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Pharmacologic stabilization of HIF-1 alpha increases hematopoietic stem cell quiescence in vivo and accelerates blood recovery after severe irradiation

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Unlabelled: Quiescent hematopoietic stem cells (HSCs) preferentially reside in poorly perfused niches that may be relatively hypoxic. Most of the cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIFs). To investigate the effects of hypoxia on HSCs, we blocked O2-dependent HIF-1α degradation in vivo in mice by injecting 2 structurally unrelated prolyl hydroxylase domain (PHD) enzyme inhibitors: dimethyloxalyl glycine and FG-4497. Injection of either of these 2 PHD inhibitors stabilized HIF-1α protein expression in the BM. In vivo stabilization of HIF-1a with these PHD inhibitors increased the proportion of phenotypic HSCs and immature hematopoietic progenitor cells in phase G0 of the cell cycle and decreased their proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. This effect was independent of erythropoietin, the expression of which was increased in response to PHD inhibitors. Finally, pretreatment of mice with a HIF-1α stabilizer before severe, sublethal 9.0-Gy irradiation improved blood recovery and enhanced 89-fold HSC survival in the BM of irradiated mice as measured in long-term competitive repopulation assays. The results of the present study demonstrate that the levels of HIF-1α protein can be manipulated pharmacologically in vivo to increase HSC quiescence and recovery from irradiation. Key points: HIF-1α protein stabilization increases HSC quiescence in vivo. HIF-1α protein stabilization increases HSC resistance to irradiation and accelerates recovery.
In vivo stabilization of HIF-1 ␣ increases the number of HSCs and HPCs in the BM. (A) Number of LKS ϩ 48-150 ϩ HSCs, LKS ϩ 48 ϩ lineage-restricted HPCs, and LKS- myeloid progenitors in mouse BM after a 6-day treatment with saline or FG-4497 in vivo. Data are shown as the means Ϯ SD from 6 mice per treatment group. (B) Number of total CFCs and CFU-GEMM after a 6-day treatment with saline or FG-4497 in vivo. Data are shown as the means Ϯ SD from 6 mice per treatment group. (C) Competitive repopulation assay after treatment with DMOG or saline for 18 days. BM cells from 10 CD45.2 ϩ donor mice per treatment group were pooled within each treatment group. A total of 200 000 CD45.2 ϩ BM cells from each treatment group were transplanted with 200 000 competitive whole BM cells from untreated congenic B6.SJL CD45.1 ϩ mice into 9 lethally irradiated CD45.1 ϩ recipients. CD45.2 ϩ donor contribution was measured in the blood 16 weeks after transplantation in total CD45 ϩ leukocytes, CD11b ϩ myeloid cells, B220 ϩ B cells, and CD3 ϩ T cells by flow cytometry (all recipients showed multilineage chimerism with over 0.5% donor CD45.2 ϩ contribution in each lineage). Each dot represents an individual recipient; the bar represents the average. (D) Percentage of CD45.2 ϩ donor leukocytes in the blood at 8, 12, and 16 weeks after transplantation. Each line represents an individual mouse (black lines are DMOG-treated donors; gray lines are saline-treated donors). (E) Number of RUs/femur from donor chimerism at 16 weeks after transplantation. Data are shown as the means Ϯ SD from 9 mice per group. Significance levels were calculated using a t test (A-B) or Mann-Whitney test (C,E).
