The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for Hemoglobin Scavenging

Abstract

The haptoglobin- (Hp-) CD163-heme oxygenase-1 (HO-1) pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO), biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6), but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.

Full text

Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume , Article ID ,  pages
http://dx.doi.org/.//
Review Article
The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for
Hemoglobin Scavenging
Jens Haugbølle Thomsen, Anders Etzerodt, Pia Svendsen, and Søren K. Moestrup
Department of Biomedicine, University of Aarhus, Ole Worms Alle 3, Building 1170, 8000 Aarhus C, Denmark
Correspondence should be addressed to Søren K. Moestrup; skm@biokemi.au.dk
Received  March ; Revised  May ; Accepted  May 
Academic Editor: Mohammad Abdollahi
Copyright ©  Jens Haugbølle omsen et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.
e haptoglobin- (Hp-) CD-heme oxygenase- (HO-) pathway is an ecient captor-receptor-enzyme system to circumvent the
hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp
leading to CD-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO--catalyzed
conversion of heme into the metabolites carbon monoxide (CO), biliverdin, and iron. e plasma concentration of Hp is a limiting
factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure
on backup protecting systems such as the hemopexin-CD-HO pathway. e Hp-CD-HO- pathway proteins are regulated by
the acute phase mediator interleukin- (IL-), but other regulatory factors indicate that this upregulation is a counteracting anti-
inammatory response during inammation. e heme metabolites including bilirubin converted from biliverdin have overall
an anti-inammatory eect and thus reinforce the anti-inammatory ecacy of the Hp-CD-HO- pathway. Future studies of
animal models of inammation should further dene the importance of the pathway in the anti-inammatory response.
1. Introduction
Erythrocytes produced in the bone marrow have a life span of
average  days []. At this time the senescent erythrocytes
have undergone changes in the phospholipid composition in
the plasma membrane and they are recognized and phagocy-
tosed by macrophages particularly in the spleen red pulp and
the bone marrow []. Alternatively, the erythrocytes can rup-
ture in the circulation before their expected recognition by
the macrophages. is intravascular hemolysis accounts for
about–percentofthetotalturnoveroferythrocytesdur-
ing normal physiological conditions. Several diseases such
as hemoglobinopathies, autoimmune disorders, malaria, and
other infections may highly increase intravascular hemolysis
thus challenging the inherent Hb scavenging from plasma [].
Hbcanalsobereleasedoutsidethevascularsystemincaseof
internal bleedings such as microvascular and macrovascular
hemorrhage. e Hb release due to intravascular hemolysis
and internal bleeding may have damaging eect on the tissues
[].
e Hb-binding Hp represents a rst defense line that
instantly reduces the Hb toxicity and facilitates its removal
by CD (Figure ). is leads to proteolytic degradation of
Hb and catabolism of the toxic heme moiety via the HO-
 pathway. In this review, we describe the proteins in this
pathway and their suggested role in the anti-inammatory
response.
2. Hp—Expression, Structure, and
BindingofHb
Hp is an abundant plasma glycoprotein(.–. g/L) secreted
primarily by hepatocytes but also by other cell types, such
as monocytes/macrophages and neutrophils [–]. It is post-
translationally cleaved into an 𝛼-anda𝛽-chain forming
a complement control protein (CCP) domain and a serine
proteinase domain, respectively []. e two domains remain
connected through disulde bridges. Furthermore, the CCP
𝛼-chain connects to another 𝛼-chain leading to the Hp 𝛼𝛽
formation, which is the basic form seen in all species. Higher
polymericformsarealsoseeninhumansbecauseofthetwo
allelic Hp variants genes Hp, and Hp [].eygiveriseto
three possible phenotypes: Hp-, Hp- and Hp-. e Hp
gene contains a duplication of a part of the Hp gene, which

Oxidative Medicine and Cellular Longevity
Haptoglobin
Hemoglobin
CD163
CD91
Hemopexin
Heme
Macrophage membrane
Cytoplasm
Receptor
recycling Ligand
endocytosis
Ligand
endocytosis
Receptor
recycling
F : e CD pathway for uptake of Hb-Hp complexes and the CD pathway for uptake of hemopexin- (Hx-) heme complexes. e
endocytosis of the ligand leads to degradation in lysosomes while the receptor recycles from the endosomes back to the plasma membrane.
results in a Hp protein with a duplicated 𝛼-chain. is causes
the formation of a range of polymeric forms of the Hp-
and Hp- phenotypes []. e phenotype is associated with
slight dierences in average plasma Hp levels (Hp- >Hp-
>Hp-) [].
Hp has dierent expression patterns in mammals and
responds to various degrees on inammation. In humans,
Hp is moderately upregulated during acute phase conditions,
where the acute phase mediators interleukin- (IL-) and IL-
further stimulate Hp synthesis in hepatocytes [,]. At sites
of inammation, Hp may be upregulated locally by release
from stored granules in activated neutrophils [].
Recent determination of the crystal structure of the
porcine Hb-Hp complex revealed a barbell-like structure
with oxygenated Hb bound to the serine proteinase domain
(𝛽-chain) of Hp. In this structure, the CCP domains were
connected by the formation of a not previously described
CCP fusion domain formed by 𝛽-strand swapping []. e
binding interface encompasses several of the amino acids
prone to oxidation in the absence of Hp, thus providing
a structural basis for the direct protective function of Hp
[]. e “loop  region” of the serine proteinase domain
previouslyshowntobeinvolvedinthebindingofHpto
CD protrudes from the complex [].
Hb released into plasma from ruptured erythrocytes
dissociates into dimers that instantly bind to Hp by a virtually
irreversible interaction []. It thereby directly “detoxies” the
oxidative Hb, prevents its ltration in the kidney [,], and
promotes the CD-mediated uptake of Hb in macrophages
[].
Hb’s toxicity relates in particular to the formation of
oxygen radicals and the scavenging of nitric oxide (NO) [].
e iron coordinated in Hb and heme reacts strongly in the
presence of hydrogen peroxide producing hydroxyl radicals
and downstream oxidation products. While sequestered in
the erythrocytes, cytoplasmic enzymes such as catalase and
superoxide dismutase limit the concentration of hydrogen
peroxide and oxygen anions and thus the oxidative reactivity

Oxidative Medicine and Cellular Longevity 
ofHb.WhenboundtoHp,theoxidativeintermediateHb-
FeIV is stabilized and rendered less kinetically active [].
Additionally, Hp protects Hb from oxidative modications
that would otherwise prevent its clearance or result in release
of free heme to the circulation [,]. Binding of NO to Hb
in plasma impairs NO signaling, which may aect platelet
aggregation and increase vascular contraction []. ese are
serioussymptomsindiseaseswithstronghemolyticcrises
such as sickle cell anemia. Hp has not yet been shown to
protect against NO scavenging directly, but the observed
protection provided by Hp against nitric oxide scavenging is
probablyduetotheacceleratedclearanceofHbasmediated
by Hp [].
e binding of Hb to Hp- leads to the formation of
an approximate  kDa complex. Much larger complexes
are formed, when Hb binds to the Hp- and Hp- forms.
