aor_1305 151..160

A Novel Hemoglobin-Based Oxygen Carrier, PolymerizedPorcine Hemoglobin, Inhibits H2O2-Induced Cytotoxicity of

Endothelial Cells

1*†Wei Zhang, 1*‡Kunping Yan, *§Penggao Dai, *Jingjing Tian, *§Hongli Zhu,and *§Chao Chen

*College of Life Science and §National Engineering Research Center for Miniaturized Detection Systems, NorthwestUniversity; ‡Shaanxi Lifegen Co. Ltd, Xi’an; and †Department of Biochemistry, Jilin Medical College, Jilin, China

Abstract: Hemoglobin-based oxygen carriers (HBOCs),with their capacity for delivering oxygen, could poten-tially function as red blood cell substitutes or primaryresuscitation solutions. However, there has been someconcern regarding redox-related safety issues of HBOCs.The present study describes a novel function of polymer-ized porcine hemoglobin (pPolyHb) in protecting ahuman umbilical vein endothelial cell line from H2O2-induced cytotoxicity. Through the examination of H2O2

consumption and ferrylhemoglobin formation, we foundthat pPolyHb exhibits antioxidant activity, suggesting that

pPolyHb may protect cells from free radical-induced celldamage. Additionally, we investigated the effect ofpPolyHb on H2O2-induced cell cytotoxicity, and foundthat pPolyHb significantly inhibits H2O2-mediated endot-helial cell damage as well as apoptosis. Thus, pPolyHbmay be developed as a new HBOC in the future. KeyWords: Hemoglobin-based oxygen carriers—Polymerizedporcine hemoglobin—Hydrogen peroxide—Cytotoxicity—Human umbilical vein endothelial cell line—Superoxidedismutase—Catalase.

Hemoglobin-based oxygen carriers (HBOCs) are apotential replacement for red blood cells (RBCs)when transfusion is needed (1). Over the past severaldecades, various hemoglobin (Hb) modificationshave been developed, studied, and brought to clinicaltrials in humans. Unfortunately, progress to date hasnot fulfilled early expectations because of unantici-pated and unwanted adverse clinical effects.A signifi-cant impediment to progress is the oxygen-bindingaffinity and cytotoxicity of HBOCs (2).

Hydrogen peroxide (H2O2) has been implicated inthe redox regulation of several physiological pro-cesses, including signal transduction (3,4), response to

oxidative stress (5–7), development (7), cell prolifera-tion (8–10), and apoptosis (7,11). H2O2, the productof spontaneous superoxide dismutation or directenzymatic reaction (e.g. amine oxidase or glucoseoxidase reactions), can be generated by a variety ofmammalian cells, including neutrophils, macroph-ages, vascular smooth muscle, and endothelial cells.The yield of H2O2 may increase under certain condi-tions, such as ischemia–reperfusion (12,13).

It has been reported that H2O2 is able to induceboth apoptotic and necrotic cell death (3,14–16), andto generate hydroxyl radicals when reacting with freeferrous iron (Fe2+), oxidizing it to ferric iron (Fe3+) ina process called the Fenton reaction. H2O2 can alsoconvert ferrous Hb (containing bound Fe2+) intoferric Hb (Fe3+), which in its oxidized form cannotdeliver O2 and is toxic to cells (17). Under someconditions, H2O2 reacts with ferric Hb as well as otherheme proteins to produce an even higher oxidationstate of the iron (ferryl, Fe4+), which can cause lipidperoxidation, carbohydrate degradation, and proteincross-linking (18–20). Ferrylhemoglobin can also

doi:10.1111/j.1525-1594.2011.01305.x

Received July 2010; revised May 2011.Address correspondence and reprint requests to Dr. Chao Chen

or Dr. Hongli Zhu, College of Life Science, Northwest University,No. 229, Taibai Northroad, Xi’an, Shaanxi 710069, China. E-mail:cchen898@nwu.edu.cn or zhuyjw1971@nwu.edu.cn

1These two authors contributed equally to this work.

Artificial Organs36(2):151–160, Wiley Periodicals, Inc.© 2011, Copyright the AuthorsArtificial Organs © 2011, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

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generate stable F2-isoprostanes through the per-oxidation of arachidonic acid. F2-isoprostanes arepotent vasoconstrictors, a fact that is particularlyinteresting, given the vasopressor effects associatedwith most HBOCs (21).

