Review Article Vasculotoxic and Proinflammatory...

Hindawi Publishing CorporationISRN Oxidative MedicineVolume 2013 Article ID 831596 9 pageshttpdxdoiorg1011552013831596

Review ArticleVasculotoxic and Proinflammatory Effects of Plasma Heme CellSignaling and Cytoprotective Responses

John D Belcher1 Karl A Nath2 and Gregory M Vercellotti1

1 Division of Hematology Oncology and Transplantation Vascular Biology Center Department of MedicineUniversity of Minnesota Medical School 420 Delaware Street SE Minneapolis MN 55455 USA

2Division of Nephrology and Hypertension Mayo Clinic Rochester MN USA

Correspondence should be addressed to John D Belcher belcherumnedu

Received 10 April 2013 Accepted 30 May 2013

Academic Editors R Fato and K Iles

Copyright copy 2013 John D Belcher et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The proinflammatory vasculotoxic effects of intravascular hemolysis are modulated by plasma hemoglobin and heme clearancevia the haptoglobinCD163 system and the hemopexinCD91 system respectively and detoxification through the hemeoxygenaseferritin system However sudden or excessive hemolysis can overwhelm these protective systems leading to hemeinteracting with cells of the vasculature Heme presents a damage-associatedmolecular pattern to the innate immune systemHemeis an extracellular inflammatory signalingmolecule with strict binding specificity for TLR4 onmonocytemacrophages endothelialand other cells The resulting TLR4 signaling cascade rapidly leads to intracellular oxidative stress and an inflammatory responseHeme also induces a cytoprotective response that includes Nrf2 responsive genes such as heme oxygenase-1 ferritin haptoglobinhemopexin and other antioxidant response genes It is the balance between the pro-inflammatoryvasculotoxic effects of plasmahemoglobinheme and the cytoprotective responses that ultimately determines the pathophysiologic outcome in patients

1 Introduction

When hemoglobin (Hb) is released from red blood cells(RBCs) into plasma it has the potential to release freeheme that can trigger severe oxidative proinflammatoryand pro-thrombotic injury Heme has several proinflamma-tory activities including leukocyte activation and migrationupregulation of adhesion molecules reactive oxygen species(ROS) production and induction of cytokine and chemokineexpression [1ndash4] Organisms have evolved intricate systemsto defend against free heme The term ldquofreerdquo heme will beused loosely in this review as heme is amphipathic mostlyinsoluble in aqueous solutions at neutral pH and likely boundto proteins andor lipids in vivo This review will focus on theproinflammatory and anti-inflammatorymolecular signalingevents that are activated by cells in response to free heme

2 Hemolysis and Plasma Defenses

Intravascular hemolysis releases Hb from an antioxidant richenvironment inside the RBC into the plasma Once free in

plasma the Hb tetramer is in equilibrium with the 120572120573 Hbdimer favored at low plasma Hb concentrations The Hbdimers bind to haptoglobin which can safely carry Hb toCD163 receptors on macrophages where the Hb is degraded[5ndash7] However during massive or chronic hemolysis thehaptoglobinCD163 system can be overwhelmed leaving freeHb in plasma When not bound to haptoglobin oxyHb inplasma can react rapidly with nitric oxide (NO) to formnitrate (NO

3

minus) and ferric Hb (metHb) To illustrate thispoint steady state metHb levels can reach as high as 5in humans inhaling 80 ppm NO for 4 hours [8] Howeversteady-state metHb levels do not accurately reflect the kineticthroughput of metHb in plasma because metHb is unstableand releases ferric heme (hemin) from the globin chain[9] The released hemin binds to plasma hemopexin (Kd lt10minus12M) albumin (Kd sim 10minus8M) or intercalates into plasmalipoproteins (Kd 10minus10M to 10minus11M) or cell membranesbecause of its amphipathic structure [10 11] Because of itshigh binding affinity hemopexin is the first line of defenseagainst hemin released from metHb Hemopexin delivers

2 ISRN Oxidative Medicine

the bulk of hemin to CD91 receptors on hepatocytes whereit is endocytosed and degraded [12]

3 Heme Stimulation of TLR4 SignalingIs Proinflammatory

Like haptoglobinCD163 the hemopexinCD91 system canbe overwhelmed during periods of excessive hemolysis Inhemopexin null mice there is enhanced clearance of hemeby nonhepatic tissues including the vessel wall [13] Recentlyheme has been shown to be an extracellular inflammatorysignaling molecule in macrophages with strict binding speci-ficity for toll-like receptor-4 (TLR4) [4]These findings implythat heme is a damage-associatedmolecular pattern (DAMP)molecule Signaling of heme through TLR4 is distinct fromthe signaling of bacterial lipopolysaccharide (LPS) throughTLR4 [4] Heme activation of TLR4 is remarkably stringentand requires the iron moiety [4 14] Unlike its analogs orprecursors heme inducesmacrophage tumor necrosis factor-alpha (TNF-120572) secretion that is dependent on TLR4 and itsadaptor molecules MYD88 and CD14 [4] By binding heminhemopexin thwarts hemin-mediated TLR4 signaling andthereby prevents the proinflammatory effects of hemolysis[13 14]

Endothelial cells also express TLR4 [15ndash18] In endothe-lium hemin- or LPS-mediated TLR4 signaling stimulatestwo parallel proinflammatoryprothrombotic pathways (1)Weibel-Palade Body (WPB) degranulation [14] and (2)nuclear factor-kappa B (NF-120581B) activation [14 19] WPBdegranulation occurs within 5 minutes of hemin (or LPS)stimulation of TLR4 WPB degranulation releases numer-ous vasoactive proteins including von Willebrand Fac-tor P-selectin endothelin-1 endothelin-converting enzymeinterleukin-8 tissue-type plasminogen activator eotaxin-3angiopoietin-2 osteoprotegerin and calcitonin gene-relatedpeptide [20] HeminTLR4-mediated degranulation ofWPBson the endothelial cell surface triggers rapid proinflamma-tory prothrombotic and vasoconstrictive responses in thevasculature

In addition to WPB degranulation hemin- or LPS-mediated TLR4 signaling leads to NF-120581B activation andthe transcription of proinflammatory genes [19] Inhibitionof TLR4 signaling or knockout of the TLR4 gene com-pletely abrogates hemin-stimulated WPB degranulation andNF-120581B activation in endothelial cells [14] Heme amplifiesLPS-induced TLR4 signaling [21] which may explain whyhemolysis and hemoglobinuria are associated with increasedmortality in septic patients [22 23]

Hemin also has procoagulant effects on endothelium (vonWillebrand factor and tissue factor expression) [14 24] thatcould be explained by hemin-mediated TLR4 signaling [14]Moreover hemin activation of TLR4 signaling in monocytesand platelets could potentially induce thrombosis via tissuefactor expression on monocytes (unpublished data) andplatelet activation

Hemin also induces oxidative stress in cells Much ofthis has been attributed to iron-catalyzed reactions [25]However there is evidence that LPS activated TLR4 in

endothelial cells interacts with NADPH oxidase 4 (Nox4)a protein related to gp91phox (Nox2) of phagocytic cellsand that Nox4 activity is required for TLR4-mediated NF-120581B activation [26] The Nox protein generates superoxide(O2

minus) by transferring electrons from NADPH to O2 These

superoxide free radicals are rapidly converted to H2O2and

O2in cells Thus heme-mediated TLR4NADPH oxidase

signaling may explain much of the cellular oxidative stressthat occurs during hemolysis

