Oxidases and Reactive Oxygen
Species During Hematopoiesis:
A Focus on Megakaryocytes
ALEXIA ELIADES,1SHINOBU MATSUURA,2,3AND KATYA RAVID1,2,3,4*
1Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts
2Department of Medicine, Boston University School of Medicine, Boston, Massachusetts
3Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts
4Evans Center for Interdisciplinary Biomedical Research, Boston University School of Medicine, Boston, Massachusetts
Reactive oxygen species (ROS), generated as a result of various reactions, control an array of cellular processes. The role of ROS during
megakaryocyte(MK) development hasbeenasubject ofinterest andresearch. The bonemarrow niche is asiteofMK differentiation and
maturation. In this environment, a gradient of oxygen tension, from normoxia to hypoxia results in different levels of ROS, impacting
cellular physiology. This article provides an overview of major sources of ROS, their implication in different signaling pathways, and their
including myelofibrosis, is also described.
J. Cell. Physiol. 227: 3355–3362, 2012. ? 2012 Wiley Periodicals, Inc.
Reactive oxygen species (ROS) may be beneficial for the
organism, as in the classic example of ROS produced by
nicotinamide adenine dinucleotide phosphate (NADPH) for
defense against pathogens. They may also arise as harmful
by-products of other oxidative reactions. ROS levels influence
a number of basic physiological processes, ranging from cell
differentiation and proliferation to cell death. The underlying
There has been increasing interest in the role of ROS and
oxygen stress in the regulation of hematopoiesis. A large body
of studies has shown that control of intracellular levels of ROS
is essential for maintenance of quiescence and self-renewal
potential of hematopoietic stem cells (HSCs). The bone
marrow (BM) niche seems to be an essential component of the
regulation of ROS in HSCs. Like HSCs, megakaryocytes (MKs)
differentiate and mature within the BM niche. These cells
undergo a unique cell cycle termed endomitosis, during which
the DNA content of the cell replicates without corresponding
cell divisions, before platelets are released into the circulation.
The present article seeks to provide an overview of the recent
literature concerning the effects of ROS on different stages of
MK development (Fig. 1). Although not a focus of this review,
important aspects of HSC biology with parallels in the MK
lineage will be noted for reference and analysis. More detailed
information on the role of ROS in HSCs and other lineages is
Sardina et al., 2011; Suda et al., 2011).
ROS and oxidases
reactions (donation/gaining of an electron). ROS can be
generated as a result of those reactions. ROS are free radicals
(highly reactive molecules with unpaired electrons in their
highest atomic orbital) containing partially reduced forms of
molecular oxygen. Examples of ROS include highly reactive
oxygen radicals, such as superoxide anions ðO??
(OH.), peroxyl ðRO?
non-radicals that are oxidizing agents and/or easily converted
into radicals, such as hypochlorous acid (HOCl), ozone (O3),
singlet oxygen (1O2), and hydrogen peroxide (H2O2; Bedard
and Krause, 2007). Although H2O2is not a free radical (having
2Þ, and alkoxyl (RO.) radicals, and
no unpaired electrons), it is treated as one because it can easily
give rise to reactive OH.(Boonstra and Post, 2004).
Sources of ROS can be either extracellular (pollutants,
tobacco smoke, ultraviolet radiation, ionizing radiation, iron
oxidase complexes, xanthine oxidases (XOs), amine oxidases,
nitric oxide synthases (NOSs), myeloperoxidase (MPO),
peroxysomes, and metabolism of arachidonic acid by
lipoxygenases and cyclooxygenases] (Turrens, 2003; Stocker
and Keaney, 2004; Bedard and Krause, 2007).
The mitochondrion is the major intracellular source of
chain is mediated by five enzyme complexes that oversee
the reduction of oxygen to water, one electron at a time.
Complex IV (cytochrome oxidase) retains all partially reduced
intermediates until full reduction is achieved. However, the
Q-cytochrome c oxireductase (Complex III), as well as the
nicotinamide adenine dinucleotide quinone (NADH-Q)
reductase (Complex I), are well-documented sources of ROS,
as they may leak electrons to oxygen, partially reducing this
molecule to O??
Apart from the mitochondria, membrane-bound NADPH
oxidase (NOX) induces production of ROS in phagocytes,
which is important for defense against pathogens (Rossi and
Zatti, 1964). XOs are key enzymes in purine metabolism
catalyzing oxidative reactions to produce uric acid, which is an
2(Turrens, 2003; Kowaltowski et al., 2009).
