomy was performed to facilitate breath-ing throughout the experiment. The right carotid arterywas cannulated for measurement of systemic arterialblood pressure using a Statham P23A pressure trans-ducer (World Precision Instruments, Sarasota, FL). Theright jugular vein was cannulated for drug administra-tion. A midline abdominal incision was made to allow

for exteriorization of a section of the mesentery from thesmall intestine. A ligature of clear vinyl tubing (I.D. 0.50mm, O.D. 0.80 mm) was placed around the superiormesenteric artery, for induction of ischemia.

The rats were positioned on a 203 30-cm plexiglasboard in a way that allowed a selected section of mes-entery to be placed over a glass slide covering a 3.533.5-cm hole centered in the board. To minimize tissuedehydration, all exposed tissue was covered with gauzesoaked with bicarbonate-buffered saline (BBS). Theboard was mounted on the stage of an upright micro-scope. The mesentery was superfused at 2.5 ml/min withBBS bubbled with 5% CO2 and 95% N2. The superfu-sate was maintained at 37°C by pumping the solutionthrough a heat exchanger warmed with a constant-tem-perature circulator. Rectal temperature was monitoredand kept between 36.5°C and 37.5°C with an infraredheat lamp.

Single unbranched venules with diameters of 25–35mm and length.150mm were observed with an uprightmicroscope using an320 objective. A color cameraattached to the microscope allowed images to be re-corded on videotape and displayed on a monitor. Venulardiameter was measured on-line with a video caliper.Centerline red blood cell velocity was measured with anoptical Doppler velocimeter (Microcirculation ResearchInstitute, Texas A&M University). Venular blood flowwas calculated from the product of mean red cell velocityand microvascular cross-sectional area, assuming cylin-drical vessel geometry. Wall shear rate (­) was calcu-lated based on the Newtonian definition­ 5 8(Vmean/diameter). The number of adherent and emigratedleukocytes was determined off-line during playback ofvideo tape images. Leukocyte adherence was defined asthe number of leukocytes that remained stationary on thevessel wall for a period of.30 s in a 100mm length ofvenule, over a 5-min observation period. Emigration wasdefined as the number of extravascular leukocytes ob-served on the monitor screen during the same 5-minperiods. Leukocyte rolling velocity was estimated fromthe time taken for a leukocyte to roll a measured lengthof venule: an average of 5 cells was taken for eachobservation period.

Experimental protocols. After stabilization period of 30min, images from the mesenteric preparation were re-corded on video tape for 5 min. Immediately after that,ischemia was induced by occluding the ligature of clearvinyl tubing placed around the superior mesenteric ar-tery. After 15 min of ischemia, the animals receivedeither saline, HSA or PNA (5 ml/kg), injected intrave-nously. After 20 min of ischemia, the ligature was gentlyremoved, allowing blood to recirculate in the gut and

154 J. RUSSELL et al.

mesentery. Repeat measurements for all parameters wereobtained after 5, 10, 20 and 30 min of reperfusion.

In vitro assessment of neutrophil-endothelial celladhesion

Cultured endothelial cells. The procedures used to obtainhuman endothelial cells was approved by the Institu-tional Review Board for Human Research at the Louisi-ana State University Medical Center. Each subject pro-vided written consent and was paid for participating inthe study.