Whatever kind of Hb-Hp complex is formed, the complex
formation eectively reduces renal ltration of Hb [,]. In
addition, it elicits a high anity site for CD recognition
leading to clearance of Hp and Hb []. As a consequence,
hemolysis leads to consumption of Hp that can be vir-
tuallyabsent,ifthereleaseofHbintoplasmaoverrides
the production of the Hp. A low Hp level in plasma is
therefore a strong and well-known biomarker for accelerated
intravascular hemolysis. Despite circulating Hp in its free
none-Hb-bound form does not bind to CD, the Hb-bound
Hp is directly involved in the binding to CD. Extensive
mutagenesis studies of Hp have identied basic residues in Hp
loop  as important residues involved in the receptor binding
[,]. It is yet not known if Hb binding is involved in CD
binding of the Hp-Hb complex.
3. Other Roles of Hp
Besides its established eect in protecting against the toxic
eectofHb,otherfunctionsofHphavebeenreported.
ese functions, which are yet less explored than the Hb-
relatedfunctionofHp,includepromotedangiogenesisandan
overall anti-inammatory eect as reviewed elsewhere [,].
Furthermore, speculations of other roles of Hp are nourished
by intriguing correlations between various diseases and Hp
phenotypes (reviewed by Levy et al. []). In diabetic patients,
the risk of cardiovascular disease is reported signicantly
higher for patients with the Hp- genotype []. In the same
group of patients, vitamin E supplementation has been shown
to be protective against these cardiovascular complications
[]. Studies of cases of subarachnoid hemorrhage also
indicate an increased risk of cerebral vasospasms in Hp-
 individuals compared to Hp-. On the other hand, Hp-
 has been proposed to have a protective function against
malaria [,]. A recent study demonstrates a link between
hemolysis-induced activation of the HO- and neutrophil
dysfunction which may be aected by the Hp concentration
and phenotype []. However, it should be noted that other
malaria studies have not conrmed signicant association
of Hp genotype on disease outcome [–]. Further epi-
demiological analyses and biochemical studies are warranted
to document and mechanistically understand associations
between Hp phenotype and disease.
4. CD163—Structure, Expression, and
Receptor Function for Hp-Hb
CD is a  kDa transmembrane glycoprotein expressed
exclusively in cells of the reticuloendothelial system. It is a
member of the “scavenger receptor cysteine rich” (SRCR)
superfamily class B. is receptor family is characterized by
containing one or more SRCR domains that are conserved
domains consisting of – amino acids and  to  cysteine
residues connected by disulde bridges []. Crystallization
of the repeat in other proteins has revealed a compact fold
of - 𝛽-sheets cradling an 𝛼-helix [–]. Class A and
classBSRCRdomainsarestructurallysimilarwithonlya
few slight dierences. Class B domains are translated from
a single exon, and class A domains are from two exons and
they contain one more disulde bridge than class B []. e
extracellular segment of CD contains nine SRCR domains
only separated by a  proline/serine/threonine-rich linker
region between domain  and  [].
Four dierent isoforms have been demonstrated, result-
ing from alternative splicing of the RNA encoding the
cytoplasmic tail []. e shortest and most abundant variant
consists of  amino acid residues, while the longest consists
ofand,respectively.erstaminoacidsaer
the transmembrane segment are identical amongst the iso-
forms and contain phosphorylation motives for casein kinase
and protein kinase C []. Other possible phosphorylation
motives are present on the longer isoforms []. Confocal
microscopy has revealed that the shortest tail variant is pri-
marily present in the cell membrane while the longer variants
are located in the endosomal/Golgi cellular compartment
[].
CD is expressed exclusively on cells of the monocyte-
macrophage cell lineage. A high expression is seen in most
mature tissue macrophages such as Kuper cells in the liver,
red pulp macrophages in the spleen, resident bone marrow
macrophages, and alveolar macrophages in the lungs [].
Cell types derived from monocytes showing low or no CD
expression include dendritic cells, Langerhans cells, and
white pulp macrophages in the spleen [,].
Several endogenous and exogenous molecules have been
shown to regulate the expression of CD in in vitro
experiments. Glucocorticoids, IL-, and interleukin- (IL-
) strongly upregulate its expression, whereas interferon-
𝛾(IFN𝛾), tumor necrosis factor-𝛼(TNF-𝛼), interleukin-
(IL-), granulocyte/macrophage colony stimulating factor
(GM-CSF), lipopolysaccharide (LPS), and CXC-chemokine
ligand  (CXCL) downregulate CD expression [,,
]. e upregulation of CD by glucocorticoids has also
been demonstrated in human volunteers following injection
with the glucocorticoid prednylidene []. Whereas IL-
 has both pro- and anti-inammatory eects [], the
overall pattern is that CD expression is induced by anti-
inammatory mediators and reduced by proinammatory
molecules.
Experimental studies have shown that CD is expressed
on macrophages matching the phenotype dened by in
vitro dierentiation in response to IL- and interleukin-
(IL-) (M/alternatively activated macrophages) despite the

Oxidative Medicine and Cellular Longevity
fact that IL- alone decreases CD expression in mono-
cyte/macrophages [,]. CD positive macrophages of a
similar phenotype are abundant in the resolution phase of the
inammatory process []. ese ndings have been used to
hypothesize that CD is a marker of an anti-inammatory
and tissue homeostatic macrophage subclass []. CD is
now widely used as a marker for the macrophage class.
Finally, a novel macrophage subtype designated Mhem is
dened by a high CD and a low mannose receptor
expression []. ese macrophages have been described in
atherosclerotic lesions and they were suggested to exhibit an
antiatherogenic phenotype when examined in vivo [].
A soluble form of CD (sCD) is present in plasma
anditisupregulatedinanumberofdiseasesinvolving
macrophagesasrecentlyreviewedbyMoller[]. It is
generated by ectodomain shedding of the extracellular part of
the receptor. Both TNF-𝛼cleaving enzyme (TACE)/ADAM
and neutrophil elastase have been reported as enzymes
responsible for the cleavage []. However, the concomitant
increase in sCD and TNF-𝛼in humans exposed to LPS
does suggest an important role of TACE/ADAM, which
is activated by LPS in macrophages []. e biological
function of sCD is not yet clear, although several possible
functions have been proposed—including opsonization of
Staph. Aureus [], inhibition of T-cell proliferation []
and inhibition of tumor necrosis factor-like weak inducer of
apoptosis (TWEAK) [].
e third SRCR domain of CD is involved in the
Ca2+-dependent binding of the Hp-Hb complex [,].
e subsequent endocytosis of the ligand bound receptor
is dependent on the endocytic motifs in the cytoplasmic
tail [,]. e various CD isoforms dier in endocytic
ecacy with the shortest variant demonstrating the fastest
uptake [].
In addition to its uptake of the Hb-Hp complex CD
can facilitate the uptake of free Hb. is allows CD to
act as its own fail-safe system in pathological situations
where Hp is depleted due to excessive intravascular hemolysis
[].Towhatdegreethisfunctionisimplicatedinhuman
disease is unknown. Studies in the mouse Hp-Hb system
have disclosed subtle dierences. In this species, Hb binds
with higher anity to CD and the binding of Hp to Hb
does not further increase anity for CD []. e CD-
mediated uptake of Hb (in complex with Hp or not) induces
the secretion of IL- and IL-, as well as it upregulates several
genes responsible for the degradation of Hb-including HO-
[,].
5. Other Potential Functions of CD163
Several functions besides the scavenging of Hb have been
proposed for CD. In rats, CD expressed on resident
bone marrow macrophages has been shown to bind erythrob-
lasts and promote growth and/or survival in erythropoiesis
[]. A recent study indicates a role of CD as a pathogen-
associated molecular pattern (PAMP) receptor []. CD
demonstrated binding to both gram-positive and negative
bacteria and the bacteria induced TNF-𝛼secretion from
human monocytes []. TWEAK has been shown to be
bound and internalized by CD, indicating CD as a
possible regulator of this cytokine []—in addition to the
regulation of TWEAK by sCD as mentioned earlier. A
high sCD and low TWEAK concentration has been shown
to correlate with intima-media thickness, cardiovascular
mortality in peripheral arterial disease, and a type  diabetes
diagnosis [,,]. Finally, porcine CD has been
implicated in the entry mechanisms of African swine fever
virus (ASFV) and the porcine reproductive and respiratory
syndrome virus (PRRSV) infecting myeloid cells [,].
6. Physiological Back-Up Systems
for the Heme-Protective Function of
the Hp-Hb Pathway
Excessive hemolysis as seen during malaria, sickle cell ane-
mia, autoimmune hemolysis, and many other conditions with
pathological intravascular hemolysis may lead to depletion of
Hp in plasma []. In such cases, Hb accumulates in plasma
with toxic consequences. Hb may then be taken up directly
by CD by a yet unknown pathway, be ltered in the kidney
or be degraded in plasma. e absence of Hp binding to Hb
leads to release of heme that then binds to heme-binding
proteins such as albumin, 𝛼-microglobin, and hemopexin.
Hemopexin binds heme with the highest anity leading to
uptake via LDL-receptor related protein  (LRP) []/CD
(Figure ), an abundant receptor in macrophages, hepatocytes
and other cells []. Studies of hemopexin-decient mice
with and without a Hp gene knockout background have
evidenced that hemopexin constitutes a backup system for
the heme-protective role of Hp [].
7. HO-1
Hb internalized through interaction with CD is trans-
ferred to early endosomes and subsequently degraded to
heme, bioactive peptides, and amino acids [,]. HO
is responsible for the further enzymatic heme catabolism
resulting in the degradation products carbonmonoxide (CO),
ferrous iron (Fe2+) and biliverdin. Biliverdin is reduced to
bilirubin by the biliverdin reductase (Figure ).
ree isoforms of HO diering in tissue distribution,
regulation and proposed function have been identied.
HO- ( kDa) is expressed in many cell types includ-
ing macrophages. It is highly inducible in response to a
wide range of factors []. HO- ( kDa) is constitutively
expressed with the highest expression in testis and brain
[]. Finally, an HO- isoform was identied in rats but later
studies suggest it may be a pseudogene with no apparent
function [,].
HO- is a monomeric enzyme anchored to the outer
membrane of a microsomal membrane by a hydrophobic C-
terminal domain []. More recently HO- has also been
identied in caveolae demonstrating direct interaction with
caveolin- []. Whether the heme oxidation takes place in
the cytosol or in endosomal vesicles is not fully outlined
[]. Proteolytic cleavage of the active site of HO- from
the membrane-anchored C-tail occurs following hypoxia or

Oxidative Medicine and Cellular Longevity 
CO
Biliverdin
Heme
Lysosome Bilirubin
CRP Fe2+
HO-1
F : e intracellular pathway for heme-degradation subse-
quent to CD and CD mediated endocytosis in macrophages.
Free heme is degraded to biliverdin, CO, and ferrous iron by the
endoplasmic reticulum enzyme HO- facing the cytosol. Electrons
are delivered by NADPH p cytochrome reductase. Biliverdin is
reduced to bilirubin by biliverdin reductase and transported to the
liver bound to albumin.
heme loading leading to translocalization of the truncated
enzyme to the nucleus. Here it promotes transcription of
antioxidative related genes including activating protein-
(AP-) []. e binding site for heme is located between
two concave 𝛼-helixes termed the proximal and distal helix,
respectively [].
e enzymatic process leading to the degradation of heme
comprises three major steps. In the rst step heme is oxidized
to hydroxyheme, and in the second step verdoheme is formed
and CO is released. e third step results in biliverdin and
Fe2+ []. e last step is rate limiting but is also the least
characterized []. During the process three molecules of
oxygen and seven electrons are consumed []. e electron
donor is NADPH cytochrome p reductase, which is
anchored alongside HO- on the endoplasmic reticulum on
the side facing the cytosol [].
e molecular mechanisms for the regulation of HO-
 have been extensively investigated and the complexities
of the pre-translational regulation are now steadily being
unraveled although hampered by major dierences between
the examined species and between cell types [,]. e
expressionofHO-isinduciblebyalonglistofendogenous
and exogenous molecules []. In the context of this review,
it should be noted that heme itself aside from functioning
as cofactor and substrate of HO- also seems as the most
potent inducer of HO- expression. Other inducers include,
but are far from being limited to, heat, ultraviolet radiation,
LPS, hydrogen peroxide, several dietary phytochemical, IL-
𝛼,TNF𝛼,andNO[–]. Interestingly, IL-, which
has a central role in the CD regulation, also stimulates
synthesis of HO- []. Some common mechanisms have
been proposed based upon shared cellular eects of some the
inducers: a transient increase in intracellular heme, increased
production of reactive oxygen species (ROS) generation, and
glutathione depletion [].
Many studies have demonstrated a role for protein phos-
phorylation dependent signaling pathways in the observed
HO- upregulation. A growing body of evidence points to
a central role for the mitogen associated protein kinases
(MAPK) family of kinases in this. MAPK proteins belong to
the serine/threonine kinase superfamily and is involved in
mediating signals for cell growth, dierentiation, and apop-
tosis and commonly activated in response to stressors [].
PI K/Akt, protein kinase A, protein kinase C, and tyrosine
kinase have also been implicated as possible mediators of
HO- induction [].
e existence of multiple HO- inducers corresponds
with the abundance of response elements and cis-acting
elements in the promoter region of Hmox- (the human HO-
 gene). e promoter region spans at least  kb from the
󸀠start of Hmox- and contains several consensus binding
motives for binding of transcription factors such as ARE
(antioxidant response element or stress-related response
element) which is found in the promoters of proteins asso-
ciated with anti-oxidative functions (also known as phase II
enzymes) [–]. Its ligands include transcriptions factors
of the basic leucine zipper-superfamily of which several have
been shown to induce HO- transcription. NF-E related
factor  (Nrf-) belongs to this family as well and a growing
body of evidence shows that it is essential for ARE-binding
and HO- induction [,].
Until recently, no direct molecular link between increased
oxidative stress and transcriptional activity was known.
e identication of the interactions between Nrf- and
Kelch-like ECH associated protein- (Keap-) has provided
such a link. Under low-stress conditions Keap- binds Nrf-
 in the cytoplasm and directs it to ubiquitin-dependent
proteasomal degradation [,]. Oxidation of specic
cysteine residues in Keap- or the phosphorylation of Nrf-
inhibits its degradation and result in nuclear translocation,
heterodimerization,and transcriptional activity of Nrf- [].
is model explains both the observed link between MAPK
activation and Nrf- transcriptional activity [,]and
the link between oxidative stress/ROS and HO- expression.
Using knock-out technology and genetic transduction a pro-
tectiveroleofHO-inavarietyofdiseasemodelsincluding
atherosclerosis, hypoxia, hyperoxia-induced lung damage,
liver failure, liver allogra, hypertension and reperfusion
injury has been shown [–].