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doi:10.1182/blood-2012-02-408419
Prepublished online December 14, 2012;
2013 121: 759-769
Jean-Pierre Levesque
Catherine E. Forristal, Ingrid G. Winkler, Bianca Nowlan, Valerie Barbier, Gail Walkinshaw and
irradiation
quiescence in vivo and accelerates blood recovery after severe
increases hematopoietic stem cellαPharmacologic stabilization of HIF-1
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by the American Society of Hematology, 2021 L St, NW, Suite 900,
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly
For personal use only. at UQ Library on January 31, 2013. bloodjournal.hematologylibrary.orgFrom
Regular Article
HEMATOPOIESIS AND STEM CELLS
Pharmacologic stabilization of HIF-1 incr eases hematopoietic stem cell
quiescence in vivo and accelerates blood r ecovery after sever e irradiation
Catherine E. Forristal,
1
Ingrid G. Winkler,
2
Bianca Nowlan,
1
Valerie Barbier,
2
Gail Walkinshaw,
3
and Jean-Pierre Levesque
1,4
1
Stem Cell Biology Group and
2
Stem Cells and Cancer Group, Mater Medical Research Institute, South Brisbane, Australia;
3
Fibrogen Inc, San Francisco, CA;
and
4
School of Medicine, University of Queensland, Brisbane, Australia
Key Points
HIF-1 protein stabilization
increases HSC quiescence
in vivo.
HIF-1 protein stabilization
increases HSC resistance to
irradiation and accelerates
recovery.
Quiescent hematopoietic stem cells (HSCs) preferentially reside in poorly perfused
niches that may be relatively hypoxic. Most of the cellular effects of hypoxia are
mediated by O
2
-labile hypoxia-inducible transcription factors (HIFs). To investigate the
effects of hypoxia on HSCs, we blocked O
2
-dependent HIF-1 degradation in vivo in
mice by injecting 2 structurally unrelated prolyl hydroxylase domain (PHD) enzyme
inhibitors: dimethyloxalyl glycine and FG-4497. Injection of either of these 2 PHD
inhibitors stabilized HIF-1 protein expression in the BM. In vivo stabilization of HIF-1
with these PHD inhibitors increased the proportion of phenotypic HSCs and immature
hematopoietic progenitor cells in phase G
0
of the cell cycle and decreased their
proliferation as measured by 5-bromo-2-deoxyuridine incorporation. This effect was
independent of erythropoietin, the expression of which was increased in response to PHD inhibitors. Finally, pretreatment of mice
with a HIF-1 stabilizer before severe, sublethal 9.0-Gy irradiation improved blood recovery and enhanced 89-fold HSC survival in
the BM of irradiated mice as measured in long-term competitive repopulation assays. The results of the present study demonstrate
that the levels of HIF-1 protein can be manipulated pharmacologically in vivo to increase HSC quiescence and recovery from
irradiation. (Blood. 2013;121(5):759-769)
Introduction
To remain in an undifferentiated state, hematopoietic stem cells
(HSCs) need be lodged in specific niches of the BM where they can
preserve their essential capacity to self-renew and reconstitute the
whole hematopoietic and immune systems on transplantation.
1-2
In
mice
3
and humans,
4
the BM contains 2 pools of HSCs: (1) a
quiescent pool that divides very infrequently approximately every
145 days to self-renew and maintain a genetic reserve and (2) an
activated pool that divides more frequently for the daily replace-
ment of hematopoietic progenitor cells (HPCs), blood leukocytes,
erythrocytes, and platelets. Molecular components of HSC niches
are critical to maintaining the correct balance among quiescence,
self-renewing proliferation, and differentiation of HSCs. It has
been found recently that, in addition to the stromal cells forming
niches and the arrays of essential mediators they secrete, the
physicochemical conditions within niches are critical to maintain-
ing HSC quiescence and self-renewal.
5
For example, the most
quiescent HSCs capable of serial reconstitution in serial transplan-
tations reside in niches very poorly perfused by the circulating
blood, whereas more active and proliferative HSCs capable of only
a single round of transplantation or reconstitution reside in more
perfused niches.
6
A direct consequence of low perfusion could be
increased local hypoxia. Indeed, the oxygenation rate of a tissue is
dependent on how rapidly oxygen solubilized in the circulating
blood perfuses into the tissue and how rapidly this oxygen is
consumed by cells in an active metabolic state.
7-8
Similar to the
BM, solid tumors are sites of rapid regeneration and cell division.