Human umbilical vein endothelial cells (HUVEC)were harvested from umbilical cords by collagenasetreatment as previously described.15 The cells wereplated in EGM (Endothelial Cell Growth Medium; Clo-netics) supplemented with 10% heat-inactivated fetalcalf serum (Hyclone Laboratories Inc., Logan, Utah),thymidine (2.4 mg/l; Sigma Chemical, St. Louis, MO),glutamine (230 mg/l, Gibco Laboratories, Gaithersburg,MD), heparin sodium (10 IU/ml, Sigma, St. Louis, MO),antibiotics (100 IU/ml penicillin, 100 mg/ml streptomy-cin, and 0.125 mg amphotericin B), and endothelial cellgrowth factor (80 mg/ml, Biomedical Technologies Inc.,Stoughton, MA). All other tissue culture reagents wereobtained from Gibco Laboratories. The cell cultures wereincubated at 37°C in a humidified atmosphere with 5%CO2 and expanded by brief trypsinization (0.25% trypsinin phosphate-buffered saline containing 0.02% ethyl-enediamine tetraacetic acid). Primary passage HUVECwere seeded into gelatin (0.1%) and fibronectin-coated(25 mg/ml) 11 mm, 48-well tissue culture plates(GIBCO) and used when confluent. Culture medium wasreplaced every second day. Only primary cultures wereused for these studies. Cells were identified as endothe-lial cells by their cobblestone appearance at confluency,positive labeling with (1) acetylated low density lipopro-tein labeled with 1,11-dioctadecyl-1 3,3,31,31,3-tetram-ethylindocarbocyanine perchlorate (Dil-Ac-LDL; Bio-medical Technologies, Inc.) and (2) mouse antihumanfactor VIII (Calbiochem, San Diego, CA).

Neutrophil isolation. Human neutrophilic polymorpho-nuclear leukocytes were isolated from venous blood ofhealthy adults using standard dextran sedimentation andgradient separation on Histopaque 1077 (Sigma).15 Thisprocedure yields a polymorphonuclear leukocyte popu-lation 95–98% viable (by trypan blue exclusion) and98% pure (by acetic acid-crystal violet staining).

Static adhesion assay. Isolated neutrophils were sus-pended in phosphate-buffered saline (PBS) and radiola-beled by incubating PMN (at a concentration of 23 107

cells/ml) with 30 mCi Na51 CrO4 per ml neutrophil

suspension at 37°C for 1 h. The cells were washed twicewith 4°C PBS, spun at 250 g for 8 min to removeunincorporated radioactivity and resuspended in plasmafree Hanks’ balanced salt solution (HBSS). Labeled neu-trophils were added to HUVEC monolayers at a neutro-phil-to-HUVEC ratio of 10:1. After co-incubation (30min), the percent added neutrophils that adhered to theHUVEC monolayers was quantified.15

Experimental protocols. In order to elicit neutrophil-endothelial cell adhesion in a manner consistent withmechanisms described for I/R-induced leukocyte adhe-sion in vivo,1 HUVEC monolayers were treated for 30min with xanthine (Sigma Chemicals, 0.1 mM) and xan-thine oxidase (Sigma Chemicals, St. Louis) at concen-trations between 0 and 20 mU/ml. Since 10 mU/mlxanthine oxidase (in the presence of 0.1 mM xanthine)significantly increased neutrophil adhesion, we used thisstimulus to test the effect of polynitroxyl-albumin (1–40mM) on the adhesion of neutrophil to HUVEC. In someexperiments, albumin (bovine; Sigma, St. Louis, MO)was tested in the in vitro model of xanthine-xanthineoxidase mediated neutrophil adhesion. The concentra-tions of albumin used in these experiments (0-0.6 g%)were selected to cover the range of albumin levels em-ployed in the PNA studies.

After incubating endothelial cells with xanthine-xan-thine oxidase6 either PNA or albumin, 13 106 51Cr-labeled PMN were added to each well and allowed tostatically adhere for an additional 30 min. Monolayerswere washed twice with 500ml of HBSS and adherentPMN lysed by the addition of 500ml 1 M NaOH.Samples were collected and radioactivity measured on agamma counter. The percentage of adherent neutrophilsis expressed as the amount of radioactivity in each welldivided by the total radioactivity for the neutrophilsadded to the well.

Statistics

All data are presented as mean6 SE and representvalues obtained from 7 animals in each experimentalgroup. Standard statistical procedures (e.g., one-wayanalysis of variance, Bartlett’s test for homogeniety ofvariances, and Bonferroni multiple comparisons test)were applied to the data.