8. The Physiological Effects of
Heme Metabolites
CO is most widely known as a toxic inhaled gas inhibiting
oxygen binding and release from Hb thus causing asphyxia-
tion. However, as mentioned earlier, it is also endogenously
produced during heme oxidation by HO-. At these relative
low levels an increasing body of evidence indicates that
CO serves several benecial physiological functions. Most
of these cellular eects are believed to be mediated by CO
binding to heme in heme-proteins []. Of notice, CO binds
andactivatessolubleguanylatecyclase(sGC)toproduce
cyclic guanosine monophosphate similarly to nitric oxide

Oxidative Medicine and Cellular Longevity
though with less ecacy []. is may mimic nitric oxide’s
well-established cytoprotective eects and this is believed
to be a major contributor to the observed cytoprotection
mediated by HO-/CO [,].
CO has also been shown to cause vasodilatation via sGC
independent activation of potassium channels in vascular
smooth muscle cells []. Furthermore, CO is believed
to modulate p MAPK in an sGC independent way
by inhibiting the expression of classical pro-inammatory
cytokines such as TNF-𝛼,IL-𝛽,andmacrophageinamma-
tory protein-𝛽while promoting the expression of the anti-
inammatory cytokine IL- []. ese anti-inammatory
eects have been demonstrated in vivo where administered
CO in nonlethal concentrations was able to reduce the
inammation induced by mechanical ventilation [,].
Cytochromecoxidase(COX)isaheme-proteininthe
inner mitochondrial membrane which transfers protons and
electrons to O2, creating water and providing energy for
transport of two protons across the membrane. CO binds
and inhibits this protein thus inhibiting O2consumption
and stimulating ROS from the accumulating electron carriers
in the mitochondria. is process is termed mitochondrial
redox signaling, and it is believed to stimulate mitochondrial
biogenesis and angiogenesis [].
e CO cleaved from hydroxyheme is primarily removed
from the body via respiration. CO diuses readily cross-
cell membranes and binds Hb with approximately -fold
higher anity than O2. Provided adequate circulation and
respiration, it is then transported to the alveoli and diuses to
the alveolar gas. Additionally, CO is slowly oxidized by COX
to CO2[]. e exhalation of CO can be used as a measure
of heme catabolism in the body [].
Iron (Fe2+)isreleasedfromhemeduringthelastenzy-
matic step of its conversion to biliverdin. A P-type ATPase
iron transporter is colocalized with HO- in the microsomal
membrane []. e importance of this iron release is
suggested because of the anemia and iron accumulation in
theliverandkidneyinHO-knock-outmiceandintherst
reported case of human HO- deciency [,,]. Most
likely, the iron released from heme enters a labile pool of
intracellular iron, available for cellular processes involving
iron or cellular export via the hepcidin-regulated ferroportin
protein in the membrane.
Intracellular iron is oxidized and bound to the ubiquitous
apoprotein ferritin. An increase in the intracellular iron
deposit aects the posttranscriptional expression of several
proteins by interaction with iron regulatory proteins and
mRNA iron response elements []. Via this mechanism
ferritin is upregulated by increased HO- activity [].
Ferritin has been shown to have antiapoptotic eects and
provide cytoprotection against oxidative damage [,].
Iron is exported from the cell by ferroportin and transported
bound to the plasma protein transferrin. Transferrin-bound
iron is taken up by cells expressing the transferrin receptor
and recycled.
e main product of heme degradation, biliverdin, is a
greenish water-soluble pigment. It is reduced to bilirubin by
biliverdin reductase. Bilirubin is a hydrophobic, yellowish
T : Major reportedc ytoprotective andantiinammatory eects
of the Hp-CD-HO- pathway.
Intravascular HpHb complex formation:
Protects against oxidative “hot spot” in Hb
Protects against heme release from Hb
Facilitates CD-mediated clearance
Prevents renal ltration of Hb and uptake in proximal tubules
Prevents NO scavenging
Cellular response on CD-mediated Hb endocytosis:
Cellular dierentiation
HO- upregulation
Nrf- activation
IL- synthesis
Other eects of heme metabolites generated by HO- activity:
Antagonism of proinammatory cytokines
ROS scavenging
Angiogenesis
Inhibition of platelet aggregation
Vaso d i l a t a t i o n
pigmentandistransportedintheplasmaboundtoalbumin.
Intheliver,bilirubinisconjugatedandexcretedinthebile.
For decades bilirubin has been considered a toxic byprod-
uct of heme degradation. Recent studies have, however,
also demonstrated potential benecial functions of bilirubin
and biliverdin in the circulation and extravascular tissues
[]. Epidemiological studies have revealed that moderately
increased plasma levels of bilirubin decrease the risk of
developing cardiovascular diseases []. In vitro studies
have demonstrated bilirubin and biliverdin as functional
antioxidants []. Biliverdin reductase has also been shown
to be at least partly responsible for HO--mediated anti-
oxidative protection []. Additionally, biliverdin reduc-
tase has been shown to promote an anti-inammatory
response in macrophages through transcriptional regulation
[].
9. Perspectives
e Hp-CD-HO- pathway for degradation of
hemoglobin is as an important and apparently to some extent
a coordinately regulated pathway that by direct hemoglobin
binding and subsequent clearance from plasma prevents
toxic and proinammatory eects of heme and hemoglobin.
In addition, the proteins in the pathway and the metabolic
heme products reinforce an anti-inammatory response.
Table  summarizes major anti-inammatory eects of this
pathway. Future studies of various inammatory conditions
in vitro and in vivo models should further delineate the
molecular mechanism and elucidate if the proteins of the
pathway have anti-inammatory eects independent of
heme.Finally,thispathwayseemsasapotentialtargetfor
stimulation of the inammatory response by small molecule
drugs.

Oxidative Medicine and Cellular Longevity 
Acknowledgment
is study was supported by ERC to the project TROJA.
References
[] D. Bratosin, J. Mazurier, J. P. Tissier et al., “Cellular and
molecular mechanisms of senescent erythrocyte phagocytosis
by macrophages. A review,” Biochimie,vol.,no.,pp.–
, .
[]M.Straat,R.vanBruggen,D.deKorte,andN.P.Juer-
mans, “Red blood cell clearance in inammation,” Trans f usio n
Medicine and Hemotherapy,vol.,no.,pp.–,.
[] R. P. Rother, L. Bell, P. Hillmen, and M. T. Gladwin, “e clinical
sequelae of intravascular hemolysis and extracellular plasma
hemoglobin: a novel mechanism of human disease,” Journal of
the American Medical Association,vol.,no.,pp.–
, .
[] J. Aronowski and X. Zhao, “Molecular pathophysiology of
cerebral hemorrhage: secondary brain injury,” Stroke,vol.,
no. , pp. –, .
[] A. P. Levy, R. Asleh, S. Blum et al., “Haptoglobin: basic and
clinical aspects,” Antioxidants and Redox Signaling,vol.,no.
, pp. –, .
[] K. eilgaard-M¨
onch, L. C. Jacobsen, M. J. Nielsen et al.,
“Haptoglobin is synthesized during granulocyte dierentiation,
stored in specic granules, and released by neutrophils in
response to activation,” Blood,vol.,no.,pp.–,.
[]C.B.Andersen,M.Torvund-Jensen,M.J.Nielsenetal.,
“Structure of the haptoglobin-haemoglobin complex,” Nature,
vol. , no. , pp. –, .
[] K. B. Wicher and E. Fries, “Evolutionary aspects of hemoglobin
scavengers,” Antioxidants and Redox Signaling,vol.,no.,pp.