The authors of this article have no conflict of interest to declare.
Contract grant sponsor: National Heart, Lung and Blood Institute
(NHLBI) of the National Institutes of Health (NIH);
Contract grant number: HL80442.
*Correspondence to: Katya Ravid, Boston University School of
Medicine, 700 Albany St. W-601, Boston, MA 02118.
Manuscript Received: 17 October 2011
Manuscript Accepted: 1 February 2012
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 13 February 2012.
? 2 0 1 2 W I L E Y P E R I O D I C A L S , I N C .
antioxidant and free-radical scavenger (Stocker and Keaney,
2004). NOS produce the free-radical nitric oxide (NO) from
oxidation of L-arginine. Three NOS isoenzymes are known:
nNOS, expressed in neuronal tissues; eNOS, expressed in
endothelial cells; and iNOS, the inducible form expressed in
of NO is involved in proinflammatory reactions, functioning as
a cytostatic and cytotoxic molecule (Knowles and Moncada,
1994). MPO is produced in myeloid phagocytic cells and its
primary function is the destruction of microorganisms. The
oxidation of chloride by MPO and H2O2results in HOCl, a
highly reactive oxidizing agent (Klebanoff, 2005). Breakdown
of fatty acid chains by b-oxidation in peroxisomes is another
source of intracellular ROS (Boonstra and Post, 2004).
Lipoxygenases catalyze the insertion of molecular oxygen into
prostaglandins, thromboxanes, and leukotrienes (Kuehl and
Although ROS are important for normal physiology,
accumulation of ROS at high levels can be harmful to the
organism, causing damage to a variety of biomolecules, such as
lipids, DNA, carbohydrates, and proteins. Consequently, a
number of defense systems have evolved to prevent such
of ROS, these defense mechanisms are not always sufficient
to regulate the intracellular ROS balance, leading to oxidative
stress with detrimental effects on cellular homeostasis.
The antioxidant systems in mammalians include enzymes
such as superoxide dismutases (SOD), glutathione (GSH),
glutathione peroxidase (GPX), glutathione S-transferases
(GST), catalase, and peroxiredoxins (Prx). SOD2 has been
reported to affect the erythrocyte lineage; no other effects
of SODs have been reported in hematopoiesis. GSH is the
hallmark redox buffer in living cellular systems. GSH is a
tripeptide of glutamic acid, cysteine and glycine and is the
predominant non-protein thiol in biological systems. The
balance between reduced glutathione (GSH) and oxidized
glutathione (GSSG) is critical in maintaining redox homeostasis
selenium for catalytic activity. GST is another member of
the glutathione-dependent antioxidant defenses, catalyzing
the thioester conjugation to glutathione. TLK 199 or Telintra
is a peptido-mimetic inhibitor of GST, which recently showed
promising results for treatment of myelodysplastic syndrome
(MDS; Grek et al., 2011). Prxs are a family of small antixodant
proteins characterized by an amino-terminal catalytic cysteine
residue that is converted to sulfenic acid via reaction with
H2O2. Deficiency of Prx I and II in mice affects erythroid
homeostasis; loss of Prx I further increases susceptibility to
several malignancies (Ghaffari, 2008). Non-enzymatic
molecules, including vitamin E, vitamin C, and flavonoids act
as antioxidants capable of neutralizing ROS (Halliwell, 1999).
Overview of Megakaryopoiesis
MKs are highly specialized blood cells residing primarily in
the BM but also in the spleen and lung capillaries (Slater et al.,
1983; Kaushansky, 2008). These cells are responsible for the
production of platelets, which are renewed on a daily basis
(Kaushansky and Drachman, 2002). In adults, MKs derive
from BM-residing HSCs. In response to physiological demand,
the HSC gives rise to early progenitor cells including the
early common myeloid progenitor (CMP); further lineage
specification leads to development of the bipotential MK/
erythrocyte progenitor (MEP; Akashi et al., 2000). Under
certain cytokine stimulations, the bipotential MEP can develop
into a highly proliferative early MK-burst-forming unit
(CFU-MK; Deutsch and Tomer, 2006). These MK progenitors
eventually lose their proliferative ability and commit to an
endomitotic cycle. This process leads to mature polyploid MKs
that grow several-fold in size. Mature MKs can obtain a DNA
content of up to 128N, with the average being 16N
(Kaushansky, 1999; Tomer, 2004). During this process, MKs
increase the production of proteins necessary for platelet
biogenesis and function (Paulus, 1970). Mature MKs form
proplatelet extensions that fragment and give rise to platelets
(Italiano et al., 2007). Figure 1 illustrates the above-described
major steps in MK development.