RESULTS

Figures 1–3 illustrate the effects of PNA on thechanges in leukocyte behavior within postcapillaryvenules elicited by I/R. At 5 min after reperfusion, asignificant increase in the number of adherent leukocytes

155Polynitroxyl albumin and leukoxyte-endothelial cell adhesion

was noted in all experimental groups (Fig. 1). As thereperfusion period continued, the number of adherentleukocytes increased progressively in the saline and al-bumin groups, which were not significantly differentfrom each other at any time point. Compared with base-line values, the increase in adherent cells after 30 min ofreperfusion was approximately 5-fold in the saline group(10.16 1.5 cells per 100 vs. 1.96 0.5, p , .001) and

approximately 4-fold in the HSA group (12.16 1.6 cellsper 100 vs. 3.06 0.7,p , .001).Despite a tendency toincrease at 5 minutes of reperfusion, leukocyte adherencein the PNA group did not change significantly through-out reperfusion.

In general, the leukocyte emigration responses to I/Rin the different experimental groups (Fig. 2) paralleledthose observed for leukocyte adherence. Compared with

Fig. 1. Effects of 20 min of ischemia and 30 min of reperfusion on the number of adherent leukocytes in rat mesenteric venules.*Significant difference relative to HSA;†significantly different from saline.

Fig. 2. Effects of 20 minutes of ischemia and 30 min of reperfusion on the number of emigrated leukocytes surrounding rat mesentericvenules. *Significant difference relative to HSA;†significantly different from saline.

156 J. RUSSELL et al.

baseline values, leukocyte emigration in the saline groupwas increased approximately 1.8-fold (7.16 2.0 cellsper field vs. 3.96 0.9) after 30 min of reperfusion, andapproximately 2.3-fold (9.46 2.9 vs. 4.16 1.4) in theHSA group. However, there was no change in leukocyteemigration in the PNA-treated group throughout the ex-periment.

Leukocyte rolling velocity tended to fall followingreperfusion in all experimental groups, with no signifi-cant differences noted between the saline, HSA, andPNA groups at any time point.

The dose of PNA and HSA employed in this studyresulted in a 0.4 to 0.5 gm% increase in total plasmaprotein concentration (from a control value of 6.9–7.0gm%).

Figure 4 summarizes the changes in venular wallshear rate caused by I/R in the different treatment groups.Although shear rate fell following reperfusion in thesaline-treated animals, no such change was noted in ratsreceiving either HSA or PNA. Red cell velocity did notchange in the HSA or PNA groups, but fell by 34% in thesaline group (3.26 0.2 mm/sec vs. 2.16 0.3 at 30 minof reperfusion,p , .05).

Figure 5 demonstrates the effects of different concen-trations of PNA on the increased neutrophil adhesion toxanthine-xanthine oxidase (X-XO) treated HUVECmonolayers. Neutrophil adhesion to HUVEC increasedover 7-fold after exposure to 10 mU/ml xanthine oxi-dase1 0.1 mM xanthine. PNA, at a concentration of 40mM (0.3 g% albumin), completely abolished the X-XOinduced neutrophil adhesion to HUVEC. A 50% reduc-tion in neutrophil adhesion was noted when a PNA

concentration of 1mM was studied. Nitroxide-free albu-min concentrations over the 1–40mM range had noeffect on X-XO induced neutrophil adhesion to HUVEC.

DISCUSSION

The narrow therapeutic window (3 h) of recentlyapproved tPA for the treatment of ischemic stroke indi-cates that an efficacious protective agent against I/Rinjury is needed in order to extend tPA treatment to alarger stroke population.16 Likewise, an effective agentfor the prevention of reperfusion injury in perioperativeI/R is also an unmet medical need.17 Since HSA isalready being used both in ischemic stroke and for vol-ume expansion in the surgical setting, it would be attrac-tive to modify the protein so as to impart SOD-mimeticand other antioxidant properties, thereby extending itsutility as an I/R therapeutic drug. The stable nitroxidefree radical, which behaves as an SOD-mimetic with abroad spectrum of antioxidant activity would appear tobe a logical choice,18 although the free nitroxide is rap-idly eliminated from the circulation, such that little com-pound can be detected in blood within 5 min after intra-venous administration (unpublished observation, Dr LiMa, SynZyme Technologies Inc., Irvine, California).However, PNA, prepared by covalently attaching multi-ple nitroxides to HSA and thereby extending its plasmahalf-life to .75 min (unpublished observation, Dr LiMa, SynZyme Technologies Inc., Irvine, California), hasbeen shown to have remarkable efficacy in the preven-tion of neurological damage in a transient focal ischemiamodel of stroke.14 Magnetic resonance imaging studies

Fig. 3. Effects of 20 min of ischemia and 30 min of reperfusion on leukocyte rolling velocity in rat mesenteric venules.