–, .
[]M.J.NielsenandS.K.Moestrup,“Receptortargetingof
hemoglobin mediated by the haptoglobins: roles beyond heme
scavenging,” Blood,vol.,no.,pp.–,.
[] J. G. Bode, U. Albrecht, D. Haussinger, P. C. Heinrich, and F.
Schaper, “Hepatic acute phase proteins—regulation by IL- and
IL--type cytokines involving STAT and its crosstalk with NF-
kappaB-dependent signaling,” European Journal of Cell Biology,
vol. , no. -, pp. –, .
[] A. I. Alayash, C. B. Andersen, S. K. Moestrup, and L. B¨
ulow,
“Haptoglobin: the hemoglobin detoxier in plasma,” Trend s in
Biotechnology,vol.,no.,pp.–,.
[] V.P.D.A.Wobeto,T.R.Zaccariotto,andM.D.F.Sonati,“Poly-
morphism of human haptoglobin and its clinical importance,”
Genetics and Molecular Biology,vol.,no.,pp.–,.
[] A. Etzerodt and S. K. Moestrup, “CD and inammation:
biological, diagnostic and therapeutic aspects,” Antioxid Redox
Signal,vol.,no.,pp.–,.
[]S.Banerjee,Y.Jia,C.J.P.Siburtetal.,“Haptoglobinalters
oxygenation and oxidation of hemoglobin and decreases prop-
agation of peroxide-induced oxidative reactions,” Free Radical
Biology and Medicine,vol.,no.,pp.–,.
[] P.W.Buehler,B.Abraham,F.Vallelianetal.,“Haptoglobinpre-
serves the CD hemoglobin scavenger pathway by shielding
hemoglobin from peroxidative modication,” Blood,vol.,no.
, pp. –, .
[] E. Tolosano, S. Fagoonee, N. Morello, F. Vinchi, and V. Fiorito,
“Heme scavenging and the other facets of hemopexin,” Antiox-
idants and Redox Signaling,vol.,no.,pp.–,.
[] I. Azarov, X. He, A. Jeers et al., “Rate of nitric oxide scavenging
by hemoglobin bound to haptoglobin,” Nitric Oxide,vol.,no.
, pp. –, .
[] M. Kristiansen, J. H. Graversen, C. Jacobsen et al., “Identica-
tion of the haemoglobin scavenger receptor,” Nature,vol.,
no.,pp.–,.
[] M. J. Nielsen, S. V. Petersen, C. Jacobsen et al., “A unique
loop extension in the serine protease domain of haptoglobin is
essential for CD recognition of the haptoglobin-hemoglobin
complex,” JournalofBiologicalChemistry,vol.,no.,pp.
–, .
[] M. J. Nielsen, C. B. F. Andersen, and S. K. Moestrup, “CD
binding to haptoglobin-hemoglobin complexes involves a dual-
point electrostatic receptor-ligand pairing,” Journal of Biological
Chemistry,.
[] A. P. Levy, I. Hochberg, K. Jablonski et al., “Haptoglobin
phenotype is an independent risk factor for cardiovascular
disease in individuals with diabetes: the strong heart study,”
JournaloftheAmericanCollegeofCardiology,vol.,no.,pp.
–, .
[] A. P. Lev y, S. Blum, M. Vardi et al., “Vitamin e reduces
cardiovascular disease in individuals with diabetes mellitus and
the haptoglobin - genotype,” Pharmacogenomics,vol.,no.,
pp. –, .
[] A. A. Elagib, A. O. Kider, B. ˚
Akerstr¨
om, and M. I. Elbashir,
“Association of the haptoglobin phenotype (-) with falc iparum
malaria in Sudan,” Transactions of the Royal Society of Tropical
Medicine and Hygiene, vol. , no. , pp. –, .
[] I.K.Quaye,F.A.Ekuban,B.Q.Gokaetal.,“Haptoglobin-is
associated with susceptibility to severe Plasmodium falciparum
malaria,” Transactions of the Royal Society of Tropical Medicine
and Hygiene,vol.,no.,pp.–,.
[] A. J. Cunnington, M. Njie, S. Correa, E. N. Takem, E. M.
Riley,andM.Walther,“Prolongedneutrophildysfunctionaer
Plasmodium falciparum malaria is related to hemolysis and
heme oxygenase- induction,” Journal of Immunology,vol.,
no. , pp. –, .
[] V.R.Mendonca,N.F.Luz,N.J.G.Santosetal.,“Association
between the haptoglobin and heme oxygenase  genetic proles
and soluble CD in susceptibility to and severity of human
malaria,” Infection and Immunity,vol.,no.,pp.–,
.
[] I. Quaye, “Haptoglobin genotypes are not associated with
resistance to severe malaria in e Gambia,” Transactions of the
Royal Society of Tropical Medicine and Hygiene,vol.,no.,p.
, .
[] B.Beiguelman,F.P.Alves,M.M.Mouraetal.,“eassociation
of genetic markers and malaria infection in the Brazilian
Western Amazonian region,” Memorias do Instituto Oswaldo
Cruz, vol. , no. , pp. –, .
[] M.Freeman,J.Ashkenas,D.J.G.Reesetal.,“Anancient,highly
conserved family of cysteine-rich protein domains revealed
by cloning type I and type II murine macrophage scavenger
receptors,” Proceedings of the National Academy of Sciences of
the United States of America,vol.,no.,pp.–,.
[] B.Rodamilans,I.G.Mu
˜
noz, E. Bragado-Nilsson et al., “Crystal
structure of the third extracellular domain of CD reveals the
fold of a group B scavenger cysteine-rich receptor domain,”
Journal of Biological Chemistry,vol.,no.,pp.–,
.
[] E. Hohenester, T. Sasaki, and R. Timpl, “Crystal structure
of a scavenger receptor cysteine-rich domain sheds light on

Oxidative Medicine and Cellular Longevity
an ancient superfamily,” Nature Structural Biology,vol.,no.,
pp. –, .
[] J. R. Somoza, J. D. Ho, C. Luong et al., “e structure of the
extracellular region of human hepsin reveals a serine protease
domain and a novel scavenger receptor cysteine-rich (SRCR)
domain,” Structure, vol. , no. , pp. –, .
[] V. G. Mart´
ınez,S.K.Moestrup,U.Holmskov,J.Mollenhauer,
and F. Lozano, “e conserved scavenger receptor cysteine-
rich super family in therapy and diagnosis,” Pharmacological
Reviews,vol.,no.,pp.–,.
[] H. Van Gorp, P. L. Delputte, and H. J. Nauwynck, “Scavenger
receptor CD, a Jack-of-all-trades and potential target forcell-
directed therapy,” Molecular Immunology,vol.,no.-,pp.
–, .
[] M.Ritter,C.Buechler,T.Langmann,E.Orso,J.Klucken,andG.
Schmitz, “e scavenger receptor CD: regulation, promoter
structure and genomic organization,” Pathobiology,vol.,no.
-, pp. –, .
[] M. Ritter, C. Buechler, M. Kapinsky, and G. Schmitz, “Interac-
tion of CD with the regulatory subunit of casein kinase II
(CKII) and dependence of CD signaling on CKII and protein
kinase C,” European Journal of Immunology,vol.,no.,pp.
–, .
[] M.J.Nielsen,M.Madsen,H.J.Møller,andS.K.Moestrup,“e
macrophage scavenger receptor CD: endocytic properties of
cytoplasmic tail variants,” Journal of Leukocyte Biology,vol.,
no. , pp. –, .