Thrombopoietin (TPO), also known as c-Mpl ligand or MK
growth and development factor (MGDF), is the key regulator
of megakaryopoiesis. In pathological conditions, such as
thrombocytopenia, where there is high demand for platelets,
TPO release to the MK microenvironment is increased
(Sungaran et al., 1997, 2000). Mutations in the regulatory
regions of the TPO gene that result in overexpression of TPO,
as well as activating mutations of the c-Mpl receptor, are
associated with familial essential thrombocythemia,
characterized by enhanced MK progenitor proliferation and
platelet production (Deutsch and Tomer, 2006).
of cytokines including platelet-derived growth factor (PDGF)
are involved is this process as well. Earlier studies in primary
murine and human BM cultures, as well as in human umbilical
1995). Interestingly, PDGF-BB upregulates the expression of
MK-associated transcription factors, such as c-Fos, GATA-1,
and NFE2 in megakaryocytic cell lines (Chui et al., 2003).
Targeted deletion of the PDGF-B polypeptide chain is
embryonic lethal. Hematological analysis of these embryos
reveals erythroblastosis, macrocytic anemia, and
thrombocytopenia (Leveen et al., 1994; Kaminski et al., 2001).
MK Differentiation: Significance of Hypoxia in the
HSCs predominantly remain in a quiescent state in the low-
oxygen environment of the osteoblastic niche (Adams and
progenitor cells actively undergo cell division for proliferation
major reported effects of ROS on MK biology, covered in this review.
MK, megakaryocyte; TPO, thrombopoietin; c-Mpl, thrombopoietin
receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF
available at http://wileyonlinelibrary.com/journal/jcp]
Effects of ROS on MK biology. Schematic illustration of the
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and further differentiation in the more oxygenic vascular niche
(Kopp et al., 2005; Li and Li, 2006). Thus, an oxygen gradient
from below 1% in the most hypoxic environment to 6% in the
sinusoidal cavity provides conditions that allow different
regulatory processes to take place, including self-renewal and
differentiation (Eliasson and Jonsson, 2010). A study by Jang
et al. indicates that HSCs that produce lower levels of ROS are
more primitive, as shown by higher self-renewal capacity. On
the other hand, ROShighHSCs exhibit significant exhaustion in
serial transplantation assays, and have increased levels of p38
MAPK and mTOR. Importantly, treatment with a p38 inhibitor
or rapamycin (mTOR inhibitor) was able to restore HSC
function in the ROShighpopulation (Jang and Sharkis, 2007).
Pallotta et al. (2009) developed an in vitro system combining
osteoblastic niche. Differentiation of human CD34þcells to
MKs in this system revealed that in hypoxic (5% O2) but not in
normoxic (20% O2) conditions, there is a progressive increase
of hematopoietic progenitors for matrix deposition and
modulation of the niche environment in hypoxic conditions.
proplatelet formation, which was further exacerbated by
hypoxia (Pallotta et al., 2009).
where there is increased oxygen tension (Li and Li, 2006; Junt
et al., 2007). There, MKs shed platelets into the sinuses, the
MKs are also present in another compartment of high oxygen
the average 5% pO2in the BM hematopoietic niche (8% pO2in
the BM sinusoids and 16% pO2in the lung capillaries), it is
reasonable to hypothesize that oxygen levels contribute to MK
maturation (Pennathur-Das and Levitt, 1987; Kietzmann et al.,
maturation showed that CD34þperipheral blood (PB) cells
5% pO2conditions. The TPO-induced increase in MK size was
1998). MK differentiation under 20% pO2also yielded higher
conditions gave greater numbers of CFU-MK (Mostafa et al.,
2000). This increase in MK maturation, ploidy and terminal
differentiation under 20% O2was also associated with elevated
expression of MK maturation-specific transcription factors
under 20% pO2culturing conditions (Mostafa et al., 2001).
Collectively, these studies indicate that the hypoxic
environment in the BM niche is favorable for maintenance of
essential for complete MK maturation and platelet production
The Role of ROS as Signaling Moieties
Fluctuating low levels of ROS are shown to be important in a
number of regulatory processes. More specifically, H2O2can
serve as a second messenger promoting cell proliferation
(Martindale and Holbrook, 2002). Although controversial, the
notion of ROS as activators of signaling processes is slowly
gaining ground (D’Autreaux and Toledano, 2007).