4 Cytoprotection by Heme Oxygenase

Once heme enters the cell membrane the next line ofdefense is heme oxygenase (HO) HO degrades heme intocarbon monoxide (CO) biliverdin and iron (Fe2+) Thereare two isoforms of HO HO-1 and HO-2 [27] HO-1 ishighly inducible by a large number of oxidative stressorsincluding heme In normal healthy tissues not involvedin ongoing RBC destruction HO-1 levels are low [27] Intissues such as the kidney HO-2 a constitutive enzyme isan important first responder to intracellular heme HO-2null mice (HO-2minusminus) compared with HO-2++ mice exhibitgreater renal dysfunction and histologic injury when admin-istered Hb [28] However within 3 hours HO-1 expressionquickly surpasses HO-2 and HO-1 becomes the principlecytoprotectant to subsequent exposures to heme [29] Themechanisms controlling HO-1 induction are complex cellspecific and tightly regulated by transcription A largenumber of kinases (eg mitogen-activated protein kinases(MAPKs) protein kinase C (PKC) and phosphatidylinositol3-kinase (PI3 K)protein kinase B (Akt)) and transcriptionfactors (eg NF-120581B activator protein-1 (AP-1) nuclear fac-tor E2-related factor 2 (Nrf2) biliverdin reductase (BVR)and BTB and CNC homologue 1 (Bach1)) are involved inregulating HO-1 expression [30] Nrf2 appears to be anespecially important regulator of HO-1 induction in responseto oxidative stress and heme [31 32] Nrf2 normally resides inthe cytoplasm bound to an inhibitor protein kelch-like ECH-associated protein-1 (Keap1) Keap1 expedites the ubiquitina-tion and proteolysis of Nrf2 Oxidation of cystine residuesto cysteine (ie disulfide cross-linking) on Keap1 releasesNrf2 prolongs Nrf2 half-life and allows Nrf2 transport to thenucleus [33] Molecular signaling by the PI3 KAkt pathwayalso plays an important role inNrf2 activation ActivatedNrf2translocates to the nucleus and binds to antioxidant responseelements (ARE) which promotes the transcription of a widevariety of antioxidantanti-inflammatory genes includingHO-1 ferritin haptoglobin and hemopexinHeme also bindsto the transcriptional repressor Bach1 in the nucleus Bach-1 is a member of the bZIP transcription factor family thatserves as a repressor of HO-1 transcription through itshigher binding affinity for multiple Maf recognition element(MARE) regions compared with Nrf2 [34] When Bach1forms a heterodimer with Maf it binds to MARE regionsand represses HO-1 transcription Heme binding to Bach1releases Bach1Maf repression allowing Nrf2 binding andHO-1 transcription

ISRN Oxidative Medicine 3

5 CO Mimics HO-1 Cytoprotection

The cytoprotective properties of HO-1 are mimicked by COwhich is released from heme by the HO reaction CO gasis produced by HO-mediated opening of the heme ringCO is a colorless odorless gas that has traditionally beenconsidered a dangerous poison This toxicity is in part dueto its high affinity for Hb (234X greater than O

2) altering O

2

transport and delivery [35] CO also can interact with otherheme proteins However likemany other compounds this gasencompasses both a toxic and a therapeutic range [36] At lowconcentrations CO is a potentmediator of cell protection andhas a number of properties that make it an attractive ther-apeutic option for treating hemolytic diseases CO mimicsmany of the protective effects of HO-1 as well as some ofthe functions of NO [3 36 37] Like NO CO activates theheme protein guanylate cyclase inhibits platelet activationand aggregation and has a possible role as a neurotransmitter[3 37] Exogenous inhaled CO at approximately 250 partsper million (ppm) and in some studies as low as 10 ppm[38] reduces inflammatory responses in several models ofoxidant injury in similar ways to HO-1 overexpression [37]CO interacts with signal transduction pathways inhibitsproinflammatory genes and augments anti-inflammatorycytokines [3 37 39 40] Specifically it selectively activatescytoprotective p38 MAPK and Akt signaling pathways ina guanylate cyclase-independent manner [37 39] CO alsoinhibits proliferation of vascular smoothmuscle cells and hasantiapoptotic effects on cells [3 37]

CO induces HO-1 expression by its action on Nrf2 thusamplifying HO-1 expression [41 42] CO initially acts viainhibition of cytochrome 119888 oxidase in the mitochondrialelectron transport chain leading to the generation of lowlevels of O

2

minus and subsequently hydrogen peroxide (H2O2)

that initiates the ensuing adaptive signaling [36] Inhaled COin mice or treatment of keratinocytes with H

2O2induces the

phosphorylationactivation of p38 MAPK and Akt [43 44]Analysis using specific inhibitors of p38 MAPK and Akt hasdemonstrated that only Akt activation is involved in HO-1 and Nrf2 expression [44] In addition PI3 K and PKCinhibitors suppressed Akt phosphorylation Nrf2 activationand HO-1 expression [44] Additional studies in knockoutanimals are warranted to further define the molecular signal-ing pathways responsible for upregulation of HO-1 by CO

Thus CO induces an antioxidant (Nrf2 responsive genes)and anti-inflammatory (eg NF-120581B suppression HO-1 andinterleukin-10 upregulation) response In addition CO mayinhibit TLR4 signal transduction by enhancing the interac-tion of TLR4 with caveolin-1 [45] and by downregulatingTLR4 expression [46]

6 Biliverdin Cytoprotection

Biliverdin is produced by the HO reaction with hemeBiliverdin reductase (BVR) catalyzes the reduction ofbiliverdin to bilirubin BVR is expressed on the exterior ofthe plasma membrane where it quickly converts biliverdinto bilirubin [47] The enzymatic conversion of biliverdin tobilirubin by BVR initiates a signaling cascade that results

in a rapid increase in phosphorylation of Akt leading tocytoprotection due in part to upregulation of interleukin-10expression [47] In addition phosphorylated Akt phosphory-lates endothelial nitric oxide synthase (eNOS) in endothelialcells leading to S-nitrosylation of BVR [47] S-nitrosylation ofBVR leads to nuclear translocation where BVR binds to AP-1sites in the TLR4 promoter and blocks transcription of TLR4[47] In addition human BVR is a SerThrTyr-kinase andupstream activator of PKC and the insulininsulin growthfactor-1 pathways [48] Thus like CO biliverdin reductionto bilirubin by BVR regulates vital homeostatic signalingpathways in response to hemolysis

7 Ferritin Heavy Chain (FHC) Cytoprotection

The induction of HO-1 is accompanied by the induction offerritin [49] Iron (Fe2+) released during the HO reactioninduces the translation of ferritin [50] Labile cellular ironstimulates the translation of ferritin mRNA through inter-action between a cytoplasmic iron regulatory protein (IRP)and a conserved nucleotide iron responsive element (IRE)present in the 51015840 noncoding region of all ferritin mRNAsTheIRE forms a stem-loop structure and when the supply of ironto the cells is inadequate the IRP is bound to the IRE andsuppresses ferritin synthesis [51] Ferritins are comprised ofvarious ratios of heavy and light chains that form a proteinshell surrounding an iron core Ferritin is cytoprotectivein cells by its capacity to bind 4500 iron molecules andthrough its FHC ferroxidase activity [52] which oxidizesredox active Fe2+ to Fe3+ for safe (redox inactive) storage inthe core of the ferritin complex FHC is protective againstheme-mediated oxidative injury to endothelial cells invitro [49] FHC mutants lacking ferroxidase activity arenot cytoprotective against heme-mediated oxidative injuryOverexpression of FHC protects tissues from ischemia-reperfusion injury [53] antagonizes TNF120572-mediatedapoptosis [54] protects cells from UV-radiation damage[55] prevents 1-methyl-4-phenyl-1236-tetrahydropyridine-(MPTP-) induced neurotoxicity [56] and protects HeLacells from H