157Polynitroxyl albumin and leukoxyte-endothelial cell adhesion

have revealed that PNA is protective during both theischemic and the reperfusion phases, as manifested bythe relative changes in tissue perfusion and lesion devel-opment.14 The present study demonstrates that the ob-served protective effect of PNA in an in vivo model of

mesenteric I/R, as well as an in vitro model of oxidant-mediated neutrophil adhesion, can be attributed to thecovalently attached nitroxide, inasmuch as HSA did notoffer similar protection.

Our findings clearly indicate that PNA attenuates theprocess of leukocyte-endothelial cell adhesion that iselicited in postcapillary venules by I/R. This anti-adhe-sion effect is manifested as a reduction in trafficking ofadherent and emigrating leukocytes that is normally ob-served in venules exposed to I/R. The absence of a rolefor hemodynamic factors in the anti-adhesive actions ofPNA is supported by our findings in an in vitro staticassay of oxidant-mediated neutrophil adhesion to cul-tured endothelial cells. PNA appears to mimic the actionsof SOD, catalase, and monoclonal antibodies directedagainst leukocyte or endothelial cell adhesion moleculesin similar in vivo10,11 and in vitro15 models of I/R-induced inflammation, oxidant stress and tissue injury.We and others have demonstrated that administration ofeither MnSOD or CuZnSOD reduces I/R-induced leuko-cyte recruitment in postcapillary venules.11,19 These ob-servations, coupled to the results of the present study,suggest that the SOD mimetic actions of PNA mayaccount for its anti-adhesive actions and may also pro-vide a basis for the protective action of PNA in differentmodels of I/R injury.

PNA may act to interfere with leukocyte-endothelialcell adhesion either by: (1) preventing the formation of

Fig. 4. Effects of 20 min of ischemia and 30 min of reperfusion on wall shear rate in rat mesenteric venules. *Significant differencerelative to HSA;†significantly different from saline.

Fig. 5. Effects of PNA on xanthine-xanthine oxidase induced adhesionof human neutrophils to monolayers of human umbilical vein endothe-lial cells. **p , .01 relative to controls;##p , .01 relative to a PNAconcentration of 0 (xanthine-xanthine oxidase alone).

158 J. RUSSELL et al.

inflammatory mediators (leukotrienes or PAF); (2) pre-venting the degradation/metabolism of an endogenousanti-adhesion molecule (e.g., nitric oxide); or (3) inhib-iting the expression of leukocyte and/or endothelial celladhesion molecules. Indeed, superoxide has been impli-cated as a modulator of all of these I/R associatedevents.1 Hence, the SOD mimetic and anti-oxidant prop-erties of PNA seem to offer a reasonable explanation forthe anti-inflammatory properties of this nitroxide-con-taining reagent. Even though PNA cannot compete withthe specific activity of SOD,20 its efficacy is neverthelesscompensated for by large quantities used as well as itsbroad antioxidant activities, such as inhibition of theFenton reaction catalyzed by reduced metal ions.18

While the issue of PNA safety in humans remainsunresolved, there is evidence suggesting that albumin-bound nitroxides are far less toxic than free nitroxides.The acute toxicity of PNA has been tested in miceaccording to the US Pharmacopoeia (1990). Acute tox-icity of intravenous PNA at a nitroxide concentration of33 mmoles/Kg body weight is well tolerated in mice overa 72 hr period.14 However, it has also been reported thatthe free nitroxide has an LD50 of 2 mmoles/Kg afterintraperitoneal injection.21 Therefore, the increase insafety of nitroxide when covalently attached to albuminis greater than 16.5-fold. The reduction of nitroxidetoxicity may result from intravascular retention of thecompound when bound to albumin.

Acknowledgements—The authors wish to thank Charles Trimble andAndy Chen for their assistance with preparation of the manuscript andgraphics.

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159Polynitroxyl albumin and leukoxyte-endothelial cell adhesion