[] M. B. Maniecki, H. J. Møller, S. K. Moestrup, and B. K. Møller,
“CD positive subsets of blood dendritic cells: the scavenging
macrophage receptors CD and CD are coexpressed on
human dendritic cells and monocytes,” Immunobiology, vol. ,
no. -, pp. –, .
[] V. Kodelja and S. Goerdt, “Dissection of macrophage dier-
entiation pathways in cutaneous macrophage disorders and in
vitro,” Experimental Dermatology,vol.,no.,pp.–,
.
[] G. Zwadlo-Klarwasser, S. Bent, H. D. Haubeck, C. Sorg, and
W. Schmutzler, “Glucocorticoid-induced appearance of the
macrophage subtype RM / in peripheral blood of man,”
International Archives of Allergy and Applied Immunology,vol.
,no.,pp.–,.
[] D. Kamimura, K. Ishihara, and T. Hirano, “IL- signal transduc-
tion and its physiological roles: the signal orchestration model,”
Reviews of Physiology, Biochemistry and Pharmacology,vol.,
pp. –, .
[] S. Gordon and F. O. Martinez, “Alternative activation of
macrophages: mechanism and functions,” Immunity,vol.,no.
, pp. –, .
[] G. Zwadlo, R. Voegeli, K. Schulze Ostho, and C. Sorg, “A mon-
oclonal antibody to a novel dierentiation antigen on human
macrophages associated with the down-regulatory phase of the
inammatory process,” Experimental Cell Biology,vol.,no.,
pp.–,.
[] A. V. Finn, M. Nakano, R. Polavarapu et al., “Hemoglobin
directs macrophage dierentiation and prevents foam cell
formationinhumanatheroscleroticplaques,”Journal of the
American College of Cardiology,vol.,no.,pp.–,.
[] J. J. Boyle, “Heme and haemoglobin direct macrophage Mhem
phenotype and counter foam cell formation in areas of
intraplaque haemorrhage,” Current Opinion in Lipidology,vol.
,no.,pp.–,.
[] H. J. Moller, “Soluble CD,” Scandinavian Journal of Clinical
& Laboratory Investigation,vol.,no.,pp.–,.
[] A. Etzerodt, M. B. Maniecki, K. Møller, H. J. Møller, and S.
K.Moestrup,“Tumornecrosisfactor𝛼-converting enzyme
(TACE/ADAM) mediates ectodomain shedding of the scav-
enger receptor CD,” Journal of Leukocyte Biology, vol. , no.
, pp. –, .
[] J. Kneidl, B. L¨
oer,M.C.Eratetal.,“SolubleCDpromotes
recognition, phagocytosis and killing of Staphylococcus aureus
via binding of specic bronectin peptides,” Cellular Microbiol-
ogy, vol. , no. , pp. –, .
[] D. Baeten, H. J. Møller, J. Delanghe, E. M. Veys, S. K. Moestrup,
and F. De Keyser, “Association of CD+ macrophages and
local production of soluble CD with decreased lympho-
cyte activation in spondylarthropathy synovitis,” Arthritis and
Rheumatism,vol.,no.,pp.–,.
[] J. A. Moreno, B. Mu˜
noz-Garc´
ıa,J.L.Mart
´
ın-Ventura et al.,
“e CD-expressing macrophages recognize and inter-
nalize TWEAK. Potential consequences in atherosclerosis,”
Atherosclerosis,vol.,no.,pp.–,.
[] M. Madsen, H. J. Møller, M. J. Nielsen et al., “Molecular char-
acterization of the haptoglobin-hemoglobin receptor CD:
ligand binding properties of the scavenger receptor cysteine-
rich domain region,” JournalofBiologicalChemistry,vol.,
no. , pp. –, .
[] C. A. Schaer, G. Schoedon, A. Imhof, M. O. Kurrer, and
D. J. Schaer, “Constitutive endocytosis of CD mediates
hemoglobin-heme uptake and determines the noninamma-
tory and protective transcriptional response of macrophages to
hemoglobin,” Circulation Research,vol.,no.,pp.–,
.
[] D. J. Schaer, C. A. Schaer, P. W. Buehler et al., “CD is
the macrophage scavenger receptor for native and chemically
modied hemoglobins in the absence of haptoglobin,” Blood,
vol. , no. , pp. –, .
[] A. Etzerodt, M. Kjolby, M. J. Nielsen, M. Maniecki, P. Svendsen,
and S. K. Moestrup, “Plasma clearance of hemoglobin and hap-
toglobin in mice and eect of CD gene targeting disruption,”
Antioxidants & Redox Signaling,vol.,no.,pp.–,
.
[] P. Philippidis, J. C. Mason, B. J. Evans et al., “Hemoglobin
scavenger receptor CD mediates interleukin- release
and heme oxygenase- synthesis: antiinammatory monocyte-
macrophage responses in vitro, in resolving skin blisters in
vivo, and aer cardiopulmonar y bypass surgery,” Circulation
Research, vol. , no. , pp. –, .
[] B. O. Fabriek, M. M. J. Poliet, R. P. M. Vloet et al., “e
macrophage CD surface glycoprotein is an erythroblast
adhesion receptor,” Blood,vol.,no.,pp.–,.
[] B. O. Fabriek, R. V. Bruggen, D. M. Deng et al., “e macrophage
scavenger receptor CD functions as an innate immune
sensor for bacteria,” Blood,vol.,no.,pp.–,.
[] L. C. Bover, M. Card´
o-Vila, A. Kuniyasu et al., “A previously
unrecognized protein-protein interaction between TWEAK
and CD: potential biological implications,” Journal of
Immunology,vol.,no.,pp.–,.
[] G.Llaurado,J.M.Gonz
´
alez-Clemente, E. Maym´
o-Masip, D.
Sub´
ıas, J. Vendrell, and M. R. Chac ´
on, “Serum levels of TWEAK
and scavenger receptor CD in type  diabetes mellitus:
relationship with cardiovascular risk factors. a case-control
study,” PLoS ONE,vol.,no.,ArticleIDe,.

Oxidative Medicine and Cellular Longevity 
[] G. Urbonaviciene, J. L. Martin-Ventura, J. S. Lindholt et al.,
“Impact of soluble TWEAK and CD/TWEAK ratio on
long-termcardiovascularmortalityinpatientswithperipheral
arterial disease,” Atherosclerosis,vol.,no.,pp.–,
.
[] C. S´
anchez-Torres, P. G´
omez-Puertas, M. G´
omez-Del-Moral et
al., “Expression of porcine CD on monocytes/macrophages
correlates with permissiveness to African swine fever infection,”
Archives of Virology,vol.,no.,pp.–,.
[] S. K. W. Welch and J. G. Calvert, “A brief review of CD and
its role in PRRSV infection,” Virus R es earch,vol.,no.-,pp.
–, .
[] J. D. Belcher, J. D. Beckman, G. Balla, J. Balla, and G. Vercellotti,
“Heme degradation and vascular injury,” Antioxidants and
Redox Signaling,vol.,no.,pp.–,.
[] V. Hvidberg, M. B. Maniecki, C. Jacobsen, P. Højrup, H. J.
Møller, and S. K. Moestrup, “Identication of the receptor
scavenging hemopexin-heme complexes,” Blood,vol.,no.,
pp. –, .