Analyses of blood perfusion and hypoxia in solid tumors have
shown that areas that are poorly perfused are stained by the hypoxia
sensitive marker pimonidazole, suggesting a hypoxic state,
9
and
contain tumor stem cells.
10-11
Similarly, the endosteal region of the
mouse BM, which is known to harbor niches containing quiescent
HSCs,
3,12-15
is also stained by pimonidazole in steady-state condi-
tions, also suggesting a hypoxic state.
16-17
A functional consequence of tissue hypoxia is the stabilization
of a family of oxygen-labile transcription factors called hypoxia-
inducing factors (HIFs). HIFs are heterodimers of an O
2
-labile
-subunit, and a stable -subunit called the aryl hydrocarbon
receptor nuclear translocator (ARNT). Once the HIF-:ARNT
complex is formed, it can then translocate to the nucleus and
activate the transcription of genes containing hypoxia-responsive
elements. Hematopoietic cells, including HSCs, express HIF-1
mRNA.
18
In hypoxic conditions with an oxygen concentration
below 2%, the HIF- protein is stable and the complex with ARNT
is formed, translocates to the nucleus, and initiates transcription of
hypoxia-responsive element–containing genes. In normoxic condi-
tions or when O
2
concentration exceeds 2%, the HIF-1 protein is
degraded within 5 minutes of formation by the proteasome,
19
Submitted February 1, 2012; accepted November 28, 2012. Prepublished
online as Blood First Edition paper, December 14, 2012; DOI 10.1182/blood-
2012-02-408419.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2013 by The American Society of Hematology
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preventing the formation of the transcription factor and its translo-
cation to the nucleus (Figure 7). The sensitization of HIF- proteins
to proteasomal degradation in the presence of O
2
is mediated by
3 prolyl hydroxylase domain (PHD) enzymes that hydroxylate
2 proline residues within the oxygen degradation domain of HIF-
proteins. These hydroxylated proline residues then bind the von
Hippel Lindau (VHL) tumor-suppressor protein to form an
E3 ubiquitin ligase complex that ubiquinates and targets HIF-
protein to the proteasome.
20
PHD enzymes are Fe(II)–dependent
and use 2-oxoglutarate as a substrate to hydroxylate proline
residues. They are inactive when O
2
is 2%, resulting in HIF-
protein stabilization.
In the present study, we treated mice with 2 structurally
unrelated PHD inhibitors, dimethyloxalylglycine (DMOG)
21
and
FG-4497,
22
to determine the effect of pharmacologic stabilization
of the HIF-1 protein on HSCs in vivo. We report herein that both
agents stabilize HIF-1 protein in vivo in the BM and increase the
proportion of HSCs and primitive HPCs into quiescence. As a
result, treatment with PHD inhibitors resulted in faster hematopoi-
etic recovery and recovery of higher numbers of transplantable BM
HSCs after full-body sublethal irradiation.
Methods
Mouse treatments
All procedures were approved by the Animal Experimentation Ethics
Committee of the University of Queensland. Seven- to 9-week-old C57BL/6
and 129Sv male mice were injected intraperitoneally daily with 400 mg/kg
of DMOG diluted in saline, 20 mg/kg of FG-4497 (at 10 mg/mL in
5% dextrose and 30mM NaOH), or saline. At specified time points, mice
were anesthetized with isoflurane before cardiac puncture to harvest blood.
Mice were then euthanized by cervical dislocation and the BM and bones
harvested. In some experiments, mice were injected subcutaneously with
1000 IU/kg of recombinant human erythropoietin (EPO; Eprex
2000; Janssen-Cilag) daily.