With respect to the mechanisms involved in the effect of
ROS on proliferation, one study points to the possibility
of H2O2and O??
cells in response to cytokine stimuli. These may act as signaling
mediators, thus promoting growth responses. Indeed,
hematopoietic growth factors that stimulate proliferation and
2being generated at sub-micromolar levels by
differentiation of HSCs and progenitor cells signal through
ROS. Studies in MO7e human megakaryoblastic leukemia cells
colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), stem
(Prata et al., 2004). This peaking of ROS, in turn, may be
essential for the activation of cytokine receptors.
The majority of signaling responses occur through the
phosphorylation of tyrosine residues. Seminal works
demonstrated that treatment of cells with H2O2induces a
global increase in proteins with phosphorylated tyrosine
residues (pTyr), thus establishing a correlation between ROS
muscle cells (VSMC) treated with PDGF, the time-course
of H2O2production was similar to that of PDGF-induced
Further studies aimed at discovering the proteins
phosphorylated in response to ROS. In MO7e cells, the b
common chain receptor for both GM-CSF and IL-3 is
phosphorylated upon H2O2stimulation, as upon stimulation
with GM-CSF. H2O2stimulation also induced tyrosine
et al., 1996). Addition of the antioxidant pyrrolidine
dithiocarbamate (PDTC) to the cultures decreases the ROS
levels and inhibits tyrosine phosphorylation induced by GM-
SCF (Sattler et al., 1999). Mediators of signaling responses are
also phosphorylated in response to ROS production. signal-
transducer and activator of transcription 5 (STAT5) which is
a mediator of TPO responses, is phosphorylated in response
to H2O2stimulation (Sattler et al., 1999). Inhibition of ROS
production hampered the activation of serine/threonine
protein kinase (AKT), STAT3 and STAT5 in an HEL cell line
stimulated with TPO (Sardina et al., 2010). Other studies have
focused on the activation of mitogen-activated protein kinases
MAP kinase (ERK) phosphorylation and activation, where cells
are exposed to H2O2, or indirectly by activating upstream
effectors such as MAP kinase/ERK kinase (MEK, stimulated
by ONOO?or H2O2), Raf-1, or protein kinase C (PKC,
stimulated by H2O2; Abe et al., 1998; Zhang et al., 2000). As a
of transcription factors, such as c-fos and c-jun (Buscher
et al., 1988; Rao, 1997). ERK activation in K562 and HEL cells
of oxygen tension and its effect on MK differentiation. pO2, oxygen
tension; HSC, hematopoietic stem cell; MK: megakaryocyte. [Color
figure can be seen in the online version of this article, available at
Spatial organization of the BM, with respect to distribution
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occurs during phorbol myristate acetate (PMA)-induced MK
differentiation, and this activation is hampered by inhibitors of
ROS production (Sardina et al., 2010).
Signaling initiated by protein phosphorylation induced by
tyrosine kinases is followed by de-phosphorylation by protein
tyrosine phosphatases (PTPs) for adequate termination of the
signaling. Recent studies indicate that perhaps the major role
of ROS in cell signaling is the modulation of PTP function.
Cys residue, which is important to catalytic activity and is
particularly susceptible to oxidation (Tonks, 2005). The
classical example is the MAP kinase phosphatase-3 (MKP-3),
a specific regulator of Erk, oxidized in vitro by H2O2(Kamata
et al., 2005). The low molecular weight PTP (LMW-PTP)
negatively regulates PDGF signaling through binding and
dephosphorylation of the receptor. LMW-PTP is oxidized
can also be effected by PDGF signaling, likely through H2O2
production. Moreover, glutathione, an antioxidant, most likely
regulates the reversibility of LMW-PTP inactivation (Chiarugi,
2001). Exposure of cells to H2O2or PDGF induces Akt
phosphorylation and oxidation of phosphatase and tensin
homolog (PTEN; Kim et al., 2011). The antioxidant PrxII, has
beendemonstrated tolocally regulatePDGF signalingin VSMC
by decreasing the oxidation of PTPs, thereby functioning as a
negative regulator of PDGF signaling (Choi et al., 2005).
IL-3 and erythropoietin (Epo) induce a transient increase
in ROS levels when added to cultures of the hematopoietic
progenitor cell line 32Dcl3. Furthermore, treatment of these
cells with the antioxidant N-acetyl-L-cysteine (NAC) inhibits
the IL-3-induced phosphorylation of JAK2, AKT, and ERK.
Moreover, upon treatment with the antioxidant, there is a
downregulation of cyclin D2 and cyclin E concomitantly with
an increase in expression of the cell cycle inhibitor p27, thus
inhibiting G1–S phase progression (Iiyama et al., 2006).
Studies in HeLa cells and primary fibroblasts demonstrate
that exposure of cells to H2O2or peroxynitrite induces Akt
activation respectively mediated by epidermal growth factor
(EGF) and PDGF receptors (Klotz et al., 2000; Wang et al.,
2000). Other pathways activated by ROS are the p38 and c-Jun
N-terminal kinases (JNK). These pathways are responsive to
oxidative stress and are associated with apoptosis and mitotic
arrest (Martindale and Holbrook, 2002).
The above ROS-affected signaling processes are clearly
involved in expansion of primary BM MKs, as STAT and MAPK
activation are important for MK proliferation (Miyakawa et al.,
et al., 1997; Eliades et al., 2010). Although the accumulated
evidence is not sufficient to establish a definitive role of ROS in
signaling affecting MK biology, the data indicates a strong effect
of ROS on the activation of tyrosine kinase signaling and MK
expansion. For PDGF signaling, there is a need to validate
current findings in the MK lineage. Further studies elucidating
the precise mechanisms of this effect are awaited.
MK Endomitosis and Polyploidization: Role of ROS and
As described above, a number of studies have investigated the
role of ROS in MK maturation. Therefore, it is of interest to
determine the source of ROS in this lineage. An important
insight came from the observation that diphenylene iodonium
inhibited platelet aggregation (Salvemini et al., 1991). NOX is
such an enzyme, and also a known enzymatic source of ROS
(Rossi and Zatti, 1964). The family of NOX proteins consists
of oxidases responsible for the transfer of electrons across
biological membranes (Bedard and Krause, 2007). Members
of the family include the phagocyte NOX2 (gp91phox), NOX1,
NOX3, NOX4, NOX5, and the dual oxidases DUOX1 and
DUOX2 (Bedard and Krause, 2007). Organizer subunits
p47phoxand NOXO1, activator subunits p67phoxand NOXA1
and modulator subunit p40 are associated with NOXs (Geiszt,
2006; Bedard and Krause, 2007). All members of the NOX
family contain six transmembrane domains, two binding sites
for heme, and conserved binding sites for NADPH and flavin
Recent studies have focused on the source of ROS in HSCs.
In PB-derived CD34þcells, a low mitochondrial oxygen
consumption rate was detected, thusqualifying HSCs as a poor
oxidative phosphorylating cell type. In addition to low
mitochondrial oxygen consumption, the authors depicted the
contribution of NOX2 and NOX4 in extra-mitochondrial
oxygen consumption (Piccoli et al., 2005). Moreover, both
catalase and the NOX pharmacological inhibitors, Apocynin
and DPI, inhibited ROS production by human BM-derived
HSCs. These cells were also shown to express at least three
different NOX isoforms—NOX1, NOX2, and NOX4—at
both the mRNA and protein level along with a set of their
regulatory subunits (Piccoli et al., 2007b). Interestingly, CD34þ
and CD133þHSCs express hypoxia-inducible factor 1a
2007a). Seno et al. (2001), using a pharmacological approach
to target NOXs, demonstrated that they are possible sources
of ROS in human platelets and in the megakaryocytic cell line
MEG01. The authors also verified the expression of the NOX
regulatory components p22phoxand p67phoxin both platelets
and MEG01 cells (Seno et al., 2001).
More recently, it was reported that Nox1 is the major Nox
expressed in primary mouse MKs and contributes to the
production of ROS in CD41þMKs. Inhibition of Nox1 by
Apocynin or DPI reduced polyploidization in wild-type MKs.
This defect was due to reduced levels of G1 cyclins D3 and E,
which have been shown to be important for MK polyploidy
(Wang et al., 1995; Eliades et al., 2010). Thus, the effect of ROS
on expression of G1 phase cyclins and G1–S cycle progression,
along with the reported effect of NOX4 in VSMC polyploidy
polyploidization. The influence of ROS on G1 phase cyclins has
been validated not only in MKs. NOX1-mediated increase in
catalase or diphylene iodonium, a potent NOX inhibitor,
level is known to increase as cells progress from G1 to S phase
et al., 2006).