2O2toxicity [57] Nuclear FHC may play an

important role in cytoprotection Identification of a DNAbinding motif for FHC raises the novel possibility of a rolefor FHC as a conventional transcription factor [58] NuclearFHC has been reported to incorporate into DNA and toprotect DNA from UV and oxidative damage FHC alsobinds with nuclear death domain-associated protein toinhibit DAXX-mediated apoptosis [59 60]

8 HO-1 and Sickle Cell Disease

Sickle cell disease is an archetypal example of a chronichemolytic disease An inherited mutation the amino acidglutamic acid is replaced with the amino acid valine at posi-tion 6 in 120573-globin (Glu6Val)The resulting Hb-S polymerizesin the de-oxygenated state forming long rigid moleculesthat stick together The polymerization of Hb-S leads tovarious shape changes in RBC including an elongated sickleor crescent shape release of heme into RBC membranes


0

20

40

60

80

100

0 5 10 15 20 25 30

Biot

in R

BC i

n bl

ood

()

Days

HbSS-TownesHbAS-TownesHbAA-TownesC57BL6

Half-lives (days)25111624

Figure 1 Red blood cell survival in C57BL6 HbAA- HbAS-and HbSS-Townes mice RBC half-lives were measured in C57BL6HbAA- HbAS- and HbSS-Townes mice (119899 = 5ndash7 micegroup)Values are means standard deviations have been omitted for claritybut coefficients of variation were less than 10 The half-lives ofcirculating RBCs were measured by biotinylating RBCs by tailvein injection of 150 120583L of 30mgmL sulfo-N-hydroxysuccinimide-biotin (Thermo Scientific Rockford IL USA) Biotinylated RBCswere labeled with streptavidin-APC and the percentages of labeledand unlabeled RBC were measured by flow cytometry RBC half-lives were estimated by interpolating between data points on eitherside of 50 survival Half-lives for each mouse model are listedbelow the figure

oxidation of RBC lipids and ultimately both intravascularand extravascular hemolysis [61] Excessive release of Hb-S from sickle RBC into plasma can lead to the depletion ofplasma haptoglobin hemopexin and NO [62 63] depositionof heme iron in cells and a proinflammatoryprothromboticphenotype that promotes episodic painful vasoocclusivecrises leading to ischemiareperfusion injury and organinfarction

Sickle cell disease patients in crisis-free steady state areexperiencing unrelenting chronic hemolysis The rate ofhemolysis can be determined by measuring the half-life ofRBC in patients or transgenic mouse models of sickle celldisease (Figure 1) The 120573murine knock-out120573S knock-in mousemodel developed by the Townes laboratory [64] can beused to illustrate this point RBC half-lives were measuredin separate cohorts of HbAA-Townes (expressing normalhuman 120573A-globin) HbAS (heterozygous for 120573A- and 120573S-globin) and HbSS (homozygous for 120573S-globin) mice andin C57BL6 mice All circulating RBC were biotinylated attime zero Five 120583L blood samples were obtained by tailvein puncture at various time intervals as indicated andthe percentages of biotin labeled and unlabeled RBC weremeasured by flow cytometry The half-lives of RBC in HbSS-Townes mice were markedly shorter (sim25 days) than that ofHbAA-Townes mice (sim16 days) RBC in the HbAS-Townesmice had a half-life that was intermediate (sim11 days) betweenAA and SS mice The mean RBC half-life in normal C57BL6

0

2

4

6

C57B

L6

HbA

A-To

wne

s

HbA

S-To

wne

s

HbS

S-To

wne

s

CO (n

mol

hg

)

Mouse strain

C57B

L6+

PHZ

lowast lowast

Figure 2 Endogenous CO production in mouse models of SCDExhaled CO was collected for 3 hours from C57BL6 HbAA-HbAS- and HbSS-Townes mice (119899 = 5ndash7 micegroup) C57BL6mice injected with phenylhydrazine an hemolysis inducing drug48 and 24 hours before collection of exhaled CO served as positivecontrols for breath CO measurements Exhaled CO was 59-foldhigher in C57BL6 mice injected with phenylhydrazine (119899 = 4) thanuntreated C57BL6 mice (119899 = 12 lowast119875 lt 0001) Exhaled CO levelswere not significantly higher than untreated C57BL6 controls in theHbAA- and Hb-AS-Townes (119899 = 4) models Exhaled CO levels werehighest in HbSS-Townes mice (119899 = 4) 69-fold higher than HbAA-Townes mice (119899 = 4 lowast119875 lt 0001) Values are means plusmn SD

mice was 24 days which was 50 longer than HbAA-Townesmice The chronic hemolysis in HbSS mice can also be seenwhen measuring expired CO (Figure 2) The heme releasedfrom Hb during hemolysis eventually reaches tissues wherethe heme is degraded by HO releasing CO that can becarried by Hb to the lungs and expired Normal C57BL6mice release sim1 nmolhg of CO When C57BL6 mice areinjected with phenylhydrazine (PHZ) to induce massivehemolysis the expired CO goes to sim5 nmolshg SimilarlyHbAA mice expire sim1 nmolshg of CO and the HbSS miceexpire 6-7 nmolshg of CO which is indicative of the chronichemolysis in theHbSSmice CO expiration in theHbASmiceis similar to HbAAmice suggesting measurement of expiredCOmay not be as sensitive in determining hemolytic rates asmeasurement of RBC turnover However the elevated levelsof CO in expired breath are consistent with HO catabolism ofheme in states of excessive hemolysis

Counteracting the toxic effects of hemolysis is the upreg-ulation of HO-1 in tissues of transgenic sickle mice (Figure 3)and human sickle patients [65 67] Further increases in HO-1 inhibit vascular inflammation and vaso-occlusion in mousemodels of sickle cell disease [65 66] Transgenic sickle micewith additional 3ndash5 fold overexpression of wild type (wt)-HO-1 in the liver using a Sleeping Beauty transposon systemhave activated nuclear phospho-p38 MAPK and phospho-Akt (Figures 4(a) and 4(b)) decreased nuclear expressionof NF-120581B p65 (Figure 4(c)) and decreased soluble vascularcell adhesion molecule-1 (sVCAM-1) in serum (Figure 4(d))[66] Pretreatment of sickle mice for 3 consecutive dayswith hemin (40120583molskg) intraperitoneally induced HO-1 in mouse tissues and mimicked the effects of wt-HO-1


LungLiver

Spleen

Normal S + S-Antilles BERK

(a)

0

2

4

6

8

Lung Liver Spleen

Fold

abov

e nor

mal

Normal

BERK

lowast

lowastlowast lowast lowast

lowast

S + S-Antilles

(b)

Figure 3 HO-1 expression is elevated in the organs of sickle mice Western blots for HO-1 were performed on organ homogenates (1120583g ofhomogenate DNA per lane) from lungs livers and spleens of untreated normal mice and S + S-Antilles and BERK sickle mice The 32 kDHO-1 bands are shown for each organ and each mouse (a) The mean HO-1 band intensities (119899 = 4) plusmn SD are expressed as fold above normalcontrol mice (b) lowast119875 lt 005 normal versus sickle Figure derived from [65] American Society for Clinical Investigation

Phospho-p38 MAPK

Total p38 MAPK

CTL LRS wt-HO-1 ns-HO-1 Hemin

(a)

Phospho-Akt

Total Akt


(b)

0

1

2

3

4

Control LRS wt-HO-1 ns-HO-1 HeminRela

tive N

F-120581

B p6

5 ex

pres

sion

in th

e nuc

leus

lowast

lowast

(c) NF-120581B p65

0

200

400

600

800

1000

1200

Control LRS wt-HO-1 ns-HO-1 Hemin

sVCA

M-1

(ng

mL)

lowast

lowast

(d) sVCAM-1

Figure 4 Cytoprotective pathways are activated in sickle mice overexpressing wild type (wt)-HO-1 Nuclear extracts were isolated from liversand 30 120583g of nuclear extract protein from each liver was run on a western blot and immunostained for phospho- and total p38 and Akt andNF-120581B p65 NF-120581B p65 protein bands at 65 kDa were quantified by densitometry Nuclear phospho-p38MAPK (a) and phospho-Akt (b) wereincreased in mice 8 weeks after hydrodynamic infusion of Sleeping Beauty (SB)-wt-HO-1 DNA or after intraperitoneal injection of heminchloride for 3 consecutive days but not in control mice given a hydrodynamic infusion of lactated Ringerrsquos solution (LRS) or SB-nonsense(ns)-HO-1 DNA Total nuclear p38 MAPK (a) and Akt (b) were not different between treatment groups NF-120581B p65 was decreased in livernuclear extracts of sickle mice injected with SB-wt-HO-1 or hemin but not in control LRS or SB-ns-HO-1 treated mice (c) Serum levelsof sVCAM-1 were measured by ELISA Serum sVCAM-1 was lower in sickle mice injected with SB-wt-HO-1 DNA (119875 lt 005) or hemin(119875 lt 005) but not in mice infused with SB-ns-HO-1 DNA when compared to mice infused with LRS (d) Values are means plusmn SEM 119899 = 2ndash4mice per treatment group lowast119875 lt 005 compared to LRS controls using one-way ANOVA Figure derived from [66]


0

5

10

15

20

25


Mea

n st

asis

()

lowast

lowast

Figure 5 Vaso-occlusion (stasis) is inhibited in the skin of sicklemice 8 weeks after hydrodynamic infusion of liverndashdirected SB-wt-HO-1 or 24 h after 3 days of intraperitoneal hemin pretreatment(40120583molskg) compared to control mice or mice given hydrody-namic infusions of LRS or enzymatically inactive ns-HO-1 Vascularstasis was measured in a dorsal skin fold chamber (DSFC) modelafter HR At baseline in room air the mice were placed under amicroscope and flowing venules were selected inside the DSFCThemice were then subjected to 1 h of hypoxia (7O

293N

2) followed

by 1 h of reoxygenation in room air After 1 h of reoxygenation thesame venules were reexamined for blood flow The number of staticvenules exhibiting no blood flow was counted and expressed as apercentage of the total number of venules examined There were 7mice and 403 venules in the control group 5 mice and 243 venulesin the LRS group 5 mice and 347 venules in the wt-HO-1 group6 mice and 325 venules in the ns-HO-1 group and 5 mice and 227venules in the hemin groupThere was aminimum of 26 venules permouse Values are mean stasis plusmn SEMThe proportions of venulesexhibiting stasis in each treatment group were compared using a 119911-test lowast119875 lt 005 compared to LRS controls Figure derived from [66]

overexpression via the Sleeping Beauty transposon system(Figures 4(a)ndash4(d)) Hypoxia-reoxygenation (HR)-inducedvaso-occlusion (stasis) a characteristic of sickle but notnormal mice is inhibited in subcutaneous skin of sicklemice despite the absence of the HO-1 transgene in theskin suggesting distal effects of HO activity in the liveron the vasculature (Figure 5) [66] No protective effects areseen in sickle mice overexpressing a nonsense rat hmox-1gene (ns-HO-1) that encodes carboxy-truncated HO-1 withlittle or no enzyme activity [66] As previously shown [65]mice pretreated for 3 days with hemin intraperitoneally toinduce HO-1 have significantly lower stasis (28) than micepretreated with lactated Ringerrsquos solution (LRS) (119875 lt 0006)[66] The cytoprotective effects of HO-1 overexpression intransgenic sickle cell mice are mimicked by administrationof inhaled CO or biliverdin [65]

In sickle mice and patients in steady state hemolysisis in balance with HO activity a biologic set point withjust enough HO-1 induction to counterbalance hemolysisHowever hyperhemolysis can occur during vasoocclusivecrises in patients with sickle cell disease [68] suggesting anincreased rate of intravascular hemolysis may tip a sicklepatientrsquos balance from steady state to vasoocclusive crisisVaso-occlusion is an inflammatory adhesion driven process[14 65 69] Vaso-occlusion can be induced in transgenicsicklemice by exposing themice toHR or by infusing a bolus

of Hb or hemin to simulate a sudden increase in intravascularhemolysis [14 65 69] Any of these insults (HR Hb andhemin) leads to stasis of blood flow in the postcapillaryvenules [14 65 69] This vaso-occlusion can be blockedby inhibiting TLR4 signaling [14] supporting the conceptthat hemin released from Hb during hemolysis promotesvaso-occlusion through hemin-induced TLR4 signaling andsubsequent inflammatory response

9 Potential Therapies for Heme-InducedInflammation and Vascular Activation

Themost effective strategy to avert heme-induced inflamma-tion and vascular activation is to prevent or inhibit hemolysisIn sickle cell disease there ismuch effort underway to developdrugs to induce fetal Hb expression in RBC because of itsability to inhibit the polymerization of HbS and subsequenthemolysis In lieu of effective strategies to stop hemolysisthe clinician must deal with the consequences of hemolysisInfusions of supplemental haptoglobin andor hemopexin areunder consideration [7] Other options include pharmaco-logic induction of the HO-1ferritin system or inhibition ofTLR4 signaling This could possibly be accomplished by theadministration of biliverdin [47 70 71] inhaled CO CO-releasing molecules or CO bound to pegylated Hb [36 4365 72]

10 Conclusions

The release of free heme during hemolysis is injurious tocells Extraordinary arrays of plasma and cellular defenseshave evolved to protect cells from free heme Failure toadequately protect against free heme can result in damageto the vasculature This damage can be stopped by a robustanti-oxidative and anti-inflammatory response by vascularcells With our current knowledge and understanding ofthese pathways it may be possible to intervene clinically tobolster the cytoprotective responses and prevent much of thevascular damage associated with hemolysis

Abbreviations

Hb HemoglobinRBC Red blood cellsROS Reactive oxygen speciesMetHb Ferric hemoglobinTLR4 Toll-like receptor-4DAMP Damage-associated molecular patternLPS LipopolysaccharideTNF-120572 Tumor necrosis factor-alphaWPB Weibel-Palade BodyNF-120581B Nuclear factor-kappa BNox NADPH oxidaseHO Heme oxygenaseMAPK Mitogen-activated protein kinasePKC Protein kinase CPI3K Phosphatidylinositol 3-kinase


Akt Protein kinase BAP-1 Activator protein-1Nrf2 Nuclear factor-erythroid 2 p45-related factor

2BVR Biliverdin reductaseBach1 BTB and CNC homologue 1Keap1 Kelch-like ECH-associated protein 1ARE Antioxidant response elementsMARE Maf recognition elementFHC Ferritin heavy chainPpm Parts per millionHIF-1120572 Hypoxia-inducible factor-1-alphaeNOS Endothelial nitric oxide synthaseIRP Iron regulatory proteinIRE Iron response elementMPTP 1-Methyl-4-phenyl-1236-

tetrahydropyridineDAXX Death domain-associated proteinPHZ PhenylhydrazineWt Wild typesVCAM-1 Soluble vascular cell adhesion molecule-1HR Hypoxia-reoxygenationNs NonsenseLRS Lactated Ringerrsquos solution

Conflict of Interests

John D Belcher and Gregory M Vercellotti receive researchfunding from Sangart Inc

Acknowledgments

This work was funded by a Grant from National HeartLung and Blood Institute National Institutes of Health (P01HL55552 and R01 HL115467-01 to Gregory M Vercellottiand John D Belcher) The authors would like to thank JuliaNguyen Julie Furne andDrMichael Levitt for collection andmeasurement of expired CO in mice Dr Chunsheng Chenand Dr TomWelsh for measurement of microvascular stasisand performing western blots Paul Marker for assistance inmeasuring RBC half-life and Dr Robert Hebbel and FuadAbdulla for breeding and phenotyping the transgenic sicklemice

References

[1] A V Graca-Souza M A B Arruda M S de Freitas C Barja-Fidalgo and P L Oliveira ldquoNeutrophil activation by hemeimplications for inflammatory processesrdquo Blood vol 99 no 11pp 4160ndash4165 2002

[2] B N Porto L S Alves P L Fernandez et al ldquoHeme inducesneutrophil migration and reactive oxygen species genera-tion through signaling pathways characteristic of chemotacticreceptorsrdquo Journal of Biological Chemistry vol 282 no 33 pp24430ndash24436 2007

[3] F A D T G Wagener H-D Volk D Willis et al ldquoDifferentfaces of the heme-heme oxygenase system in inflammationrdquoPharmacological Reviews vol 55 no 3 pp 551ndash571 2003

[4] R T Figueiredo P L Fernandez D S Mourao-Sa et alldquoCharacterization of heme as activator of toll-like receptor 4rdquoJournal of Biological Chemistry vol 282 no 28 pp 20221ndash20229 2007

[5] P W Buehler and F DrsquoAgnillo ldquoToxicological consequencesof extracellular hemoglobin biochemical and physiologicalperspectivesrdquo Antioxidants and Redox Signaling vol 12 no 2pp 275ndash291 2010

[6] M J Nielsen H J Moslashller and S K Moestrup ldquoHemoglobinand heme scavenger receptorsrdquo Antioxidants and Redox Signal-ing vol 12 no 2 pp 261ndash273 2010

[7] D J Schaer P W Buehler A I Alayash J D Belcher andG M Vercellotti ldquoHemolysis and free hemoglobin revisitedexploring hemoglobin and hemin scavengers as a novel class oftherapeutic proteinsrdquo Blood vol 121 no 8 pp 1276ndash1284 2013

[8] M T Gladwin G J Kato D Weiner et al ldquoNitric oxide forinhalation in the acute treatment of sickle cell pain crisis arandomized controlled trialrdquo Journal of the American MedicalAssociation vol 305 no 9 pp 893ndash902 2011

[9] H F Bunn and J H Jandl ldquoExchange of heme amonghemoglobins and between hemoglobin and albuminrdquo Journalof Biological Chemistry vol 243 no 3 pp 465ndash475 1968

[10] P Ascenzi A Bocedi P Visca et al ldquoHemoglobin and hemescavengingrdquo IUBMB Life vol 57 no 11 pp 749ndash759 2005

[11] E Tolosano S Fagoonee N Morello F Vinchi and V FioritoldquoHeme scavenging and the other facets of hemopexinrdquo Antiox-idants and Redox Signaling vol 12 no 2 pp 305ndash320 2010

[12] M Kristiansen J H Graversen C Jacobsen et al ldquoIdentifica-tion of the haemoglobin scavenger receptorrdquo Nature vol 409no 6817 pp 198ndash201 2001

[13] F Vinchi L de Franceschi A Ghigo et al ldquoHemopexin therapyimproves cardiovascular function by preventing heme-inducedendothelial toxicity in mouse models of hemolytic diseasesrdquoCirculation vol 127 no 12 pp 1317ndash1329 2013

[14] J D Belcher J Nguyen C Chen et al ldquoPlasma hemoglobin andheme triggerWeibel Palade body exocytosis and vaso-occlusionin transgenic sickle micerdquo Blood vol 118 article 896 2011

[15] G Andonegui C S Bonder F Green et al ldquoEndothelium-derived toll-like receptor-4 is the key molecule in LPS-inducedneutrophil sequestration into lungsrdquo Journal of Clinical Investi-gation vol 111 no 7 pp 1011ndash1020 2003

[16] J Fan R S Frey andA BMalik ldquoTLR4 signaling induces TLR2expression in endothelial cells via neutrophil NADPH oxidaserdquoJournal of Clinical Investigation vol 112 no 8 pp 1234ndash12432003

[17] W Wang M Deng X Liu W Ai Q Tang and J HuldquoTLR4 activation induces nontolerant inflammatory responsein endothelial cellsrdquo Inflammation vol 34 no 6 pp 509ndash5182011

[18] G Zanotti M Casiraghi J B Abano et al ldquoNovel criticalrole of Toll-like receptor 4 in lung ischemia-reperfusion injuryand edemardquoAmerican Journal of PhysiologymdashLung Cellular andMolecular Physiology vol 297 no 1 pp L52ndashL63 2009

[19] E M Palsson-McDermott and L A J OrsquoNeill ldquoSignal trans-duction by the lipopolysaccharide receptor Toll-like receptor-4rdquo Immunology vol 113 no 2 pp 153ndash162 2004

[20] K M Valentijn J E Sadler J A Valentijn J Voorberg and JEikenboom ldquoFunctional architecture ofWeibel-Palade bodiesrdquoBlood vol 117 no 19 pp 5033ndash5043 2011

[21] P L Fernandez F F Dutra L Alves et al ldquoHeme amplifiesthe innate immune response to microbial molecules through


spleen tyrosine kinase (Syk)-dependent reactive oxygen speciesgenerationrdquo Journal of Biological Chemistry vol 285 no 43 pp32844ndash32851 2010

[22] E Griffiths A Cortes N Gilbert P Stevenson S MacDonaldand D Pepper ldquoHaemoglobin-based blood substitutes andsepsisrdquoThe Lancet vol 345 no 8943 pp 158ndash160 1995

[23] D Su R I Roth M Yoshida and J Levin ldquoHemoglobinincreases mortality from bacterial endotoxinrdquo Infection andImmunity vol 65 no 4 pp 1258ndash1266 1997

[24] B N Y Setty S G Betal J Zhang and M J Stuart ldquoHemeinduces endothelial tissue factor expression potential rolein hemostatic activation in patients with hemolytic anemiardquoJournal of Thrombosis and Haemostasis vol 6 no 12 pp 2202ndash2209 2008

[25] J D Belcher J D Beckman G Balla J Balla and G VercellottildquoHeme degradation and vascular injuryrdquo Antioxidants andRedox Signaling vol 12 no 2 pp 233ndash248 2010

[26] H S Park H Y Jung E Y Park J Kim W J Lee and Y SBae ldquoCutting edge direct interaction of TLR4 with NAD(P)Hoxidase 4 isozyme is essential for lipopolysaccharide-inducedproduction of reactive oxygen species and activation of NF-120581BrdquoJournal of Immunology vol 173 no 6 pp 3589ndash3593 2004

[27] A Loboda A Jazwa A Grochot-Przeczek et al ldquoHemeoxygenase-1 and the vascular bed frommolecular mechanismsto therapeutic opportunitiesrdquoAntioxidants andRedox Signalingvol 10 no 10 pp 1767ndash1812 2008

[28] K A Nath J P Grande G Farrugia et al ldquoAge sensitizes thekidney to heme protein-induced acute kidney injuryrdquoAmericanJournal of PhysiologymdashRenal Physiology vol 304 no 3 ppF317ndashF325 2013

[29] E M Sikorski T Hock N Hill-Kapturczak and A AgarwalldquoThe story so far molecular regulation of the heme oxygenase-1 gene in renal injuryrdquo American Journal of PhysiologymdashRenalPhysiology vol 286 no 3 pp F425ndashF441 2004

[30] Y-M Kim H-O Pae J E Park et al ldquoHeme oxygenase in theregulation of vascular biology from molecular mechanisms totherapeutic opportunitiesrdquo Antioxidants and Redox Signalingvol 14 no 1 pp 137ndash167 2011

[31] T Ishii K Itoh S Takahashi et al ldquoTranscription factor Nrf2coordinately regulates a group of oxidative stress-induciblegenes inmacrophagesrdquo Journal of Biological Chemistry vol 275no 21 pp 16023ndash16029 2000

[32] J J Boyle M Johns J Lo et al ldquoHeme induces heme oxygenase1 via Nrf2 role in the homeostatic macrophage responseto intraplaque hemorrhagerdquo Arteriosclerosis Thrombosis andVascular Biology vol 31 no 11 pp 2685ndash2691 2011

[33] S Singh S Vrishni B K Singh I Rahman and P KakkarldquoNrf2-ARE stress response mechanism a control point inoxidative stress-mediated dysfunctions and chronic inflamma-tory diseasesrdquo Free Radical Research vol 44 no 11 pp 1267ndash1288 2010

[34] K Igarashi and J Sun ldquoThe heme-Bach1 pathway in the regula-tion of oxidative stress response and erythroid differentiationrdquoAntioxidants and Redox Signaling vol 8 no 1-2 pp 107ndash1182006

[35] C L Townsend and R L Maynard ldquoEffects on health of pro-longed exposure to low concentrations of carbon monoxiderdquoOccupational and Environmental Medicine vol 59 no 10 pp708ndash711 2002

[36] R Motterlini and L E Otterbein ldquoThe therapeutic potential ofcarbonmonoxiderdquoNature Reviews Drug Discovery vol 9 no 9pp 728ndash743 2010

[37] L E Otterbein M P Soares K Yamashita and F H BachldquoHeme oxygenase-1 unleashing the protective properties ofhemerdquo Trends in Immunology vol 24 no 8 pp 449ndash455 2003

[38] J K Sarady B S Zuckerbraun M Bilban et al ldquoCarbonmonoxide protection against endotoxic shock involves recipro-cal effects on iNOS in the lung and liverrdquo The FASEB Journalvol 18 no 7 pp 854ndash856 2004

[39] S W Ryter L E Otterbein D Morse and A M K ChoildquoHeme oxygenasecarbon monoxide signaling pathways reg-ulation and functional significancerdquo Molecular and CellularBiochemistry vol 234-235 pp 249ndash263 2002

[40] K Ohta and A Yachie ldquoDevelopment of vascular biology overthe past 10 years heme oxygenase-1 in cardiovascular home-ostasisrdquo Journal of Endovascular Therapy vol 11 supplement 2pp II140ndashII150 2004

[41] C A Piantadosi M S Carraway A Babiker andH B SulimanldquoHeme oxygenase-1 regulates cardiac mitochondrial biogenesisvia nrf2-mediated transcriptional control of nuclear respiratoryfactor-1rdquo Circulation Research vol 103 no 11 pp 1232ndash12402008

[42] B Wang W Cao S Biswal and S Dore ldquoCarbon monoxide-activated Nrf2 pathway leads to protection against permanentfocal cerebral ischemiardquo Stroke vol 42 no 9 pp 2605ndash26102011

[43] J D Beckman J D Belcher J VVineyard et al ldquoInhaled carbonmonoxide reduces leukocytosis in a murine model of sickle celldiseaserdquoAmerican Journal of PhysiologymdashHeart andCirculatoryPhysiology vol 297 no 4 pp H1243ndashH1253 2009

[44] C N Nguyen H-E Kim and S-G Lee ldquoCaffeoylsero-tonin protects human keratinocyte HaCaT cells against H

2O2-

induced oxidative stress and apoptosis through upregulation ofHO-1 expression via activation of the PI3KAktNrf2 pathwayrdquoPhytotherapy Research 2013

[45] X M Wang H P Kim K Nakahira S W Ryter and A MK Choi ldquoThe heme oxygenase-1carbon monoxide pathwaysuppresses TLR4 signaling by regulating the interaction ofTLR4 with caveolin-1rdquo Journal of Immunology vol 182 no 6pp 3809ndash3818 2009

[46] A Goldberg M Parolini B Y Chin et al ldquoToll-like receptor 4suppression leads to islet allograft survivalrdquoThe FASEB Journalvol 21 no 11 pp 2840ndash2848 2007

[47] B Wegiel C J Baty D Gallo et al ldquoCell surface biliverdinreductase mediates biliverdin-induced anti-inflammatoryeffects via phosphatidylinositol 3-kinase and Aktrdquo Journal ofBiological Chemistry vol 284 no 32 pp 21369ndash21378 2009

[48] P E Gibbs C Tudor and M D Maines ldquoBiliverdin reductasemore than a namesakemdashthe reductase its Peptide fragmentsand biliverdin regulate activity of the three classes of proteinkinase Crdquo Frontiers in Pharmacology vol 3 p 31 2012

[49] G Balla H S Jacob J Balla et al ldquoFerritin a cytoprotectiveantioxidant strategem of endotheliumrdquo Journal of BiologicalChemistry vol 267 no 25 pp 18148ndash18153 1992

[50] J L Casey M W Hentze D M Koeller et al ldquoIron-responsiveelements regulatory RNA sequences that control mRNA levelsand translationrdquo Science vol 240 no 4854 pp 924ndash928 1988

[51] R D Klausner T A Rouault and J B Harford ldquoRegulating thefate of mRNA the control of cellular ironmetabolismrdquoCell vol72 no 1 pp 19ndash28 1993

[52] P M Harrison and P Arosio ldquoThe ferritins molecular proper-ties iron storage function and cellular regulationrdquo Biochimicaet Biophysica Acta vol 1275 no 3 pp 161ndash203 1996


[53] P O Berberat M Katori E Kaczmarek et al ldquoHeavy chainferritin acts as an antiapoptotic gene that protects livers fromischemia reperfusion injuryrdquoThe FASEB Journal vol 17 no 12pp 1724ndash1726 2003

[54] C Xie N Zhang H Zhou et al ldquoDistinct roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induceddeath in L929 cellsrdquoMolecular and Cellular Biology vol 25 no15 pp 6673ndash6681 2005

[55] M Goralska B L Holley and M C McGahan ldquoOverex-pression of H- and L-ferritin subunits in lens epithelial cellsFe metabolism and cellular response to UVB irradiationrdquoInvestigative Ophthalmology and Visual Science vol 42 no 8pp 1721ndash1727 2001

[56] D Kaur F Yantiri S Rajagopalan et al ldquoGenetic or pharma-cological iron chelation prevents MPTP-induced neurotoxicityin vivo a novel therapy for Parkinsonrsquos diseaserdquoNeuron vol 37no 6 pp 899ndash909 2003

[57] A Cozzi B Corsi S Levi P Santambrogio A Albertini andP Arosio ldquoOverexpression of wild type and mutated humanferritinH-chain inHeLa cells in vivo role of ferritin ferroxidaseactivityrdquo Journal of Biological Chemistry vol 275 no 33 pp25122ndash25129 2000

[58] A A Alkhateeb and J R Connor ldquoNuclear ferritin a new rolefor ferritin in cell biologyrdquo Biochimica et Biophysica Acta vol1800 no 8 pp 793ndash797 2010

[59] C X Cai D E Birk and T F Linsenmayer ldquoNuclear ferritinprotects DNA from UV damage in corneal epithelial cellsrdquoMolecular Biology of the Cell vol 9 no 5 pp 1037ndash1051 1998

[60] F Liu Z-Y Du J-L He X-Q Liu Q-B Yu and Y-XWang ldquoFTH1 binds to Daxx and inhibits Daxx-mediated cellapoptosisrdquoMolecular Biology Reports vol 39 no 2 pp 873ndash8792012

[61] R P Hebbel ldquoReconstructing sickle cell disease a data-based analysis of the ldquohyperhemolysis paradigmrdquo for pul-monary hypertension from the perspective of evidence-basedmedicinerdquo American Journal of Hematology vol 86 no 2 pp123ndash154 2011

[62] U Muller-Eberhard J Javid H H Liem A Hanstein and MHanna ldquoPlasma concentrations of hemopexin haptoglobin andheme in patientswith various hemolytic diseasesrdquoBlood vol 32no 5 pp 811ndash815 1968

[63] C D Reiter X Wang J E Tanus-Santos et al ldquoCell-freehemoglobin limits nitric oxide bioavailability in sickle-celldiseaserdquo Nature Medicine vol 8 no 12 pp 1383ndash1389 2002

[64] L-C Wu C-W Sun T M Ryan K M Pawlik J Ren and TM Townes ldquoCorrection of sickle cell disease by homologousrecombination in embryonic stem cellsrdquo Blood vol 108 no 4pp 1183ndash1188 2006

[65] J D Belcher H Mahaseth T E Welch L E Otterbein RP Hebbel and G M Vercellotti ldquoHeme oxygenase-1 is amodulator of inflammation and vaso-occlusion in transgenicsickle micerdquo Journal of Clinical Investigation vol 116 no 3 pp808ndash816 2006

[66] J D Belcher J V Vineyard C M Bruzzone et al ldquoHemeoxygenase-1 gene delivery by Sleeping Beauty inhibits vascularstasis in a murine model of sickle cell diseaserdquo Journal ofMolecular Medicine vol 88 no 7 pp 665ndash675 2010

[67] M L Jison P J Munson J J Barb et al ldquoBlood mononu-clear cell gene expression profiles characterize the oxidanthemolytic and inflammatory stress of sickle cell diseaserdquo Bloodvol 104 no 1 pp 270ndash280 2004

[68] S K Ballas and M J Marcolina ldquoHyperhemolysis during theevolution of uncomplicated acute painful episodes in patientswith sickle cell anemiardquo Transfusion vol 46 no 1 pp 105ndash1102006

[69] J D Belcher H Mahaseth T E Welch et al ldquoCritical role ofendothelial cell activation in hypoxia-induced vasoocclusion intransgenic sickle micerdquo American Journal of PhysiologymdashHeartand Circulatory Physiology vol 288 no 6 pp H2715ndashH27252005

[70] R Ollinger H Wang K Yamashita et al ldquoTherapeutic appli-cations of bilirubin and biliverdin in transplantationrdquo Antioxi-dants and Redox Signaling vol 9 no 12 pp 2175ndash2185 2007

[71] R Ollinger K Yamashita M Bilban et al ldquoBilirubin andbiliverdin treatment of atherosclerotic diseasesrdquo Cell Cycle vol6 no 1 pp 39ndash43 2007

[72] K D Vandegriff M A Young J Lohman et al ldquoCO-MP4a polyethylene glycol-conjugated haemoglobin derivative andcarbonmonoxide carrier that reduces myocardial infarct size inratsrdquo British Journal of Pharmacology vol 154 no 8 pp 1649ndash1661 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the bulk of hemin to CD91 receptors on hepatocytes whereit is endocytosed and degraded [12]

3 Heme Stimulation of TLR4 SignalingIs Proinflammatory

Like haptoglobinCD163 the hemopexinCD91 system canbe overwhelmed during periods of excessive hemolysis Inhemopexin null mice there is enhanced clearance of hemeby nonhepatic tissues including the vessel wall [13] Recentlyheme has been shown to be an extracellular inflammatorysignaling molecule in macrophages with strict binding speci-ficity for toll-like receptor-4 (TLR4) [4]These findings implythat heme is a damage-associatedmolecular pattern (DAMP)molecule Signaling of heme through TLR4 is distinct fromthe signaling of bacterial lipopolysaccharide (LPS) throughTLR4 [4] Heme activation of TLR4 is remarkably stringentand requires the iron moiety [4 14] Unlike its analogs orprecursors heme inducesmacrophage tumor necrosis factor-alpha (TNF-120572) secretion that is dependent on TLR4 and itsadaptor molecules MYD88 and CD14 [4] By binding heminhemopexin thwarts hemin-mediated TLR4 signaling andthereby prevents the proinflammatory effects of hemolysis[13 14]

Endothelial cells also express TLR4 [15ndash18] In endothe-lium hemin- or LPS-mediated TLR4 signaling stimulatestwo parallel proinflammatoryprothrombotic pathways (1)Weibel-Palade Body (WPB) degranulation [14] and (2)nuclear factor-kappa B (NF-120581B) activation [14 19] WPBdegranulation occurs within 5 minutes of hemin (or LPS)stimulation of TLR4 WPB degranulation releases numer-ous vasoactive proteins including von Willebrand Fac-tor P-selectin endothelin-1 endothelin-converting enzymeinterleukin-8 tissue-type plasminogen activator eotaxin-3angiopoietin-2 osteoprotegerin and calcitonin gene-relatedpeptide [20] HeminTLR4-mediated degranulation ofWPBson the endothelial cell surface triggers rapid proinflamma-tory prothrombotic and vasoconstrictive responses in thevasculature

In addition to WPB degranulation hemin- or LPS-mediated TLR4 signaling leads to NF-120581B activation andthe transcription of proinflammatory genes [19] Inhibitionof TLR4 signaling or knockout of the TLR4 gene com-pletely abrogates hemin-stimulated WPB degranulation andNF-120581B activation in endothelial cells [14] Heme amplifiesLPS-induced TLR4 signaling [21] which may explain whyhemolysis and hemoglobinuria are associated with increasedmortality in septic patients [22 23]

Hemin also has procoagulant effects on endothelium (vonWillebrand factor and tissue factor expression) [14 24] thatcould be explained by hemin-mediated TLR4 signaling [14]Moreover hemin activation of TLR4 signaling in monocytesand platelets could potentially induce thrombosis via tissuefactor expression on monocytes (unpublished data) andplatelet activation

Hemin also induces oxidative stress in cells Much ofthis has been attributed to iron-catalyzed reactions [25]However there is evidence that LPS activated TLR4 in

endothelial cells interacts with NADPH oxidase 4 (Nox4)a protein related to gp91phox (Nox2) of phagocytic cellsand that Nox4 activity is required for TLR4-mediated NF-120581B activation [26] The Nox protein generates superoxide(O2

minus) by transferring electrons from NADPH to O2 These

superoxide free radicals are rapidly converted to H2O2and

O2in cells Thus heme-mediated TLR4NADPH oxidase

signaling may explain much of the cellular oxidative stressthat occurs during hemolysis

4 Cytoprotection by Heme Oxygenase

Once heme enters the cell membrane the next line ofdefense is heme oxygenase (HO) HO degrades heme intocarbon monoxide (CO) biliverdin and iron (Fe2+) Thereare two isoforms of HO HO-1 and HO-2 [27] HO-1 ishighly inducible by a large number of oxidative stressorsincluding heme In normal healthy tissues not involvedin ongoing RBC destruction HO-1 levels are low [27] Intissues such as the kidney HO-2 a constitutive enzyme isan important first responder to intracellular heme HO-2null mice (HO-2minusminus) compared with HO-2++ mice exhibitgreater renal dysfunction and histologic injury when admin-istered Hb [28] However within 3 hours HO-1 expressionquickly surpasses HO-2 and HO-1 becomes the principlecytoprotectant to subsequent exposures to heme [29] Themechanisms controlling HO-1 induction are complex cellspecific and tightly regulated by transcription A largenumber of kinases (eg mitogen-activated protein kinases(MAPKs) protein kinase C (PKC) and phosphatidylinositol3-kinase (PI3 K)protein kinase B (Akt)) and transcriptionfactors (eg NF-120581B activator protein-1 (AP-1) nuclear fac-tor E2-related factor 2 (Nrf2) biliverdin reductase (BVR)and BTB and CNC homologue 1 (Bach1)) are involved inregulating HO-1 expression [30] Nrf2 appears to be anespecially important regulator of HO-1 induction in responseto oxidative stress and heme [31 32] Nrf2 normally resides inthe cytoplasm bound to an inhibitor protein kelch-like ECH-associated protein-1 (Keap1) Keap1 expedites the ubiquitina-tion and proteolysis of Nrf2 Oxidation of cystine residuesto cysteine (ie disulfide cross-linking) on Keap1 releasesNrf2 prolongs Nrf2 half-life and allows Nrf2 transport to thenucleus [33] Molecular signaling by the PI3 KAkt pathwayalso plays an important role inNrf2 activation ActivatedNrf2translocates to the nucleus and binds to antioxidant responseelements (ARE) which promotes the transcription of a widevariety of antioxidantanti-inflammatory genes includingHO-1 ferritin haptoglobin and hemopexinHeme also bindsto the transcriptional repressor Bach1 in the nucleus Bach-1 is a member of the bZIP transcription factor family thatserves as a repressor of HO-1 transcription through itshigher binding affinity for multiple Maf recognition element(MARE) regions compared with Nrf2 [34] When Bach1forms a heterodimer with Maf it binds to MARE regionsand represses HO-1 transcription Heme binding to Bach1releases Bach1Maf repression allowing Nrf2 binding andHO-1 transcription




2) altering O

2



2



2O2induces the













0

20

40

60

80

100

0 5 10 15 20 25 30

Biot

in R

BC i

n bl

ood

()

Days






0

2

4

6

C57B

L6

HbA

A-To

wne

s

HbA

S-To

wne

s

HbS

S-To

wne

s

CO (n

mol

hg

)

Mouse strain

C57B

L6+

PHZ

lowast lowast





LungLiver

Spleen


(a)

0

2

4

6

8

Lung Liver Spleen

Fold

abov

e nor

mal

Normal

BERK

lowast


lowast

S + S-Antilles

(b)


Phospho-p38 MAPK

Total p38 MAPK


(a)

Phospho-Akt

Total Akt


(b)

0

1

2

3

4


tive N

F-120581

B p6

5 ex

pres

sion

in th

e nuc

leus

lowast

lowast

(c) NF-120581B p65

0

200

400

600

800

1000

1200


sVCA

M-1

(ng

mL)

lowast

lowast

(d) sVCAM-1



0

5

10

15

20

25


Mea

n st

asis

()

lowast

lowast


293N

2) followed







10 Conclusions


Abbreviations








Acknowledgments


References















































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2) altering O

2



2



2O2induces the













0

20

40

60

80

100

0 5 10 15 20 25 30

Biot

in R

BC i

n bl

ood

()

Days






0

2

4

6

C57B

L6

HbA

A-To

wne

s

HbA

S-To

wne

s

HbS

S-To

wne

s

CO (n

mol

hg

)

Mouse strain

C57B

L6+

PHZ

lowast lowast





LungLiver

Spleen


(a)

0

2

4

6

8

Lung Liver Spleen

Fold

abov

e nor

mal

Normal

BERK

lowast


lowast

S + S-Antilles

(b)


Phospho-p38 MAPK

Total p38 MAPK


(a)

Phospho-Akt

Total Akt


(b)

0

1

2

3

4


tive N

F-120581

B p6

5 ex

pres

sion

in th

e nuc

leus

lowast

lowast

(c) NF-120581B p65

0

200

400

600

800

1000

1200


sVCA

M-1

(ng

mL)

lowast

lowast

(d) sVCAM-1



0

5

10

15

20

25


Mea

n st

asis

()

lowast

lowast


293N

2) followed







10 Conclusions


Abbreviations








Acknowledgments


References















































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

0 5 10 15 20 25 30

Biot

in R

BC i

n bl

ood

()

Days






0

2

4

6

C57B

L6

HbA

A-To

wne

s

HbA

S-To

wne

s

HbS

S-To

wne

s

CO (n

mol

hg

)

Mouse strain

C57B

L6+

PHZ

lowast lowast





LungLiver

Spleen


(a)

0

2

4

6

8

Lung Liver Spleen

Fold

abov

e nor

mal

Normal

BERK

lowast


lowast

S + S-Antilles

(b)


Phospho-p38 MAPK

Total p38 MAPK


(a)

Phospho-Akt

Total Akt


(b)

0

1

2

3

4


tive N

F-120581

B p6

5 ex

pres

sion

in th

e nuc

leus

lowast

lowast

(c) NF-120581B p65

0

200

400

600

800

1000

1200


sVCA

M-1

(ng

mL)

lowast

lowast

(d) sVCAM-1



0

5

10

15

20

25


Mea

n st

asis

()

lowast

lowast


293N

2) followed







10 Conclusions


Abbreviations








Acknowledgments


References















































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LungLiver

Spleen


(a)

0

2

4

6

8

Lung Liver Spleen

Fold

abov

e nor

mal

Normal

BERK

lowast


lowast

S + S-Antilles

(b)


Phospho-p38 MAPK

Total p38 MAPK


(a)

Phospho-Akt

Total Akt


(b)

0

1

2

3

4


tive N

F-120581

B p6

5 ex

pres

sion

in th

e nuc

leus

lowast

lowast

(c) NF-120581B p65

0

200

400

600

800

1000

1200


sVCA

M-1

(ng

mL)

lowast

lowast

(d) sVCAM-1



0

5

10

15

20

25


Mea

n st

asis

()

lowast

lowast


293N

2) followed







10 Conclusions


Abbreviations








Acknowledgments


References















































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

25


Mea

n st

asis

()

lowast

lowast


293N

2) followed







10 Conclusions


Abbreviations








Acknowledgments


References















































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References















































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































2O2-




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Review Article Vasculotoxic and Proinflammatory...

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