Western blotting
For quantification of the HIF-1 protein by Western blotting, C57BL/6
mice were injected with a single dose of 400 mg/kg of DMOG, 20 mg/kg of
FG-4497, or saline. BM cells from one femur were flushed at 2, 6, and
12 hours after injection with 1 mL of urea cell lysis buffer (7M urea,
10% glycerol, 1% SDS, 5mM EDTA, and 20mM Tris-HCl, pH 6.8)
containing complete protease inhibitors (Roche) supplemented with 200M
PMSF. These cell lysates were frozen immediately on dry ice and stored at
80°C. For each time point, equal concentrations of protein were mixed
with 5 loading buffer containing 10mM dithiothreitol and boiled for
3 minutes. Proteins were separated on an 8% SDS-PAGE gel and
transferred onto a nitrocellulose membrane. Membranes were blocked in
PBS containing 0.1% Tween-20 (PBST) and 5% nonfat powdered milk for
1 hour at room temperature. After washing with PBST, membranes were
incubated overnight with rabbit anti–mouse HIF-1 Ab (NB100-134;
Novus Biologicals) diluted 1/500, and then washed with PBST and
incubated with IRD800-conjugated donkey F(ab)2 fragment anti–rabbit
IgG (Rockland Immunochemicals) at a 1/10 000 dilution. For normaliza-
tion of the results, blots were then probed with a rabbit anti–-actin loading
control Ab (Novus Biologicals). Protein expression was detected and
quantified on the Odyssey Infra-Red Imaging System (Li-COR Bioscience)
equipped with 2 solid-phase lasers at 700 and 800 nm.
23
HIF-1 flow cytometry staining
C57BL/6 mice were injected twice with 20 mg/kg of FG-4497 or saline
12 and 2 hours before euthanasia. After cervical dislocation, one femur was
flushed into ice-cold PBS with 2% FCS and 20 mg/mL of FG-4497.
BM cells were stained with FITC-lineage antibodies (CD3, CD5,
B220, CD11b, Gr1, CD41, and Ter119), anti–Sca1-PECy7, anti–Kit-
PacBlue, CD48-PerCPCy5.5, and CD150-PE (supplemental Table 1, avail-
able on the Blood Web site; see the Supplemental Materials link at the top of
the online article). Washed cells were then fixed using the FIX & PERM kit
from Caltag Invitrogen and then permeabilized with 0.05% saponin.
Dylight 650-conjugated mouse anti–human HIF-1 (NB 100-134C; Novus
Biologicals) was added to the permeabilization buffer at a 1/400 dilution for
30 minutes before washing. Fluorescence was analyzed on a CyAn ADP7c
flow cytometer.
BrdU incorporation and cell-cycle analyses
C57BL/6 or 129Sv mice were injected with 400 mg/kg/d of DMOG,
20 mg/kg/d of FG-4497, 1000 IU/kg of EPO, or saline. For the last 3 days of
the experiment, mice were administered 5-bromo-2-deoxyuridine (BrdU)
in the drinking water (0.5 mg/mL). After cervical dislocation, femora,
tibiae, pelvic bones, and spines were removed and crushed in a mortar/
pestle containing ice-cold PBS with 2% FCS and stained for cell-surface
antigens and then for BrdU or Ki67 antigen and DNA content as described
previously.
24
Briefly, BM leukocytes were enriched for Kit
cells by MACS
using mouse CD117 microbeads (Miltenyi Biotec). For BrdU staining,
enriched Kit
cells were stained with biotinylated lineage antibodies
(CD3, CD5, B220, CD11b, Gr-1, CD41, and Ter119), anti–Sca1-PECy7,
anti–Kit-APC, CD48-PacBlue, and CD150-PE, followed by streptavidin/
Alexa Fluor 700 (supplemental Table 1). Cells were washed with PBS, and
then fixed and stained for BrdU incorporation using reagents and instruc-
tions from the BrdU-FITC flow kit from BD Pharmingen.