MK Maturation and Platelet Formation: The Role of ROS
Endogenous ROS are detected in MKs from mouse BM
(McCrann et al., 2009a). TPO induces a rapid increase in ROS
that is necessary for the megakaryocytic differentiation of
human HSCs (Sardina et al., 2010). ROS can control gene
expression through the activation of redox-sensitive
transcription factors, which in turn coordinate the expression
of a number of downstream target genes with antioxidant
roles. The classical example is the NF-kB transcription factor,
activated by H2O2and intermediates of oxygen radicals
(Schreck et al., 1991; Schmidt et al., 1996). p45NF-E2 is a
member of the cap ‘‘n’’ collar-basic leucine zipper family of
transcriptional activators, with a restricted expression in
hematopoietic cells. This family is known to bind antioxidant
response elements (ARE) in DNA, which are responsible for
to complications of hemorrhage. Absolute thrombocytopenia
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was detected in those animals. MK ploidization was not
affected, indicating that the lack of platelets was not due to
differentiation defects in MKs, but rather to a defect in
formation of platelet territories in the cytoplasm of MKs
(Shivdasani et al., 1995). Recently, work by Motohashi et al.
(2010) revealed that NF-E2 p45 promotes ROS accumulation
that results in enhanced MK maturation. This is achieved by
competition with Nrf2, a key activator of stress-responsive
genes (Motohashi et al., 2010).
MK Senescence and Autophagy: The role of ROS
In conditions of hyperoxia and elevated ROS, cells resort to
a decrease in proliferation, cell cycle arrest, and senescence
(Boonstra and Post, 2004; Shao et al., 2011). Hyperoxia
increases the levels of p21, a cell cycle inhibitor that belongs
to the family of Cip/Kip proteins (Cazzalini et al., 2010).
This increase is mediated by p53 (Helt et al., 2001). In HSCs,
2006). Recently, studies on UT-7/TPO cells treated with
TPO have suggested that cell cycle arrest and senescence
participate in the process of MK maturation. This process was
accompanied by upregulation of the senescence marker p21,
and mediated by phosphorylation of ERK (Besancenot et al.,
Autophagy is a catabolic pathway in which cells sequester
organelles, such as mitochondria into lysosomes for
degradation. The process of autophagy is an important cellular
survival mechanism, which can be activated in response to
multiple physiological conditions, including starvation,
hormonal imbalanceandoxidativestress (Watsonet al., 2011).
The mammalian target of rapamycin (mTOR) is the central
molecular component of autophagy. The Atg1–13 protein
complex initiates autophagy, activated by the absence of
signaling from mTOR. Mice deficient for Atg7 in the
hematopoietic system develop myeloproliferation/MDS and
exhibit high mitochondrial content and ROS in HSCs
(Mortensen et al., 2011). Interestingly, autophagy has been
observed in the megakaryocytic differentiation of the K562 cell
line (Colosetti et al., 2009), and mTOR is reported as a
regulator of MK differentiation (Drayer et al., 2006; Raslova
et al., 2006); these findings collectively suggest the involvement
of ROS-dependent autophagic processes in MK.
Other Effects of ROS in MKs
One of the functions of 15d-PGJ2, a J-type prostaglandin, is
induced formation of ROS and increased platelet production.
effect of 15d-PGJ2(O’Brien et al., 2008).
Toll-like receptor 2 (TLR2)
production of ROS in the Meg-01 cell line and affected MK-
MKs in vitro and in vivo (Beaulieu et al., 2011).
Nitric oxide (NO)
The addition of NO donors sodium nitroprusside (SNP) or
ium-1,2-diolate (DETA/NO) tocultures ofhumanCD34þcells
has toxic effects on both total cell number and TPO-induced
MK differentiation (as measured by total number of MK and
percentage of CD41 expression; Schattner et al., 2000). The
most likely effect of NO on MKs is induction of apoptosis, as
demonstrated in detail in megakaryocytic cell lines (Battinelli
(Schattner et al., 2000). Stimulation of CD34þcells with tumor
necrosis factor-a (TNF-a) and interferon-g (IFN-g) increased
endogenous NO levels and suppressed MK growth (Schattner
et al., 2000), whereas treatment with TPO suppressed the
induction of apoptosis by NO (Battinelli and Loscalzo, 2000).
ROS in MK Pathology
Although there are no reports associating ROS with specific
megakaryocytic disorders, oxidative stress has beenimplicated
in a variety of BM failure conditions, such as MDS and
myelofibrosis, in which platelet deficiency requires therapeutic
ROS in myelodysplastic syndrome (MDS)
MDS is a clonal stem cell disorder characterized by ineffective
maturation of the erythroid, granulocytic, or megakaryocytic
lineage. The natural history of the disease is progression from
cytopenia to myeloid leukemia. MDS is a disease of the elderly,
with a mean age of 70 years at diagnosis (Corey et al., 2007).
Age itself is an established critical factor for accumulation of
oxidative damage in HSCs (Finkel and Holbrook, 2000).
Moreover, accumulating evidence has suggested a major
causative role for ROS in the pathogenesis of MDS (Farquhar
and Bowen, 2003), beginning with a seminal study that
demonstrated the presence of oxidative DNA damage in
CD34þcells from MDS patients (Tauro et al., 2001). A high
superoxide concentration has also been detected in
supernatant from MDS stroma compared to normal stroma
(Tauro et al., 2001). Exposure to cigarette smoke and benzene
are reported to be associated with risk of MDS. The
progression to disease is also likely to be associated with a
deficiency in the detoxification system, such as the
NAD(P)H:quinine oxidoreductase (NQO1) deficiency in the
HSCs (Rothman et al., 1997). An experimental model of
cigarette smoke exposure in guinea pigs demonstrated the
correlation between NQO1 deficiency, cigarette smoke
exposure, and progression to MDS (Das et al., 2011).
Moreover, as noted in our discussion of senescence and
autophagy, mice deficient for Atg7 progress to a
myeloproliferative disorder/MDS (Mortensen et al., 2011).
of defective mitochondrial autophagy have been observed in
and mutations of mitochondrial DNA are commonly detected
of autophagy in MDS is still under discussion (Watson et al.,
Lysyl oxidase (LOX) in myelofibrosis
LOX is a copper-dependent amine oxidase that catalyzes the
oxidative deamination of lysine and hydroxylysine residues on
collagen and elastin precursors. The resulting semialdehydes
form covalent cross-linkages, thus stabilizing the extracellular
matrix fiber deposits (Lucero and Kagan, 2006). LOX is
synthesized as a 50kDa glycosylated precursor (pro-LOX)
which is then secreted and undergoes proteolytic cleavage
by pro collagen C-proteinases, including bone morphogenetic
protein 1 (BMP1), to release a catalytically active 30kDa
enzyme (LOX) and an 18kDa propeptide (LOX-PP; Trackman
was demonstrated in LOX knockout mice, which die soon
after birth due to aortic rupture, incomplete diaphragm
development, and cardiovascular dysfunction (Maki et al.,
mobilizes monocytes, fibroblasts, and VSMCs (Nelson et al.,
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1988; Lazarus et al., 1995; Li et al., 2000). LOX has been
associated with various pathologies, including cardiovascular
diseases (Rodriguez et al., 2008), neurodegenerative disorders
(Gilad et al., 2001, 2005), tumor progression, and metastasis
(Payne et al., 2005; Erler et al., 2006; Min et al., 2009). An
interesting insight into the regulation of MKs by LOX came
oxidize cell surface proteins, including PDGFR-a, in rat aortic
smooth muscle cells (Lucero et al., 2008). This effect was
blocked by a-aminopropionitrile (BAPN), an inhibitor of LOX
enzyme activity (Tang et al., 1983; Lucero et al., 2008). The
BAPN-mediated inhibition of PDGFR oxidation diminished the
binding affinity for its correspondent ligand, PDGF-BB. This
inhibition resulted in an accelerated rate of decay of
phosphorylated downstream effectors of PDGFR signaling,
was also identified in MKs, showing dependency of PDGF-BB
on an active LOX to promote MK proliferation. Furthermore,
LOX is primarily expressed in low-ploidy MKs (Eliades et al.,
Our laboratory also uncovered an important role for LOX
in controlling MK-induced BM fibrosis. The term myelofibrosis
indicates BM deposition of reticulin, collagen, or both.