[] S. K. Moestrup, J. Gliemann, and G. Pallesen, “Distribu-
tion of the 𝛼-macroglobulin receptor/low density lipoprotein
receptor-related protein in human tissues,” Cell and Tissue
Research,vol.,no.,pp.–,.
[] E. Tolosano, S. Fagoonee, E. Hirsch et al., “Enhanced
splenomegaly and severe liver inammation in
haptoglobin/hemopexin double-null mice aer acute
hemolysis,” Blood,vol.,no.,pp.–,.
[]I.Fruitier,I.Garreau,A.Lacroix,A.Cupo,andJ.M.Piot,
“Proteolytic degradation of hemoglobin by endogenous lysoso-
mal proteases gives rise to bioactive peptides: hemorphins,” e
FEBS Letters,vol.,no.,pp.–,.
[] C. Y. Wang and L. Y. Chau, “Heme oxygenase- in cardiovascu-
lar diseases: molecular mechanisms and clinical perspectives,”
Chang Gung Medical Journal,vol.,no.,pp.–,.
[] M.L.Wu,Y.C.Ho,C.Y.Lin,andS.F.Yet,“Hemeoxygenase-
in inammation and cardiovascular disease,” American Journal
of Cardiovascular Disease,vol.,no.,pp.–,.
[] S. Hayashi, Y. Omata, H. Sakamoto et al., “Characterization of
rat heme oxygenase- gene. Implication of processed pseudo-
genes derived from heme oxygenase- gene,” Gene,vol.,no.
, pp. –, .
[] W. K. Mccoubrey, T. J. Huang, and M. D. Maines, “Isolation
and characterization of a cDNA from the rat brain that encodes
hemoprotein heme oxygenase-,” European Journal of Biochem-
istry,vol.,no.,pp.–,.
[] T. Yoshida and M. Sato, “Posttranslational and direct integration
of heme oxygenase into microsomes,” Biochemical and Biophys-
ical Research Communications,vol.,no.,pp.–,
.
[] N. H. Jung, H. P. Kim, B. R. Kim et al., “Evidence for
heme oxygenase- association with caveolin- and - in mouse
mesangial cells,” IUBMB Life,vol.,no.,pp.–,.
[] C. Delaby, C. Rondeau, C. Pouzet et al., “Subcellular localization
of iron and heme metabolism related proteins at early stages of
erythrophagocytosis,” PLoS ONE,vol.,no.,ArticleIDe,
.
[]Q.Lin,S.Weis,G.Yangetal.,“Hemeoxygenase-protein
localizes to the nucleus and activates transcription factors
important in oxidative stress,” JournalofBiologicalChemistry,
vol. , no. , pp. –, .
[] M.Unno,T.Matsui,andM.Ikeda-Saito,“Structureandcatalytic
mechanism of heme oxygenase,” Natural Product Reports,vol.
,no.,pp.–,.
[] M. Unno, T. Matsui, and M. Ikeda-Saito, “Crystallographic
studies of heme oxygenase complexed with an unstable reaction
intermediate, verdoheme,” Journal of Inorganic Biochemistry,
vol. , pp. –, .
[] T. Matsui, A. Nakajima, H. Fujii et al., “O2-andH
2O2-
dependent verdoheme degradation by heme oxygenase: reac-
tion mechanisms and potential physiological roles of the dual
pathway degradation,” JournalofBiologicalChemistry,vol.,
no.,pp.–,.
[] M. Linnenbaum, M. Busker, J. R. Kraehling, and S. Behrends,
“Heme oxygenase isoforms dier in their subcellular tracking
during hypoxia and are dierentially modulated by cytochrome
P reductase,” PLoS ONE,vol.,no.,ArticleIDe,.
[] S. W. Ryter, J. Alam, and A. M. K. Choi, “Heme oxygenase-
/carbon monoxide: from basic science to therapeutic applica-
tions,” Physiological Reviews,vol.,no.,pp.–,.
[] C. L. Hartseld, J. Alam, and A. M. K. Choi, “Dierential
signaling pathways of HO- gene expression in pulmonary and
systemic vascular cells,” American Journal of Physiolog y,vol.,
no. , pp. L–L, .
[] J. Alam, S. Shibahara, and A. Smith, “Transcriptional activation
of the heme oxygenase gene by heme and cadmium in mouse
hepatoma cells,” Journal of Biological Chemistry,vol.,no.,
pp. –, .
[] L. A. Applegate, A. Noel, G. Vile, E. Frenk, and R. M.
Tyrrell, “Two genes contribute to dierent extents to the
heme oxygenase enzyme activity measured in cultured human
skin broblasts and keratinocytes: implications for protection
against oxidant stress,” Photochemistr y and Photobiology,vol.,
no.,pp.–,.
[] M. Rizzardini, M. Carelli, M. R. Cabello Porras, and L. Cantoni,
“Mechanisms of endotoxin-induced haem oxygenase mRNA
accumulation in mouse liver: synergism by glutathione deple-
tion and protection by N-acetylcysteine,” Biochemical Journal,
vol. , no. , pp. –, .
[] K. M. Stuhlmeier, “Activation and regulation of Hsp and
Hsp,” European Journal of Biochemistry,vol.,no.,pp.
–, .
[] K. S. Callahan, “Eect of tumor necrosis factor-a and
interleukin-la on heme oxygenase- expression in human
endothelial cells,” American Journal of Physiology,vol.,no.
, part , pp. H–H, .
[] R. Foresti, J. E. Clark, C. J. Green, and R. Motterlini, “iol com-
pounds interact with nitric oxide in regulating heme oxygenase-
 induction in endothelial cells: involvement of superoxide and
peroxynitrite anions,” Journal of Biological Chemistr y,vol.,
no. , pp. –, .
[] Y.J.Surh,J.K.Kundu,andH.K.Na,“Nrfasamasterredox
switch in turning on the cellular signaling involved in the
induction of cytoprotective genes by some chemopreventive
phytochemicals,” Planta Medica,vol.,no.,pp.–,
.
[] T. S. Lee and L. Y. Chau, “Heme oxygenase- mediates the anti-
inammatory eect of interleukin- in mice,” Nature Medicine,
vol. , no. , pp. –, .
[] J. M. Kyriakis and J. Avruch, “Mammalian MAPK signal
transduction pathways activated by stress and inammation: a
-year update,” Physiological Reviews,vol.,no.,pp.–
, .

 Oxidative Medicine and Cellular Longevity
[] N. Hill-Kapturczak, C. Voakes, J. Garcia, G. Visner, H. S. Nick,
and A. Agarwal, “A cis-acting region regulates oxidized lipid-
mediated induction of the human heme oxygenase- gene in
endothelial cells,” Arteriosclerosis, rombosis, and Vascular
Biology,vol.,no.,pp.–,.
[] E. M. Sikorski, T. Hock, N. Hill-Kapturczak, and A. Agarwal,
“e story so far: molecular regulation of the heme oxygenase-
 gene in renal injury,” American Journal of Physiology,vol.,
no. , pp. F–F, .
[] A. M. K. Choi and J. Alam, “Heme oxygenase-: function,
regulation, and implication of a novel stress-inducibleprotein in
oxidant-induced lung injury,” American Journal of Respirator y
Cell and Molecular Biology,vol.,no.,pp.–,.
[] S. J. Chapple, R. C. Siow, and G. E. Mann, “Crosstalk between
Nrf and the proteasome: therapeutic potential of Nrf inducers
in vascular disease and aging,” International Journal of Biochem-
istry & Cell Biology,vol.,no.,pp.–,.
[] K. Itoh, J. Mimura, and M. Yamamoto, “Discovery of the nega-
tive regulator of Nrf, keap: a historical overview,” Antioxidants
and Redox Signaling, vol. , no. , pp. –, .
[  ] R . Yu , C . Ch en , Y. Y. M o e t al . , “Act i v at i on o f m i to g e n- ac t iv at e d
protein kinase pathways induces antioxidant response element-
mediated gene expression via a Nrf-dependent mechanism,”
JournalofBiologicalChemistry,vol.,no.,pp.–,
.
[]J.Alam,C.Wicks,D.Stewartetal.,“Mechanismofheme
oxygenase- gene activation by cadmium in MCF- mammary
epithelial cells: role of p kinase and Nrf transcription factor,”
JournalofBiologicalChemistry,vol.,no.,pp.–
, .
[] S. F. Yet, M. A. Perrella, M. D. Layne et al., “Hypoxia induces
severe right ventricular dilatation and infarction in heine
oxygenase- null mice,” Journal of Clinical Investigation,vol.,
no. , pp. R–R, .
[] S. F. Yet, M. D. Layne, X. Liu et al., “Absence of heme oxygenase-
 exacerbates atherosclerotic lesion formation and vascular
remodeling,” e FASEB Journal,vol.,no.,pp.–,
.
[] K. Ishikawa, D. Sugawara, X. P. Wang et al., “Heme oxygenase-
 inhibits atherosclerotic lesion formation in LDL-receptor
knockout mice,” Circulation Research,vol.,no.,pp.–,
.
[]X.Liu,J.Wei,D.H.Peng,M.D.Layne,andS.F.
Yet, “Absence of heme oxygenase- exacerbates myocardial
ischemia/reperfusion injury in diabetic mice,” Diabetes,vol.,
no. , pp. –, .
[] B. S. Zuckerbraun, T. R. Billiar, S. L. Otterbein et al., “Carbon
monoxide protects against liver failure through nitric oxide-
induced heme oxygenase ,” JournalofExperimentalMedicine,
vol. , no. , pp. –, .
[]B.Ke,J.W.Kupiec-Weglinski,R.Buelowetal.,“Hemeoxy-
genasegenetransferpreventsCD/Fasligand-mediated
apoptosis and improves liver allogra survival via carbon
monoxide signaling pathway,” Human Gene erapy,vol.,no.
, pp. –, .
[] L. E. Otterbein, J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and
A. M. K. Choi, “Exogenous administration of heme oxygenase-
by gene transfer provides protection against hyperoxia-induced
lung injury,” Journal of Clinical Investigation,vol.,no.,pp.
–, .
[]R.A.Johnson,F.J.Teran,W.Durante,K.J.Peyton,andF.
K. Johnson, “Enhanced heme oxygenase-mediated coronary
vasodilation in Dahl salt-sensitive hypertension,” American
Journal of Hypertension,vol.,no.,pp.–,.
[] F. Gullotta, A. di Masi, M. Coletta, and P. Ascenzi, “CO
metabolism, sensing, and signaling,” Biofactors,vol.,no.,
pp. –, .
[] S. W. Ryter and L. E. Otterbein, “Carbon monoxide in biology
and medicine,” BioEssays, vol. , no. , pp. –, .
[] H.O.Pae,Y.Son,N.H.Kim,H.J.Jeong,K.C.Chang,and
H. T. Chung, “Role of heme oxygenase in preserving vascular
bioactive NO,” Nitric Oxide,vol.,no.,pp.–,.
[] S.E.Williams,S.P.Brazier,N.Babanetal.,“Astructuralmotifin
the C-terminal tail of slo confers carbon monoxide sensitivity
to human BKCa channels,” Pugers Archiv European Journal of
Physiology,vol.,no.,pp.–,.
[] L. E. Otterbein, F. H. Bach, J. Alam et al., “Carbon monoxide
has anti-inammatory eects involving the mitogen-activated
protein kinase pathway,” Nature Medicine,vol.,no.,pp.–
, .
[] A. Hoetzel, T. Dolinay, S. Vallbracht et al., “Carbon monoxide
protects against ventilator-induced lung injury via PPAR-𝛾and
inhibition of Egr-,” American Journal of Respirator y and Critical
Care Medicine,vol.,no.,pp.–,.
[] T. Dolinay, W. Wu, N. Kaminski et al., “Mitogen-activated
protein kinases regulate susceptibility to ventilator-induced
lung injury,” PLoS ONE,vol.,no.,ArticleIDe,.
[] R. Gajd´
ocsy and I. Horv´
ath, “Exhaled carbon monoxide in
airway diseases: from research ndings to clinical relevance,”
Journal of Breath Research,vol.,no.,ArticleID,.
[] D. E. Baranano, H. Wolosker, B. I. Bae, R. K. Barrow, S. H.
Snyder,andC.D.Ferris,“AmammalianironATPaseinducedby
iron,” Journal of Biological Chemistry,vol.,no.,pp.–
, .
[] A. Yachie, Y. Niida, T. Wada et al., “Oxidative stress causes
enhanced endothelial cell injury in human heme oxygenase-
deciency,” Journal of Clinical Investigation,vol.,no.,pp.
–, .
[] K. D. Poss and S. Tonegawa, “Heme oxygenase  is required
for mammalian iron reutilization,” Proceedings of the National
Academy of Sciences of the United States of America,vol.,no.
, pp. –, .
[] R. S. Eisenstein and H. N. Munro, “Translational regulation of
ferritin synthesis by iron,” Enzyme,vol.,no.–,pp.–,
.
[]G.F.VileandR.M.Tyrrell,“Oxidativestressresultingfrom
ultraviolet A irradiation of human skin broblasts leads to
a heme oxygenase-dependent increase in ferritin,” Journal of
Biological Chemistry,vol.,no.,pp.–,.
[] P. O. Berberat, M. Katori, E. Kaczmarek et al., “Heavy chain
ferritin acts as an antiapoptotic gene that protects livers from
ischemia reperfusion injury,” e FASEB Journal,vol.,no.,
pp.–,.
[] G. F. Vile, S. Basu-Modak, C. Waltner, and R. M. Tyrrell, “Heme
oxygenase  mediates an adaptive response to oxidative stress in
humanskinbroblasts,”Proceedings of the National Academy of
Sciences of the United States of America,vol.,no.,pp.–
, .
[] J. Kapitulnik and M. D. Maines, “e role of bile pigments in
health and disease: eects on cell signaling, cytotoxicity, and
cytoprotection,” Frontiers in Pharmacology,vol.,p.,.
[] L. Vitek, “e role of bilirubin in diabetes, metabolic syndrome,
and cardiovascular diseases,” Frontiers in Pharmacology,vol.,
p. , .

Oxidative Medicine and Cellular Longevity 
[] R. Stocker, “Antioxidant activities of bile pigments,” Antioxi-
dants and Redox Signaling,vol.,no.,pp.–,.
[] T. Jansen and A. Daiber, “Direct antioxidant properties of
bilirubin and biliverdin. Is there a role for biliverdin reductase?”
Frontiers in Pharmacology,vol.,p.,.
[] B. Wegiel and L. E. Otterbein, “Go green: the anti-inammatory
eects of biliverdin reduct ase,” Frontiers in Pharmacology,vol.,
p. , .