For cell-cycle analyses, enriched Kit
cells were surface stained with
biotinylated lineage antibodies, anti–Sca-1-PECY7, anti–KIT-APC, and
CD48-PE followed by streptavidin/Alexa Fluor 700 (supplemental Table
1). Washed cells were then fixed and permeabilized using the FIX & PERM
kit. FITC-conjugated mouse anti–human Ki67 was added to the permeabili-
zation buffer for 30 minutes. After washing, cells were incubated in 1 mL of
PBS containing 1 g/mL of RNase A, 0.05% saponin, and 20M Hoechst
33342 for 15 minutes before analysis. Data were acquired on an LSRII flow
cytometer (BD Biosciences).
Colony assays
For myeloid colony assays, 10 L of whole blood or leukocyte suspension
from BM or spleen was deposited in 35-mm Petri dishes and covered with
1 mL of IMDM supplemented with 16% methylcellulose and 35% FCS.
Optimal concentrations of mouse IL-3, IL-6, and soluble kit ligand were
added as conditioned media from stably transfected BHK cell lines.
Colonies were counted after 7 days of culture.
Sublethal irradiation
After treatment with DMOG, FG-4497, or saline, C57BL/6 mice were
administered a split dose of 9.0 Gy of irradiation (2 4.5 Gy 4 hours apart).
Mice were given Bactrim and Diflucan antibiotics in their drinking water
for 2 weeks after irradiation. Recovery was monitored by weekly tail bleeds
in which blood leukocytes, RBCs, platelets, and hemoglobin levels were
measured. Remaining blood was then lysed and stained with CD3-FITC,
B220-APCCy7, CD11b-PECy7, anti–Ly6G-PE, anti–F4/80-Alexa Fluor
647, and anti–Ly6C-PacBlue.
Long-term competitive repopulation transplantations
Our long-term competitive repopulation transplantation protocol has been
described previously.
25
Briefly, on the day of transplantation, one femur per
donor mouse was gently crushed in PBS with 2% FCS with a mortar and
pestle. BM cells from all mice within each treatment group were pooled.
A total of 200 000 BM cell aliquots were taken from this pool and mixed
with 200 000 competitive whole BM cells from untreated congenic B6.SJL
CD45.1
mice in a total volume of 200 L and injected retroorbitally into
each lethally irradiated recipient (11.0 Gy split dose 4 hours apart).
Recipients were maintained with antibiotics for the first 3 weeks after
transplantation and then tail bled 8, 12, and 16 weeks after transplantation to
760 FORRISTAL et al BLOOD, 31 JANUARY 2013
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determine CD45.2 (test donor mobilized BM contribution) versus CD45.1
(competitive BM contribution) expression on myeloid, B-, and T-lineages
by flow cytometry.
25
Chimerism was considered positive when the contribu
-
tion of CD45.2
donor cells was above 0.5% for each blood lineage.
Content in repopulation units (RUs) was calculated for each individual
recipient mouse according to CD45.2
donor blood chimerism at 16 weeks
after transplantation using the following formula: RU D C/(100 D),
where D is the percentage of donor CD45.2
B cells and myeloid cells, and
C is the number of competing CD45.1
BM repopulation units. In most
cases, C 2 because 2 10
5
competing BM cells were cotransplanted (a
RU is defined as the HSC content in 10
5
BM cells
26
).
Statistics
Differences were analyzed using a 2-tailed t test or a nonparametric
Mann-Whitney test depending on distribution normality. A value of
P .05 was considered significant. Data are presented as means SD.
Results
PHD inhibition stabilizes HIF-1 protein in HSCs in the BM
Because HIF-1 mRNA is known to be expressed ubiquitously, we
first tested HIF-1 protein expression in mouse BM. The HIF-1
protein was very low in the BM from saline-injected animals
(Figure 1A far right lane), which is consistent with our previous
findings.
16
A single dose of the PHD inhibitor DMOG stabilized
HIF-1 protein for up to 6 hours in BM leukocytes before
approaching background levels at 12 hours. With a single dose of
FG-4497, a more potent and selective PHD inhibitor, HIF-1
protein persisted over 12 hours in BM leukocytes. We next
investigated whether treatment with the PHD inhibitor was effec-
tive in stabilizing HIF-1 in HSCs, multipotent progenitor cells,
Figure 1. PHD inhibitors stabilize HIF-1 in vivo. (A) Mice were injected with a single dose of 20 mg/kg of FG-4497, 400 mg/kg of DMOG, or saline and BM cells were
harvested at 2, 6, and 12 hours after injection. Cell lysates from 8 10
4
BM cells were run on a SDS-PAGE gel for each time point. After electro-transfer, membrane was blotted
with rabbit anti–HIF-1 and anti–-actin antibodies. (B-C) Mice were injected twice with saline or 20 mg/kg of FG-4497 at 12 and 2 hours prior to harvesting. (B) Typical plots
showing the gating strategy to measure intracellular HIF-1 in BM cells. Intracellular HIF-1 protein in HSPC populations was measured by flow cytometry (C). Data are shown
as means SD (4 mice per group) of the mean fluorescence intensities of HIF-1 fluorescence profiles in total BM leukocytes, LKS
CD48
lineage-restricted HPCs,
LKS
CD48
CD150
multipotent progenitors, and LKS
CD48
CD150
HSCs. Significance levels were calculated using a t test.
HIF-1 STABILIZATION INCREASES HSC QUIESCENCE 761BLOOD, 31 JANUARY 2013
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and myeloid progenitors. C57BL/6 mice were treated twice with
FG-4497 or saline 12 and 2 hours before killing and intracellular
HIF-1 protein was measured by flow cytometry (Figure 1B). The
geometric mean fluorescence intensity of HIF-1 staining was
significantly increased in whole BM leukocytes, Lin
Kit
Sca1
(LKS
) myeloid progenitors, lineage-restricted Lin
Sca1
Kit
CD48
(LKS
48
) HPCs, and Lin
Sca1
Kit
CD48
CD150
(LKS
48-150
) HSCs in FG-4497–treated mice (P .05; Figure
1C). This suggests that FG-4497 stabilizes in vivo a proportion of
cellular HIF-1 protein indiscriminately in all BM leukocytes,
including HSPCs.
PHD inhibition reduces HSC proliferation
Because dormant HSCs with the highest self-renewal capacity
reside in poorly perfused niches and are therefore presumably
hypoxic,
5,6
we next evaluated the effect of pharmacologic stabiliza
-
tion of the HIF-1 protein with a course of DMOG or FG-4497 on
HSC proliferation in vivo. 129Sv mice were administered
400 mg/kg/d of DMOG and HSC proliferation was measured by
BrdU incorporation for the last 3 days of the experiment. An 18-day
DMOG treatment halved the proportion of LKS
CD48
HSCs and
LKS
CD48
lineage-restricted HPCs that incorporated BrdU
(Figure 2A-B). Therefore, a significantly higher proportion of
HSPCs did not progress through the S phase during the last 3 days
of the experiment. Similarly, cell-cycle analysis showed that
DMOG treatment significantly increased the proportion of HSPCs
in the G
0
phase of the cell cycle (Figure 2C-D), with a reduction of
HSCs and HPCs in phase G
1
.
Treatment of C57BL/6 mice with 400 mg/kg/d of DMOG for
18 days also gave similar results, demonstrating that this effect was
not strain dependent (supplemental Figure 1). Furthermore, the
effect of pharmacologic stabilization of the HIF-1 protein was not
Figure 2. PHD inhibitors decrease HSPC proliferation in vivo. 129Sv mice were administered DMOG (black columns) or saline (white columns) daily for 18 days. BrdU was
administered in the drinking water for the last 3 days of the experiment. (A) Percentage of BrdU
cells. Data are shown as means SD from 5 mice per group. (B) Typical BrdU
incorporation flow cytometry profiles for saline-treated (gray) and DMOG-treated (black) mice in LKS
48
HSCs, LKS
48
and LKS
HPCs. (C) Cell-cycle analysis on
LKS
48
HPCs and LKS
48
HSCs from mice treated with DMOG or saline. (D) