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Catherine E. Forristal and Jean-Pierre Levesque
Targeting the Hypoxia-Sensing Pathway in Clinical Hematology
doi: 10.5966/sctm.2013-0134 originally published online December 26, 2013
2014, 3:135-140.Stem Cells Trans Med
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Perspectives
Targeting the Hypoxia-Sensing Pathway in Clinical Hematology
CATHERINE E. FORRISTAL,JEAN-PIERRE LEVESQUE
Stem Cell Biology Group, Mater Research Institute–University of Queensland, Woolloongabba, Queensland, Australia
SUMMARY
Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain
(PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly
heterogeneou s in terms of microvasculature an d thus oxygen concentration. Th e importance of hypoxia and HIFs in the hematopoie tic
environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be
pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoi-
esis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in
hemodialysis patients. Pharmac ological stabilization o f HIF-1aprotein with PHD inhib itors is also emerging as an impo rtant regulator of
HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the
bone marrow in viv o, thereby protecting them from hi gh doses of irradiation and accel erating hematological recove ry. Recent findings
also show that stabilization of HIF-1aincreases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerix-
afor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation.
These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology. STEM CELLS TRANSLATIONAL
MEDICINE 2014;3:135–140
INTRODUCTION
Maintenance of oxygen homeostasis is critical for the survival of
organisms. On exposure to hypoxic conditions, a cellular response
is mounted by hypoxia-inducible factors (HIFs). HIFs are a family of
three transcription factors composed of one of three oxygen-
sensitive asubunits—HIF-1a, HIF-2a, and HIF-3a—and a constitu-
tively expressed bsubunit HIF-1b, also called aryl hydrocarbon
receptor nuclear translocator (ARNT). Once the HIF-a:ARNT com-
plex is formed, it translocates to the nucleus and activates the tran-
scription of genes containing hypoxia-responsive elements (HREs)
[1, 2]. Hematopoietic cells including hematopoietic stem cells
(HSCs) express HIF-1amRNA, which is expressed ubiquitously by
all cells. In hypoxic conditions with oxygen (O
2
) concentration be-
low 2%, HIF-aproteins are stabilized and complex with ARNT to
translocate to the nucleus and initiate transcription of HRE-
containing genes. In normoxic conditions or when O
2
concentra-
tion exceeds 2%, HIF-1aprotein is degraded within 5 minutes by
the proteasome [3], preventing the formation of the transcription
factor and its translocation to the nucleus. The sensitization of
HIF-aproteins to proteasomal degradation in the presence of
O
2
is mediated by three prolyl hydroxylase domain (PHD) enzymes
that hydroxylate two proline residues within the oxygen-degradation
domain of HIF-aproteins (Fig. 1A) [4, 5]. These hydroxylated proline
residues then bind the von Hippel-Lindau tumor-suppressor protein
to form an E3 ubiquitin ligase complex that ubiquinates and targets
HIF-aprotein to the proteasome (Fig. 1B) [6, 7]. PHD enzymes
are iron(II)-dependent and utilize 2-oxoglutarate and O
2
as sub-
strates to hydroxylate proline residues [8]. In cultured cells, PHDs
areinactivewhenO
2
is ,2% in the extracellular milieu, resulting
in HIF-aprotein stabilization.
As noted previously, the expression of HIF-asubunits is predom-
inantly regulated by PHD-mediated proline hydroxylation. There
are three well known PHD isoforms, called PHD1, PHD2, and
PHD3, and all are reported to hydroxylate HIF-asubunits [9]. They
are encoded by three distinct genes: Egln2 for PHD1, Egln1 for
PHD2, and Egln3 for PHD3. A fourth PHD enzyme is also thought
to be involved in regulating HIF-asubunits and has been reported
to play a potential role in erythropoiesis [10, 11].
ROLE OF HIFSINCONTROLLING HEMATOPOIETIC STEM AND
PROGENITOR CELLS
HIF Expression in Hematopoietic Stem and Progenitor Cells
The importance of HIFs in development and hematopoiesis has
been demonstrated by genetic deletion of ARNT, which abro-
gates the function of both HIF-1aand HIF-2a. In the developing
embryo, ARNT is essential for multilineage hematopoietic pro-
genitors, vasculogenesis, and angiogenesis [12, 13]. HIF-1a
mRNA is ubiquitously expressed [14]. In steady state, HIF-1apro-
tein is detected only in the endosteal region of the bone marrow
(BM)andinsomediscretecellsinthecentralBM[15].Conse-
quently, HIF-1aprotein is generally below detection in whole
BM lysates [15, 16]; however, when HSCs are mobilized in the pe-
ripheral blood by administering granulocyte colony-stimulating
factor (G-CSF) or cyclophosphamide, HIF-1aprotein is stabilized
and found throughout the BM cavity [15].
Correspondence: Jean-Pierre Levesque, Ph.D., Mater Research, Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland 4102, Australia.
Telephone: 61-7-3443-7571; E-Mail: jplevesque@mmri.mater.org.au Received July 26, 2013; accepted for publication September 18, 2013; first published
online in SCTM EXPRESS December 26, 2013. ©AlphaMed Press; http://dx.doi.org/10.5966/sctm.2013-0134
STEM CELLS TRANSLATIONAL MEDICINE 2014;3:135–140 www.StemCellsTM.com ©AlphaMed Press 2014
P
ERSPECTIVES
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Unlike HIF-1a,HIF-2amRNA expression is restricted. HIF-2a
is expressed by vascular endothelium, hepatocytes, and inter-
stitial and glomerular cells of the kidney. In the BM, HIF-2a
mRNA is primarily expressed by hematopoietic lineage-negative
cells [14]. HIF-2amRNA is detected at very low levels in HSCs;
however, in these cells, HIF-2aprotein is mainly localized to
the cytoplasm [14], suggesting that it is not transcriptionally ac-
tive [17].
The expression profile of HIF-3ahas been largely uncharacter-
ized; however, in the BM, HIF-3ais most highly expressed in HSCs
and is expressed at low levels in more differentiated progeny [14].
The function of HIF-3ais unknown because, unlike HIF-1aand
HIF-2a, HIF-3adoes not contain a DNA-binding domain. Further-
more, HIF-3acontains many splice variants, the most studied of
which is known as inhibitory PAS domain, which functions as
a dominant-negative regulator of the other two HIFs’mediated
gene induction [18, 19].
HIFs Regulate Proliferation and Self-Renewal of
Hematopoietic Stem and Progenitor Cells
The importance of hypoxia and HIF-1ain the proliferation and
self-renewal of HSCs is well established. Indeed, culture of hu-
man and mouse HSCs in hypoxic conditions results in an accumu-
lation in phase G
0
of the cell cycle as a result of HIF-1aprotein
stabilization, which enhances expression of p21
cip
,p27
Kip1
,and
p57
Kip2
and reduces mitochondrial oxidative phosphorylation
[20, 21]. This results in increased frequency of long-term repo-
pulating HSCs in hypoxic cultures compared with cultures in air
[22, 23]. Conversely, conditional deletion of the Hif1a gene in
hematopoietic cells results in exaggerated HSC proliferation
in vivo, with premature exhaustion of HSC self-renewal poten-
tial, loss of serial reconstitution potential, and increased sen-
sitivity to cytotoxics [14]. Pharmacological stabilization of HIF-
1aprotein with PHD inhibitors or genetic stabilization by con-
ditional deletion of the VHL gene increases HSC quiescence in
vivo [14, 16].
HIF-1aprotein is difficult to detect in the mouse BM in steady
state because only a very small proportion of the BM cavity is
hypoxic in these conditions [15, 16]; however, HIF-1aprotein
can be stabilized in murine HSCs in the BM by administering
small compounds (e.g., dimethyloxalylglycine or the isoquino-
line dipeptidyl derivative FG-4497; Fig. 2) that inhibit PHDs by
competing with their physiological substrate 2-oxoglutarate
[16]. This pharmacological stabilization of HIF-1aprotein in-
creases quiescence and decreases proliferation of HSCs in the
BM in vivo [16].
Hematopoietic progenitor cells alsorequire the hypoxia-sensing
pathway to regulate cell cycling. Pharmacological stabilization of
HIF-1aprotein with PHD inhibitors decreases hematopoietic pro-
genitor cell (HPC) proliferation in vivo [16]. Unexpectedly, condi-
tional deletion of the Egln1 gene (encoding PHD2) in cells
expressing CD68 results in an increase in proliferation of HPCs in
steady state in vivo in an HIF-1a- and SMAD7-dependent manner
[24]; however, without lineage-tracking experiments to determine
which cells have the active CD68 gene promoter to conditionally
delete the Egln1 gene, it is not possible to conclude whether the
proliferative effect of PHD2 deletion on HPCs was cell intrinsic or
extrinsically mediated by CD68
1
macrophages or other cells of
the BM stroma.
Figure 1. Regulation of HIF-aproteins. (A): Hydroxylation of two
distinct proline residues is catalyzed by PHDs. (B): Regulation of
the HIF-aprotein under hypoxic and normoxic conditions. PHD
inhibitors block HIF-aproline hydroxylation and subsequent ubiq-
uitination. HIF-aproteins are stabilized. Abbreviations: ARNT, aryl
hydrocarbon receptor nuclear translocator; ATM, ataxia telangiec-
tasia mutated; DMOG, dimethyloxalylglycine;HRE, hypoxia-responsive
elements; PHD, prolyl hydroxylase domain; pVHL, von Hippel-Lindau
protein.
Figure 2. Chemical structures of 2-oxoglutarate, the physiological
substrate of PHD enzymes, and PHD inhibitors DMOG, FG-4592,
and Amgen compound 12{1,1,2} [44]. Additional structures of PHD
inhibitors based on the dipeptidyl-quinolone backbone can be found
in [44]. The structure of FG-4592 was found on the website for Sell-
eckchem Inc. (Houston, TX, http://www.selleckchem.com). Ab-
breviation: DMOG, dimethyloxalylglycine; PHD, prolyl hydroxylase
domain.
136 HIF-Stabilizing Therapies in Hematology
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HIFs Regulate Erythropoiesis by Erythropoietin
Production in the Kidney
Erythropoietin(EPO) expressionis regulated primarily by thePHD2-
HIF-2aaxis. Renal EPO production is regulated primarily at the
transcriptional level and is markedly induced by anemia or global
hypoxia encountered at high altitudes. EPO has been shown to
be releasedby interstitialrenal cells in rodentsand by renal progen-
itor cells located in the renal inner medulla in humans [25]. HIF-2a,
expressed in the endothelial, interstitial, and glomerular cells of
the kidney and hepatocytes, plays an important role in EPO pro-
duction, whereas HIF-1adoes not appear to play a role [9, 26,
27]. The major role of PHD2 in controlling HIF-2aprotein and
EPO production in the kidney has been recently confirmed in
mice with conditional deletion of the Egln1 gene in kidneys
and macrophages. In the absence of PHD2, severe erythrocytosis
with a 10-fold increase of EPO concentration in kidney extracts
and blood plasma was observed in an HIF-2a-dependent manner
[28]. In support of this notion, positive selection of adaptive poly-
morphism in genes encoding for HIF-2aand PHD2 has been noted
in human populations living at high altitude, such as Tibetans and
Andeans [29, 30].
ARE HEMATOPOIETIC STEM AND PROGENITOR CELL NICHES IN THE
BM HYPOXIC?
The BM is highly heterogeneous in terms of microvasculature
[31–37]. Consequently, HSCs are exposed to varying levels of ox-
ygen perfusion depending on their location in the BM. Serially
reconstituting, quiescent HSCs tend to reside in areas with very
low blood perfusion, whereas more proliferative HSCs with
a lower reconstitution potential tend to reside in areas that
are more perfused by blood [38]. Furthermore, quiescent HSCs
are more frequent in the endosteal region of the BM, at two cell
diameters, or on average 10 mm, from the interface with the
compact bone [36, 39, 40]. This endosteal region is thought to
be hypoxic because of binding of pimonidazole [14, 15], a com-
pound that covalently binds to protein adducts when O
2
concen-
tration is below 10 mmHg or when the oxidative state is low;
however, most HSCs reside in proximity to endothelial sinuses,
even in the endosteal region of the BM. Moreover, different
cells can demonstrate differing pimonidazole binding regardless
of direct oxygen concentration, depending on their metabolic
profile. Indeed, two recent reports indicate that HSCs and HPCs
can bind pimonidazole independently of their location in the BM
[36, 37]. Consequently, HSCs may be more correctly referred to
as “low oxidative phosphorylation cells”rather than “hypoxic
cells,”and the so-called osteoblastic niche should rather be re-
ferredtoasan“osteovascular”niche [37]. Nevertheless, a pro-
portion of phenotypic HSCs reside immediately adjacent to
endothelial cells forming blood sinuses and vessels and are away
from the endosteum [41, 42].
Because of this heterogeneity of the HSC microenvironment,
not all HSCs and progenitor cells are exposed to the same oxi-
dative conditions in steady state. Approximately 50%–60% of
long-term reconstituting HSCs defined by the lineage-negative
Kit
1
Sca1
1
CD48
2
CD150
1
phenotype, and approximately
20%–40% of more heterogeneous lineage-negative Kit
1
Sca1
1
cells reside in poorly perfused, low-oxidative regions of
the BM in steady state, as demonstrated by Hoechst 33342 in
vivo perfusion and pimonidazole staining [14, 38]. Interestingly
these proportions coincide with the proportion of quiescent
HSPCs in the BM: Approximately 60% of lineage-negative Kit
1
Sca1
1
CD48
2
CD150
1
HSCs and approximately 30% of lineage-
negative Kit
1
Sca1
1
cells are quiescent in phase G
0
of the cell
cycle, as measured by Ki67 staining [16]. Small synthetic PHD
inhibitors mimic hypoxia throughout the entire BM, regardless
of perfusion and oxygen levels, by stabilizing HIF-1ain an oxygen-
independent manner. Consequently, PHD inhibitors pharmacolog-
ically stabilize HIF-1ain HSCs located in more perfused vascular
niches as well as hypoxic regions. Thus, following treatment with
PHD inhibitors, approximately 85% of long-term reconstituting
HSCs and 70% of lineage-negative Kit
1
Sca1
1
cells become quies-
cent [16].
PHD INHIBITORS AND HIFS
As described previously, the expression of HIF-asubunits is pre-
dominantly regulated by PHD-mediated hydroxylation. PHD inhib-
itors block the enzymatic activity or interfere with its substrate
binding site. There are well-known commercially available chemi-
cals that block PHD in vivo such as dimethyloxalylglycine and
N-oxalylglycine thatmimic the PHD cosubstratestructure of 2-oxo-
glutarate[43], thus blockingenzyme activity [6, 7](Fig. 2). More re-
cently, several companies have developed PHD inhibitors with
different chemical structures. Although the chemical structures
of most of these compounds are not described in the publically
available literature, some are. FibroGen (San Francisco, CA,
http://www.fibrogen.com), for instance, has developed a line of
compounds derived from isoquinoline bound to a dipeptide that,
similarly to oxalylglycine derivatives, mimic 2-oxoglutarate and
block PHD enzymatic activity with much higher efficacy [16] (Fig.
2). Amgen (Thousand Oaks, CA, http://www.amgen.com) has de-
veloped similar dipeptides bound to quinolone scaffolds, some
of which with half maximal inhibitory concentration values for
PHD2 as low as 35 nM and on the order of 5 nM for PHD1 and
PHD3 [44] (Fig. 2).
THERAPEUTIC INTERVENTION WITH PHD INHIBITORS
PHD inhibitors have been mostly trialed to correct anemia caused
by kidney diseases, with 22 clinical trials registered at the
ClinicalTrials.gov website (http://www.clinicaltrials.gov) at the
time of this review (Table 1). To date, no toxic effects have been
reported.
Anemia
Because EPO transcription and production are directly controlled
by HIF-2aprotein in the kidney, the proerythropoietic effect of
PHD inhibitors mediated by HIF-2aprotein stabilization and in-
creased EPO production is well documented in all mammalian
species tested. This elevated endogenous EPO production in-
creases red cell and hemoglobin blood concentration [45]. Conse-
quently, PHD inhibitors are being tested for treatment of anemia
in chronic renal disease, in which renal failure dramatically
reduces EPO production [46, 47]. Five orally available PHD inhib-
itors (i.e., AKB-6548, BAY85-3934, GSK1278863, FG-4592, and
FG-2216) are being or have been tested in clinical trials for this
specific application (as shown at ClinicalTrials.gov) (Table 1). FG-
2216 hasdemonstratedefficacy in increasing endogenous EPOcon-
centration in rhesus macaques [48] and in a phase II clinical trial on
hemodialysis patients with kidneys or who are anephric [47].
Forristal, Levesque 137
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In addition to boosting EPO production, PHD inhibitors such as
FG-4497 have also been found to improve acute kidney injury in
rats [49]. Pretreatment with the PHD inhibitor FG-4497 reduced
renal tissue injury and apoptosis following kidney ischemia, thus
suggesting a renoprotective role. The PHD inhibitor GSK1278863
is in a clinical trial to test efficacy to reduce ischemia injury during
surgery to repair aortic aneurysm (Table 1).
Because EPO has also been reported to have a number of
effects on nonhematopoietic cells and organs such as the brain,
glial cells, endothelial cells, skin, and the digestive tract [50], it
is possible that PHD inhibitors could have protective effects in
a number of other tissues [51–53].
It must also be noted that the erythropoietic effect of PHD
inhibitors is bringing new challenges to sports medicine because
they are possibly being used as doping agents. Because the use of
performance enhancement with erythropoiesis-enhancing drugs
is prohibited, mass spectrometry methods are being developed to
detect PHD inhibitors in athletes [54].
Protection Against Irradiation
Quiescent cells in G
0
are more radiation resistant, with radiation
sensitivity peaking in phase G2/M of the cell cycle [55]. Pharmaco-
logical stabilization of HIF-1awith PHD inhibitors also increases
HSC resistance to severe irradiation (9.0 Gy), as measured by the
gold standard long-term competitive transplantation assay. Mice
treated with the PHD inhibitor dimethyloxalylglycine or FG-4497
prior to irradiationhad 100% of their HSCssurviving in the BM after
9.0-Gyirradiation,whereas in controlsaline-treatedmice, the num-
ber of long-termcompetitive repopulating HSCswas decreased 27-
fold by 9.0-Gy irradiation. Consequently, PHD inhibitor-treated
mice recovered significantly faster from radiation-induced neutro-
penia and thrombocytopenia and had less profound anemia than
irradiated control mice [16]. Importantly, increased HSC and HPC
quiescence in response to PHD inhibitors was not mediated by el-
evated endogenous EPO because administration of EPO has no ef-
fect on HSC and HPC cycling [16]. Consequently, treatment with
PHD inhibitors can protect HSCs from high, sublethal doses of irra-
diation; can accelerate hematological recovery; and can prevent
the rapid exhaustion of the hematopoietic system [16]. This sug-
gests that PHD inhibitors could be used preventatively in persons
at risk of being exposed to severe irradiation. This could be partic-
ularly useful in the management of catastrophic nuclear accidents
like Chernobyl or Fukushima. Additional studies are necessary to
determine whether PHD inhibitors administered shortly after irra-
diation would still be radioprotective.
HSC Mobilization
An important proportion of cancer patients fail to mobilize suffi-
cient numbers of HSCs in response to G-CSF precluding subsequent
autologous HSC transplantation. Plerixafor, a small inhibitor of the
chemokine receptor CXCR4, used for 4 days in combination with
G-CSF enables this minimal threshold to be reached in up to 60%
patients who previously failed to mobilize in response to G-CSF
alone; however, the remainder of the 40% of patients who failed
to mobilize in response to G-CSF alone still fail to mobilize ade-
quately with the combination of G-CSF plus plerixafor.
As mentioned previously, the endosteal region of the BM is
thought to be hypoxic because of binding of pimonidazole and
HIF-1aprotein expression [14, 15] in steady state; however, at
the peak of mobilization induced by G-CSF, the entire BM cavity
becomeshypoxic becauseof extensive myeloid progenitor prolifer-
ation, which leads to enhanced O
2
consumption without a detect-
able increase in blood supply [15, 38]. Coadministration of PHD
inhibitor FG-4497 with G-CSF further enhanced HIF-1aprotein sta-
bilization in mouse BM. Administration of FG-4497 in combination
with G-CSF and plerixafor increased 20-fold the number of long-
term repopulating HSCs mobilized into the peripheral blood [56].
This highlights the importance of HIF-1ain HSC mobilization and
provides a novel therapeutic strategy for increasing HSC mobiliza-
tion above levels obtained with combinations of G-CSF and plerix-
afor.Thus, PHD inhibitorscould be usefulagents in patients whostill
fail to mobilize in response to G-CSF and plerixafor.
CONCLUSION
BloodperfusionandhypoxiaviaHIF-1aare key regulators of HSCs in
their BM niches. Similarly, the hypoxia-sensing pathway via HIF-2ais
a key regulator of EPO production by the kidney and thus of eryth-
ropoiesis from HSCs. Consequently, the recent discovery of small
molecules that stabilize HIFs independently of tissue oxygenation
opens the possibility of pharmacological interventions in the hema-
topoietic system, particularly to treat anemia, to enhance HSC mo-
bilization for transplantation, and to increase HSC radioresistance.
HIFs also play a critical role in inflammation [57], expansion
of myeloid-derived suppressor cells [58], and differentiation
of antigen-presenting cells [59, 60]; therefore, PHD inhibitors
Table 1. Clinical trials using prolyl hydroxylase domain inhibitors registered on ClinicalTrials.gov
Company Drug Condition Status Phase
Akebia Therapeutics AKB-6548 Anemia caused by chronic
kidney disease
2 completed, 1 recruiting II
Bayer BAY85-3934 Anemia caused by chronic
kidney disease
3 completed I
FibroGen FG-2216 Renal anemia 1 completed II
FibroGen FG-4592 Anemia caused by chronic
kidney disease
7 completed, 2 active, 3
recruiting
II
GlaxoSmithKline GSK1278863 Ischemia reduction during
elective surgery of aortic
aneurysm
1 not yet recruiting II
GlaxoSmithKline GSK1278863 Anemia caused by chronic
kidney disease
2 completed II
138 HIF-Stabilizing Therapies in Hematology
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may be useful to treat immune diseases or to modulate immune
responses, particularly in the context of allogeneic transplantations.
ACKNOWLEDGMENTS
This work was supported by a project grant (604303) from the Na-
tional Health and Medical Research Council of Australia. J.-P.L.
is supported by a senior research fellowship (APP1044091) and
C.E.F is supported by a project grant (APP1046590) from the Na-
tional Health and Medical Research Council of Australia.
AUTHOR CONTRIBUTIONS
C.E.F.: conception and design, manuscript writing and editing;
J.-P.L.: conception and design, financial support, manuscript writ-
ing and editing, final approval of the manuscript.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
REFERENCES
1Webb JD, Coleman ML, Pugh CW. Hypoxia,
hypoxia-inducible factors (HIF), HIF hydroxy-
lases and oxygen sensing. Cell Mol Life Sci
2009;66:3539–3554.
2Covello KL, Simon MC. HIFs, hypoxia, and
vascular development. In: Gerald PS, ed. Cur-
rent Topics in Developmental Biology. Wal-
tham, MA: Academic Press, 2004:37–54.
3Jiang BH, Semenza GL, Bauer C et al.
Hypoxia-inducible factor 1 levels vary exponen-
tially over a physiologically relevant range of O2
tension. Am J Physiol 1996;271:C1172–C1180.
4Bruick RK, McKnight SL. A conserved fam-
ily of prolyl-4-hydroxylases that modify HIF. Sci-
ence 2001;294:1337–1340.
5D’Angelo G, Duplan E, Boyer N et al. Hyp-
oxia up-regulates prolyl hydroxylase activity: A
feedback mechanism that limits HIF-1 responses
during reoxygenation. J Biol Chem 2003;278:
38183–38187.
6Jaakkola P, Mole DR, Tian YM et al. Target-
ing of HIF-alpha to the von Hippel-Lindau ubiq-
uitylation complex by O2-regulated prolyl
hydroxylation. Science 2001;292:468–472.
7Ivan M, Kondo K, Yang H et al. HIFalpha tar-
geted for VHL-mediated destruction by proline
hydroxylation: Implications for O2 sensing. Sci-
ence 2001;292:464–468.
8Bruegge K, Jelkmann W, Metzen E. Hy-
droxylation of hypoxia-inducible transcription
factors and chemical compounds targeting
the HIF-alpha hydroxylases. Curr Med Chem
2007;14:1853–1862.
9Takeda K, Aguila HL, Parikh NS et al. Regu-
lation of adult erythropoiesis by prolyl hydrox-
ylase domain proteins. Blood 2008;111:3229–
3235.
10 LaitalaA, Aro E, WalkinshawG et al. Trans-
membrane prolyl 4-hydroxylase is a fourthprolyl
4-hydroxylase regulating EPO production and
erythropoiesis. Blood 2012;120:3336–3344.
11 Oehme F, Ellinghaus P, Kolkhof P et al.
Overexpression of PH-4, a novel putative proline
4-hydroxylase, modulates activity of hypoxia-
inducible transcription factors. Biochem Biophys
Res Commun 2002;296:343–349.
12 Ram´
ırez-Bergeron DL, Runge A, Adelman
DM et al. HIF-dependent hematopoietic factors
regulate the development of the embryonic
vasculature. Dev Cell 2006;11:81–92.
13 Adelman DM, Maltepe E, Simon MC.
Multilineage embryonic hematopoiesis re-
quires hypoxic ARNT activity. Genes Dev 1999;
13:2478–2483.
14 Takubo K, Goda N, Yamada W et al. Reg-
ulation of the HIF-1alpha level is essential for
hematopoietic stem cells. Cell Stem Cell 2010;
7:391–402.
15 L´
evesque JP, Winkler IG, Hendy J et al.
Hematopoietic progenitor cell mobilization
results in hypoxia with increased hypoxia-
inducible transcription factor-1 alpha and
vascular endothelial growth factor A in bone
marrow. STEM CELLS 2007;25:1954–1965.
16 Forristal CE, Winkler IG, Nowlan B et al.
Pharmacologic stabilization of HIF-1aincreases
hematopoietic stem cell quiescence in vivo and
accelerates blood recovery after severe irradia-
tion. Blood 2013;121:759–769.
17 Park SK, Dadak AM, Haase VH et al.
Hypoxia-induced gene expression occurs solely
through the action of hypoxia-inducible factor
1alpha (HIF-1alpha): Role of cytoplasmic trap-
ping of HIF-2alpha. Mol Cell Biol 2003;23:
4959–4971.
18 Hara S, Hamada J, Kobayashi C et al.
Expression and characterization of hypoxia-
inducible factor (HIF)-3alpha in human kidney:
Suppression of HIF-mediated gene expression
by HIF-3alpha. Biochem Biophys Res Commun
2001;287:808–813.
19 Maynard MA, Evans AJ, Hosomi T et al.
Human HIF-3alpha4 is a dominant-negative reg-
ulator of HIF-1 and is down-regulated in renal
cell carcinoma. FASEB J 2005;19:1396–1406.
20 Eliasson P, Rehn M, Hammar P et al. Hyp-
oxia mediates low cell-cycle activity and increases
the proportion of long-term-reconstituting hema-
topoietic stem cells during in vitro culture. Exp
Hematol 2010;38:301–310.e2.