An important concern in this field is the inherentredox activity of Hb and its potentially deleteriousconsequences. Recently, it has been reported thatHb can attenuate H2O2-induced oxidative stress byacting under certain circumstances as an antioxida-tive peroxidase (22), suggesting promise for the prac-tical use of HBOCs in the future.

Polymerized porcine hemoglobin (pPolyHb), anewly developed HBOC with superoxide dismutase(SOD) and catalase (CAT) activities, was obtained byglutaraldehyde cross-linking of porcine Hb. In pre-vious studies, both rat exchange transfusion andshock models were used to investigate the effect ofpPolyHb on rat tissues and organ recovery. After90% of the blood was replaced with either pPolyHbor hydroxyethyl starch (a clinically used volumeexpander), it was shown that the survival time forthe pPolyHb group was much longer than for thehydroxyethyl starch group. The blood gas and hemo-dynamic results, as well as physiological indicatorssuch as blood pressure and heart rate, were also muchbetter in the pPolyHb group than in the hydroxyethylstarch group. Similar results were also observed inthe shock model (unpublished data).

In the present study, we found for the first time thatpPolyHb exhibits high antioxidative activity againstH2O2-induced cytotoxicity in human blood vesselendothelial cells (EVC-304), and that H2O2-mediatedferrylhemoglobin formation was inhibited bypPolyHb. The anti-apoptotic effect of pPolyHb wasalso investigated.

MATERIALS AND METHODS

Reagents and animalsRPMI 1640 Medium and fetal bovine serum (FBS)

were purchased from Gibco (Grand Island, NY,USA. Propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),horseradish peroxidase (HRP), 4-aminoantipyrine,3,5-dichloro-2-hydroxybenzenesulfonic acid, sodiumsulfide (Na2S), H2O2, and SOD kits were purchasedfrom Sigma (Sigma-Aldrich Co., St. Louis, MO,USA). Hoechst 33258 kits were purchased fromKeygen of Nanjing. All experimental procedures inour lab were approved by the Human Subjects andAnimal Care Committee at Northwest University(China).

Preparation of porcine stroma-free hemoglobin(pSFHb) and purified porcine hemoglobin (pHb)

Porcine blood was freshly collected from a slaugh-terhouse and stored at 4°C with a citric acid/sodiumcitrate buffer as the anticoagulant. Erythrocytes werethoroughly washed and lysed. Membrane-associatedlipids and Hb were separated by filtration to obtainpSFHb (used only for positive control purposes).Subsequent pasteurization of the deoxyhemoglobinsolution was performed at 60°C for 10 h, whichsignificantly reduced macromolecular impuritiesand viral contamination. The average phospholipidcontent of pHb was lower than 1.0 mg/mL. Thecontent of endotoxin was lower than 0.2 EU/mL, andthe final purity of pHb was greater than 99.5%according to high-performance size-exclusion chro-matography analysis. A 0.22-mm filter membrane wasused for final sterilization. This ultrapure Hb (usedonly for negative control purposes) has been charac-terized extensively and is free of antioxidants.

Preparation of pPolyHbThe porcine Hb cross-linked by glutaraldehyde

(Lifegen 100) was prepared as described previously,with certain modifications (23,24), and was a kindgift from Lifegen Co. Ltd. (Xi’an, Shaanxi, China)Briefly, Hb from fresh porcine blood was purifiedthrough specific steps, and then cross-linked byglutaraldehyde. Small molecules, including excessglutaraldehyde and tetrameric hemoglobin, wereremoved by ultrafiltration. The polymerization tech-nique allowed the retention of SOD and CATactivities. The physiochemical characteristics andstructural properties of pPolyHb are listed in Table 1.The protein sample was stored at 4°C under nitrogengas until use. Additional details regarding pPolyHbare currently being withheld because of pendingpatents.