However, regardless of the pathogenic background,
MK-induced myelofibrotic conditions share a common
denominator: defective MK development and a dense
extracellular matrix (Kuter et al., 2007). BM fibrosis, in the
context of acute megakaryocytic leukemia (AMKL) and
myeloproliferative disorders usually involves deposition of
reticulin and/or collagen fibers (McCarthy, 1985; Kuter et al.,
2007). The evolving hypothesis is that MKs release growth
factors, such as transforming growth factor-b (TGF-b), PDGF,
and fibroblast growth factor (FGF), which accentuate the
production of collagen by fibrogenic cells (Terui et al., 1990;
Reilly et al., 1993; Le Bousse-Kerdiles and Martyre, 1999;
Vannucchi et al., 2002; Le Bousse-Kerdiles et al., 2008). LOX
not only affects the proliferative effect of PDGF, but through
its ability to cross-link the extracellular matrix, is a major
regulator of the fibrotic phenotype in the above pathologies.
Pharmacological inhibition of LOX significantly attenuated
matrix deposition and myelofibrosis in GATA-1lowmice
(Eliades et al., 2011). In GATA-1lowmice, the abrogation of the
distal promoter of GATA-1 and the DNAse hypersensitive
region leads to downregulated GATA-1. These mice display
myelofibrosis with a significantly increased number of MKs
arrested between the stage of megakaryoblast and immature
MK and decrease in total marrow cellularity (Vannucchi et al.,
2002; Centurione et al., 2004). The effects of LOX on MKs are
summarized in Figure 3.
Finally, a study analyzing the levels of oxidative stress in
patients with primary myelofibrosis detected significantly
raised ROS concentrations and significantly lowered total
antioxidant capacity (Vener et al., 2010). Added to the finding
LOX in endothelial cells (Guadall et al., 2011), the data
accumulated so far suggest a novel and central role of ROS
and LOX in the progression of marrow fibrotic disorders.
Therapeutic Targeting of ROS
Deregulation of ROS homeostasis has been reported in many
hematological disorders. Together with evidence of ROS
involvement in regulation of critical cellular events, such as
proliferation, differentiation, and survival, targeting ROS for
therapeutic purposes is a promising approach for further
development. However, since ROS are also important for
homeostasis of normal hematopoietic cells, specific targeting
of malignant cells has proven trickier than initially hoped.
Two basic approaches are possible: the pro-oxidant and anti-
target cells (Grek et al., 2011; Hole et al., 2011; Sardina et al.,
The rationale of the pro-oxidant approach is that since
malignant cells already have high levels of ROS, increasing their
ROS to toxic levels is more easily achievable than with normal
cells. This effect may be achievable by inhibiting intracellular
anti-oxidants. TLK 199, Telintra, is a peptido-mimetic inhibitor
of an isoform of GST, GSTP (p). In preclinical mouse studies
Telintra raised circulation of blood cells of all lineages, an effect
associated with increase in BM progenitor cells. Telintra has
shown positive results in an ongoing Phase II clinical trial for
MDS; multilineage hematologic improvement has been
observed, including decreased requirements for red blood cell,
platelet, and growth factor support (Grek et al., 2011).
The anti-oxidant amifostine has been used with traditional
anti-cancer drugs as a cytoprotective agent. Amifostine is
converted to its active metabolite by the membrane-bound
enzyme alkaline phosphatase. The active metabolite prevents
free-radicals, donating hydrogen ions to free-radicals and
directly binding and inactivating cytotoxic drugs. Because
normal tissues generally have higher levels of alkaline
phosphatase, better vascularization, and higher pH, the active
metabolite preferentially locates there. Studies have also
indicated that amifostine stimulates HSCs. These features have
inspired ongoing clinical use of amifostine in MDS patients
(Grek et al., 2011; Hole et al., 2011).
There is increasing evidence that oxygen tension in the
microenvironment affects MK differentiation, maturation,
polyploidy, and proplatelet fragmentation. Further research is
needed to better understand how different oxygen tensions
translate into signals that control MK biology. As outlined
above, an array of oxidases, including NOX and LOX, affect
ExtracellularLOX stimulates PDGF-mediated Erk andAkt signaling,
contributing to MK progenitor proliferation. Inhibition of LOX by
through inhibition of the catalytic activity of LOX, hampering
progression of myelofibrosis. [Color figure can be seen in the online
version of this article, available at http://wileyonlinelibrary.com/
Effect of LOX on MK biology and progression of BM fibrosis.
JOURNAL OF CELLULAR PHYSIOLOGY
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the BM niche and its matrix, as well as cell cycle properties.
Together, these influences have a major impact on lineage
development and cell propagation. Developing selective
inducers or inhibitors of specific oxidases to control ROS in
the BM niche will aid investigations at both the basic and
is an established Investigator with the American Heart
Association. We apologize to all authors whose important
work in the field could not be cited because of word count
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