21 Suda T, Takubo K, Semenza GL. Metabolic
regulation of hematopoietic stem cells in the
hypoxic niche. Cell Stem Cell 2011;9:298–310.
22 Cipolleschi MG, Dello Sbarba P, Olivotto
M. The role of hypoxia in the maintenance of
hematopoietic stem cells. Blood 1993;82:2031–
2037.
23 Danet GH, Pan Y, Luongo JL et al. Expan-
sion of human SCID-repopulating cells under
hypoxic conditions. J Clin Invest 2003;112:
126–135.
24 Singh RP, Franke K, Kalucka J et al. HIF
prolyl hydroxylase 2 (PHD2) is a critical regula-
tor of hematopoietic stem cell maintenance
during steady-state and stress. Blood 2013;
121:5158–5166.
25 Bussolati B, Lauritano C, Moggio A et al.
Renal CD133(1)/CD73(1) progenitors produce
erythropoietin under hypoxia and prolyl hy-
droxylase inhibition. J Am Soc Nephrol 2013;
24:1234–1241.
26 Scortegagna M, Ding K, Zhang Q et al.
HIF-2alpha regulates murine hematopoietic
development in an erythropoietin-dependent
manner. Blood 2005;105:3133–3140.
27 Warnecke C, Zaborowska Z, Kurreck J
et al. Differentiating the functional role of
hypoxia-inducible factor (HIF)-1alpha and
HIF-2alpha (EPAS-1) by the use of RNA inter-
ference: Erythropoietin is a HIF-2alpha target
gene in Hep3B and Kelly cells. FASEB J 2004;
18:1462–1464.
28 Franke K, Kalucka J, Mamlouk S et al. HIF-1a
is a protective factor in conditional PHD2-deficient
mice suffering from severe HIF-2a-induced
excessive erythropoiesis. Blood 2013;121:1436–
1445.
29 Bigham A, Bauchet M, Pinto D et al. Iden-
tifying signatures of natural selection in Tibetan
and Andean populations using dense genome
scan data. PLoS Genet 2010;6:e1001116.
30 Bigham AW, Wilson MJ, Julian CG et al.
Andean and Tibetan patterns of adaptation to
high altitude. Am J Hum Biol 2013;25:190–197.
31 Branemark P-I. Experimental investiga-
tion of microcirculation in bone marrow. Angi-
ology 1961;12:293–305.
32 Hooper AT, Butler JM, Nolan DJ et al. En-
graftment and reconstitution of hematopoiesis
is dependent on VEGFR2-mediated regenera-
tion of sinusoidal endothelial cells. Cell Stem
Cell 2009;4:263–274.
33 Kopp HG, Avecilla ST, Hooper AT et al.
The bone marrow vascular niche: Home of
HSC differentiation and mobilization. Physiol-
ogy (Bethesda) 2005;20:349–356.
34 Kopp HG, Hooper AT, Avecilla ST et al.
Functional heterogeneity of the bone marrow
vascular niche. Ann N Y Acad Sci 2009;1176:
47–54.
35 Li XM, Hu Z, Jorgenson ML et al.High levels
of acetylated low-density lipoproteinuptake and
low tyrosine kinase with immunoglobulin and
epidermal growth factor homology domains-2
(Tie2) promoter activity distinguish sinusoids
from other vessel types i n murine bone marrow.
Circulation 2009;120:1910–1918.
36 Nombela-Arrieta C, Pivarnik G, Winkel B
et al. Quantitative imaging of haematopoietic
stem and progenitor cell localization and hyp-
oxic status in the bone marrow microenviron-
ment. Nat Cell Biol 2013;15:533–543.
37 Lassailly F, Foster K, Lopez-Onieva L et al.
Multimodal imaging reveals structural and
functional heterogeneity in different bone mar-
row compartments: Functional implications on
hematopoietic stem cells. Blood 2013;122:
1730–1740.
38 Winkler IG, Barbier V, Wadley R et al. Po-
sitioning of bone marrow hematopoietic and
stromal cells relative to blood flow in vivo: Seri-
ally reconstituting hematopoietic stem cells re-
side in distinct nonperfused niches. Blood 2010;
116:375–385.
39 Xie Y, Yin T, Wiegraebe W et al. Detec-
tion of functional haematopoietic stem cell
niche using real-time imaging. Nature 2009;
457:97–101.
Forristal, Levesque 139
www.StemCellsTM.com ©AlphaMed Press 2014
by guest on May 6, 2014http://stemcellstm.alphamedpress.org/Downloaded from
40 K¨
ohler A, Schmithorst V, Filippi MD et al.
Altered cellular dynamics and endosteal loca-
tion of aged early hematopoietic progenitor
cells revealed by time-lapse intravital imaging
in long bones. Blood 2009;114:290–298.
41 Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM
family receptors distinguish hematopoietic
stem and progenitor cells and reveal endothe-
lial niches for stem cells. Cell 2005;121:1109–
1121.
42 Sugiyama T, Kohara H, Noda M et al.
Maintenance of the hematopoietic stem cell
pool by CXCL12-CXCR4 chemokine signaling in
bone marrow stromal cell niches. Immunity
2006;25:977–988.
43 Baader E, Tschank G, Baringhaus KH et al.
Inhibition of prolyl 4-hydroxylase by oxalyl
amino acid derivatives in vitro, in isolated
microsomes and in embryonic chicken tissues.
Biochem J 1994;300:525–530.
44 Murray JK, Balan C, Allgeier AM et al.
Dipeptidyl-quinolone derivatives inhibit hyp-
oxia inducible factor-1aprolyl hydroxylases-1,
-2, and -3 with altered selectivity. J Comb Chem
2010;12:676–686.
45 Safran M, Kim WY, O’Connell F et al.
Mouse model for noninvasive imaging of HIF
prolyl hydroxylase activity: A ssessment of an oral
agent thatstimulates erythropoietin production.
Proc Natl Acad Sci USA 2006;103:105–110.
46 Fraisl P, Aragon´
es J, Carmeliet P. Inhibi-
tion of oxygen sensors as a therapeutic strategy
for ischaemic and inflammatory disease. Nat
Rev Drug Discov 2009;8:139–152.
47 Bernhardt WM, Wiesener MS, Scigalla P
et al. Inhibition of prolyl hydroxylases increases
erythropoietin production in ESRD. J Am Soc
Nephrol 2010;21:2151–2156.
48 Hsieh MM, Linde NS, Wynter A et al. HIF
prolyl hydroxylase inhibition results in endoge-
nous erythropoietin induction, erythrocytosis,
and modest fetal hemoglobin expression in rhe-
sus macaques. Blood 2007;110:2140–2147.
49 Bernhardt WM, Cˆ
ampean V, Kany S et al.
Preconditional activation of hypoxia-inducible
factors ameliorates ischemic acute renal failure.
J Am Soc Nephrol 2006;17:1970–1978.
50 Sorg H, Harder Y, Krueger C et al. The non-
hematopoietic effects of erythropoietin in skin
regeneration and repair: From basic research to
clinical use. Med Res Rev 2013;33:637–664.
51 AdamcioB,SperlingS,HagemeyerNetal.
Hypoxia inducible factor stabilization leads to last-
ing improvement of hippocampal memory in
healthy mice. Behav Brain Res 2010;208:80–84.
52 Marchbank T, Mahmood A, Harten S
et al. Dimethyloxalyglycine stimulates the early
stages of gastrointestinal repair processes
through VEGF-dependent mechanisms. Lab In-
vest 2011;91:1684–1694.
53 Robinson A, Keely S, Karhausen J et al.
Mucosal protection by hypoxia-inducible factor
prolyl hydroxylase inhibition. Gastroenterology
2008;134:145–155.
54 Beuck S, Sch¨
anzer W, Thevis M. Hypoxia-
inducible factor stabilizers and other small-
molecule erythropoiesis-stimulating agents in
current and preventive doping analysis. Drug
Test Anal 2012;4:830–845.
55 Boothman DA, Burrows HL, Yang CR et al.
Damage-sensing mechanisms in human cells af-
ter ionizing radiation. STEM CELLS 1997;15
(suppl 2):27–42.
56 Forristal CE, Nowlan B, Barbier V et al.
FG-4497, a pharmacological stabilizer of HIF-1
{alpha} protein, synergistically enhances hema-
topoietic stem cells (HSC) mobilization in re-
sponse to G-CSF and plerixafor. Blood 2012;
120:216a.
57 Cramer T, Yamanishi Y, Clausen BE et al.
HIF-1alpha is essential for myeloid cell-mediated
inflammation. Cell 2003;112:645–657.
58 Corzo CA, Condamine T, Lu L et al. HIF-
1aregulates function and differentiation of
myeloid-derived suppressor cells in the tumor
microenvironment. J Exp Med 2010;207:2439–
2453.
59 Bosco MC, Pierobon D, Blengio F et al.
Hypoxia modulates the gene expression profile
of immunoregulatory receptors in human ma-
ture dendritic cells: Identification of TREM-1
as a novel hypoxic marker in vitro and in vivo.
Blood 2011;117:2625–2639.
60 Mancino A, Schioppa T, Larghi P et al. Di-
vergent effects of hypoxia on dendritic cell func-
tions. Blood 2008;112:3723–3734.
Watch for the next Pe rspectives, “The Decision on the ‘Optimal’Human Pluripotent Stem Cell”by
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140 HIF-Stabilizing Therapies in Hematology
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