Measurement of SOD and CAT activitiesSOD activity was determined using a commercially

available kit. The perborate titration method was

TABLE 1. Physiochemical and structural characteristicsof pPolyHb

Polymerized porcine hemoglobin 10.5 � 0.5 g/dLMethemoglobin <5%Endotoxin <1.0 EU/mLOsmolality 300–330 mOsmpH 7.4 � 0.05P50 28 � 3 mm HgAverage molecular weight of pPolyHb 600 � 50 kD64 kD tetramer <2%Na+ 135–155 mmol/LK+ 3.0–5.0 mmol/LCa2+ 1–3 mmol/LCl- 140–160 mmol/L

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used to measure CAT activity (25). CAT units wereexpressed as milliequivalents of perborate decom-posed per milligram of blood sample, and then con-verted to units per gram of Hb.

H2O2 measurementReaction volumes (3 mL) containing the HRP/4-

aminoantipyrine/3,5-dichloro- 2-hydroxybenzenesulfonic acid reagent solution (1.2 mL), pPolyHb, water,and H2O2 were prepared (26). The final concentra-tions of HRP, 4-aminoantipyrine, 3,5-dichloro-2-hydroxybenzenesulfonic acid were 500 kU/L, 0.99mM,and 0.6 mM,respectively. Identical mixtures con-taining additional water instead of H2O2 served ascontrols. The mixture stood for 2 min at 22°C, andthen, the absorbance at 510 nm was recorded. H2O2

and the reagent solution participated in a peroxidase-catalyzed reaction to form a dye which can be mea-sured at this wavelength. The molar ratio betweenheme/H2O2 ranged from 1:10 to 3:1.

Ferric and ferrylhemoglobin concentrationmeasurement

Spectral analysis of Hb was performed using aSPD-10AVP plus UV-VIS spectrophotometer at37°C.The concentration of ferrichemoglobin was cal-culated according to Chinese National Clinical Labo-ratory Procedures (27). Twenty microliters of eachsample was added in cyanmethemoglobin (HiCN)reagent, mixed thoroughly, and allowed to stand for5 min. Absorbance was determined by spectropho-tometry (wavelength 540 nm, optical path: 1.0 cm),adjusting to zero with HiCN before determination.The Hb concentration was calculated from thestandard curve, using the equation: hemoglobin(M) = A ¥ 5.0 ¥ 10-3. The level of ferrylhemoglobinwas detected by its reaction with Na2S to formsulfhemoglobin. The conversion of ferrylhemoglobinto sulfhemoglobin was monitored by determining theabsorbance of the reaction mixture at 620 nm. Theconcentration of sulfhemoglobin was calculatedusing the extinction coefficient of sulfheme (e620–604 nm = 10.5 mM-1 cm-1).

Cell cultureEVC-304 cells were a gift from the Fourth Military

Medical University, and the cells were harvested withpermission from the Institutional Review Board forthe Protection of Human Subjects. Cells were grownin RPMI 1640 Medium with 10% FBS and main-tained in 100-mm dishes at 37°C in a humidifiedatmosphere of 95% air and 5% CO2. For experi-ments, cells were plated in 60 mm dishes and reachedconfluence within 2 to 3 days. For harvesting, cells

were washed with N-2-hydroxyethylpiperazine-N′-2′-ethanesulfonic acid (HEPES)-buffered saline andincubated with 0.025% trypsin–0.01% ethylenedi-aminetetraacetic acid for 5 min at 37°C.After detach-ment, a trypsin-neutralizing agent was added andcells were centrifuged at 800 g for 5 min at 4°C. Cellcounts were obtained using a Coulter counter.

Experimental cell treatmentsEarly passage EVC-304 cells were grown in com-

plete RPMI 1640 for 24 h before the medium wasreplaced with fresh complete RPMI 1640. Cells weresubsequently washed with Hank’s buffered salt solu-tion (HBSS) and incubated with 3 mL FBS-freesupplemented medium or medium containing H2O2,or with three types of Hb solution combined withH2O2.

After 12 h incubation, cell morphology was viewedby phase contrast microscopy using an Olympusmodel Bx61/Bx62 inverted microscope equippedwith a MCA-85001 camera (Olympus, Tokyo, Japan).

MTT assayCells (~8 ¥ 104/well) were seeded in 96-well cell

culture plates. After treatment, 20 mL MTT (5 mg/mL) was added to the media and incubated for 3 h at37°C.The MTT solution was replaced by 150 mL dim-ethyl sulfoxide (DMSO), and the cells were agitatedfor 10 min. The absorbance was then read at 570 nm.

Fluorescence microscopyCells were treated with 150 mM pPolyHb, 50 mM

H2O2, or both. After 12 h incubation, they werewashed twice with PBS, and then treated according tothe Hoechst 33258 kit instructions. Three hours later,the cells were viewed by phase contrast microscopyusing an Olympus model Bx61/Bx62 inverted micro-scope equipped with a MCA-85001 camera.

Annexin V–PI assayCells were treated with 50 mM H2O2, or 150 mM

pPolyHb and 50 mM H2O2, for 12 h. The media wasthen removed and cells were washed with HBSSwithout calcium or magnesium and trypsinized. Cellswere suspended in 1¥ annexin V binding buffercontaining 10 mM HEPES/NaOH, 140 mM NaCl,2.5 mM CaCl2, pH 7.4. Cells were transferred to aculture tube, and annexin V and PI were added.Aftergentle vortexing, the cells were incubated for 15 minat room temperature in the dark. After adding350 mL assay buffer to each tube, cells were analyzedby flow cytometry (Beckman Coulter-Elite, Fuller-ton, CA, USA). Data (collected from a minimum of

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10 000 cells per sample) were analyzed using Expo32(AV) analysis software (Beckman Coulter, Miami,FL, USA). Stained cell populations were defined aseither B2, that is, necrotic or late apoptotic cells(annexin V+, P+), or B4, that is, cells undergoing earlyapoptosis (annexin V+, PI–).

Statistical analysisData were represented as mean � SD for replicate

experiments. The differences between treatmentgroups were assessed by one-way ANOVA followedby unpaired Student’s t-test. Statistical significancewas defined as P < 0.05 to reject a null hypothesis.Allstatistical calculations were performed with JMPversion 3.2 for the Macintosh (SAS Institute, Cary,NC, USA).

RESULTS AND DISCUSSION

Results

SOD and CAT activity of pPolyHbSuperoxide radicals play a key role in conditions of

oxidative stress. To investigate the degradation ofsuperoxide radicals by pPolyHb, the activity of SODin pPolyHb was assayed. SOD catalyzes the dismuta-tion of superoxide radicals into H2O2 and elementaloxygen, thus providing an important defense againstthe toxicity of the superoxide radical. As shown inFig. 1A, the SOD activity of pPolyHb was compa-rable to that of RBC and pSFHb, a product originat-ing from RBCs. However, its SOD activity is muchhigher than that of pHb. We also compared the CATactivity of pPolyHb with RBC and pSFHb (Fig. 1B),and the results showed that pPolyHb CAT activity iscomparable to that of RBC and pSFHb.

H2O2 consumption by pPolyHbH2O2 is an endogenous oxidant, which is believed

to be cytotoxic when it reacts with Hb under condi-tions of oxidative stress. To determine the H2O2 con-sumption, or peroxidase activity, of pPolyHb, weadded a series of amounts of pPolyHb to 200 mMH2O2 solution and then measured the remainingH2O2 (Fig. 2). These results showed that 80 mMpPolyHb can consume a significant amount of H2O2

and that the H2O2 level decreased to a basal physi-ological level when 150 mM pPolyHb was added tothe solution.

Formation of ferrylhemoglobin intermediate wasinhibited by pPolyHb

Ferrylhemoglobin is a cytotoxic agent produced bythe interaction of ferrous Hb or ferric Hb with H2O2

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FIG. 1. Determination of pPolyHb SOD and CAT activities. (A)SOD activity in RBC lysate, pSFHb, pPolyHb, and pHb; (B) CATactivity in RBC lysate, pSFHb, pPolyHb, and pHb. Data representmean � SD of one representative experiment of at least fiveindependent experiments. Statistical significance indicated by**P < 0.001.

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FIG. 2. Dose-dependent H2O2 consumption by pPolyHb. Serialconcentrations of pPolyHb were incubated with 200 mM H2O2

at room temperature for 2 min. The amount of H2O2 remainingwas measured by reaction with a HRP/4-aminoantipyrine/3,5-dichloro-2-hydroxybenzenesulfonic acid solution. Data shownare mean � SD of one representative experiment of at least fiveindependent experiments. Statistical significance is indicated by*P < 0.05 for the 80-mM pPolyHb reaction, and ** P < 0.01 for the100 and 150 mM pPolyHb reactions.

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under conditions of oxidative stress. It can bedetected by reaction with Na2S to produce sulfhemo-globin, which absorbs light at 620 nm (28). To furtherconfirm the antioxidant activity of pPolyHb, H2O2

and either pSFHb, pPolyHb, or purified Hb wereincubated for up to 60 min; after which, the concen-tration of ferrylhemoglobin was measured. The spec-trometry results revealed no significant changes in

the absorbance spectrum of pPolyHb with H2O2 overa time course of 60 min. However, the pHb caused avisible spectrum change, as seen in Fig. 3A. Consis-tent with the spectrometry results, no ferrylhemoglo-bin was formed from the incubation of pSFHb orpPolyHb and H2O2 (Fig. 3C), as measured by a fer-rylhemoglobin concentration assay.These results sug-gested that pPolyHb exhibits antioxidant activity and

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FIG. 3. pPolyHb inhibition of ferrichemoglobin or ferrylhemoglobin formation. pSFHb, pPolyHb, or pHb solutions were incubated withH2O2 at room temperature for 60 min. (A) Spectral analysis of hemoglobin with a SPD-10AVP plus UV-VIS spectrophotometer; (B) and(C) Ferrichemoglobin and ferrylhemoglobin formation. Each curve represents measurements taken every 2 min for the first 10 min,followed by every 5 min for the next 50 min.

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does not produce the cytotoxic agent ferrylhemoglo-bin when exposed to H2O2. The accumulation of fer-richemoglobin was also investigated, and the resultsshowed that the formation of ferrichemoglobin wassignificantly inhibited by pPolyHb, relative to pHb(Fig. 3B).

pPolyHb plays a protective role in H2O2-inducedcell damage

In order to verify that pPolyHb has a potentialprotective role against cell damage induced by H2O2,two different assays were used to examine the effect

of pPolyHb on cell viability. Initially, a cell viabilityand cytotoxicity assay was performed using a MTTcell viability kit. EVC-304 cells were treated withH2O2, or H2O2 combined with pSFHb, pPolyHb, orpHb, and the cell viability was determined.The molarratio of heme/H2O2 was 3:1. The results of the cellviability assays from EVC-304 cells were confirmedusing phase contrast microscopy. The drastic declinein viability was associated with visible rounding ofthe cells and detachment when H2O2 alone wasadded to the cells (Fig. 4). In contrast, the cellstreated with pSFHb and H2O2, or pPolyHb and H2O2,did not show visible changes in cell morphology and

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FIG. 4. pPolyHb inhibition of H2O2-induced cell damage. Cells were incubated with FBS-free medium, H2O2, pSFHb, pPolyHb, or pHbcombined with H2O2 as indicated. (A) Cell morphology, as detected by phase contrast microscopy; (B) MTT assay analysis of living cells.Statistical significance indicated by **P < 0.01 for pPolyHb vs. H2O2; #P < 0.05 for pPolyHb vs. pHb.

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cell number (Fig. 4), indicating that pSFHb andpPolyHb protect against H2O2-induced cell damage.Figure 4A also shows the accumulation of debrisfrom dead cells or insoluble precipitates in cellstreated with pHb with H2O2, which possibly containoxidized Hb products.

pPolyHb inhibited H2O2-induced cell apoptosisThe oxidative stress exerted by H2O2 exposure

eventually induces cell death by apoptosis or necrosis

(10).To confirm that pPolyHb plays a role in prevent-ing H2O2-induced cell apoptosis, we tracked cellviability using nuclear staining and annexin Vstaining. EVC-304 cells were incubated with H2O2

alone or H2O2 together with pPolyHb. Nuclei werestained with Hoechst 33258, a cell-permeable bluefluorescent DNA dye, to detect nuclear condensa-tion and fragmentation, which are characteristics ofapoptosis. As shown in Fig. 5A, H2O2-treated cellswere characterized by condensed bright Hoechst

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FIG. 5. pPolyHb inhibition of H2O2-induced cell apoptosis. Cells were incubated with FBS-free medium, H2O2, or pPolyHb combined withH2O2, as indicated. (A) Fluorescence microscopic examination of nuclear staining with Hoechst 33258; (B) flow cytometric analysis ofannexin V-stained cells; (C) quantitative analysis of data in (B). Statistical significance indicated by **P < 0.01.

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staining, indicating that the cells underwent apopto-sis, while the combination of pPolyHb and H2O2 pre-vented the apoptotic phenomena. These cells showeda normal Hoechst staining pattern, similar to thecontrol cells. To confirm this result, we stained thecells with annexin V, an early apoptotic marker, andPI for detection of late apoptosis. Flow cytometrywas used to quantify fluorescent cells, and showedthat pPolyHb significantly reduced the percentage ofcells in both early and late stages of apoptosis (Fig.5B,C).

DISCUSSION AND CONCLUSIONS

HBOCs have been extensively studied as potentialblood substitutes, and several products have beenbrought to clinical trials in humans (29). However,redox-related safety issues are still among the majorconcerns in this field. As mentioned above, mostHBOC products have been shown to be cytotoxic tocells through a mechanism that involves the oxidantactivity of H2O2 (30,31). pHb or HBOC preparationsin their reduced forms are not, by themselves, cyto-toxic to endothelial cells (32,33). However, when oxi-dized by H2O2, the accumulation of highly reactiveFe4+ can induce significant cytotoxicity through amechanism that involves the production of toxicoxygen intermediates such as ferryl-Hb (HbFe4+ = O)and a globin tyrosyl-based radical (•HbFe4+ = O).•OH and Hb Fe4+ = O can initiate lipid peroxidation,while •HbFe4+ = O is an effective modifier of low-density lipoproteins. Ferryl-Hb species may also con-tribute to many other pathological events, includingdamage to large molecules and apoptosis (20,34–36).The molar ratio between H2O2 and Hb was shown tobe a critical factor for the formation of ferryl-Hb.Simoni et al. reported that a high molar ratio of H2O2

to Hb increased ferryl-Hb formation by about 15%,and would likely increase the damage to humanendothelium (37).

However, we have shown here that pPolyHb has acytoprotective function and thus is a potential solu-tion to the problems mentioned above. Several linesof evidence support our conclusion. First, an H2O2

consumption assay showed that pPolyHb is ableto neutralize exogenously added H2O2, indicatingthat pPolyHb can decrease H2O2-mediated cytoto-xicity. Second, ferrylhemoglobin formation was notobserved as a result of incubation of pPolyHb andH2O2, suggesting a lack of toxic product formation bypPolyHb. Third, MTT and cell morphology assaysshowed that cell death caused by H2O2 could be pre-vented by the presence of pPolyHb. Fourth, flowcytometry and immunostaining with an apoptotic

marker revealed that pPolyHb inhibited apoptosisinduced by H2O2. Consistent with the involvement ofthe pPolyHb in preventing H2O2-induced cell cyto-toxicity, the SOD and CAT activities of pPolyHb aremuch higher than those of purified Hb, which mayresult from the polymerization technique used in thepreparation of pPolyHb.

Redox-related safety issues underlie some of theadverse effects of Hb. pPolyHb is able to overcomethese adverse effects due to its antioxidant proper-ties, which are similar to those of RBCs. In the case ofblood exchange or hemorrhagic shock, colloid orcrystal fluid resuscitation will dilute the RBCs anddecrease the antioxidant effects. In contrast, resusci-tation of pPolyHb with antioxidant properties shouldallow retention of most antioxidant capability, andthus be superior to other resuscitation fluids in thisrespect.

The increasing awareness that the toxicity of Hbmay limit the safety or efficacy of first-generationHb-based therapies has prompted the design of newstrategies aimed at reducing this toxicity (38). Cur-rently, several strategies have been explored, includ-ing chemical cross-linking, alteration of the hemepocket to reduce nitric oxide (NO) reactivity, andcoadministration of antioxidants or antiferryl com-pounds to limit HBOC toxicity (20,39). The strategyfor preparing the pPolyHb used in this experimentresults in a product that retains relatively high SODand CAT activities. These enzymes convert super-oxide radicals into H2O2, which in turn is convertedinto water and oxygen. Thus, H2O2 is consumed bypPolyHb, and cellular toxicity is lowered by pPolyHb.Although the mechanisms underlying these activitiesare still unknown, inhibition of cell cycle arrest byextracellular CAT and the loss of thiols stronglysuggest the involvement of an initial extracellularoxidative event that triggers the subsequent cellularresponses (19,32).

Our present study showed that pPolyHb is non-toxic in vitro. While it is not yet clear whether thisproduct will yield benefits in vivo, additional in vivostudies, such as ischemia–reperfusion experiments,should be performed to examine the effects ofpPolyHb in the circulatory blood. pPolyHb is beingdeveloped for eventual use in humans. Some HBOCs(PolyHeme, Hemolink, Hemospan) use outdatedhuman blood as Hb sources. However, due to thesevere limitation of human Hb sources, other Hbsources have also been developed. For example,Biopure Co. Ltd. uses bovine blood as the raw mate-rial for Hemopure. pPolyHb is derived from porcinesources. Due to its abundant supply and high homol-ogy with human Hb, as well as its freedom from

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contamination with human viruses such as HIV andhepatitis virus, porcine Hb could be a potentialmaterial in large-scale production of HBOCs. Morestudies will need to be performed on pPolyHb in theareas of immunogenicity and pathogen removal priorto its use in humans.

Ferrylhemoglobin has been shown to be toxic to anumber of different cell types, including endothelialcells (19,40). It is known to be formed via the reac-tion of oxy- and methemoglobin with H2O2,although we still do not know which cellular oxi-dants react with Hb to produce the ferryl-Hb oxi-dation state. Under our experimental conditions,ferrylhemoglobin formation after the addition ofpPolyHb and H2O2 was not observed, suggestingthat pPolyHb has no potential cytotoxicity. We arecurrently investigating ferrylhemoglobin formationin animal models, which will provide us with aphysiological condition for understanding thepotential toxicity of pPolyHb.

H2O2 can induce apoptosis or necrosis in a varietyof cell types (7). H2O2 treatment caused toxic effectsto EVC-304 cells as previously reported, while theaddition of pPolyHb significantly increased thenumber of live cells. This effect was shown to be dosedependent (unpublished data). In the presence ofpPolyHb, we do not detect H2O2-induced apoptosis,indicating either that H2O2 is immediately consumedby the peroxidase activity of pPolyHb or that apop-tosis signaling is impaired by the addition ofpPolyHb. The bolus addition of pPolyHb to highmicromolar concentrations of H2O2 decreased theamount of H2O2 detected, suggesting that the protec-tive effect of pPolyHb is due to H2O2 consumption.We do not yet have any evidence to show thatpPolyHb participates in the apoptotic signalingpathway. The investigation of pPolyHb involvementin other chemically induced apoptotic pathways willprovide further evidence for the protective mecha-nism of pPolyHb.

Although the exact molecular mechanism bywhich pPolyHb prevents H2O2-induced cell deathhas yet to be determined, the demonstration thatpPolyHb is involved in cellular protection suggests adirection for further studies. The fact that polymer-ized placental Hb significantly inhibits the expres-sion of cleaved caspase-3 as well as the ratio ofBax/Bcl-2 points to a potential anti-apoptoticmechanism for the cytoprotective function ofpPolyHb (41). While this mechanism is attractive,currently, we do not have evidence indicatingpPolyHb involvement in the caspase-3 signalingpathway, although related experiments are under-way. Regardless of whether pPolyHb participates in

this signaling pathway, the present study has notonly uncovered a novel function of pPolyHb, buthas also set the direction for the development of anew generation of blood substitutes.

Acknowledgments: This work was supported bygrants from the National 863 Program of China(grant nos. 2006AA02A143 and 2009AA022710) andthe “Special Research Foundation” of the EducationDepartment of Shaanxi Province (grant nos. 08JK469and 09JK784) as well as grants from Shaanxi Scienceand Technology Department (grant nos. 2011K12-03-10). We would like to express our gratitude to Dr.Ning Dan for his help and discussion in experimentdesign and Dr. Simoni for his constructive advice.

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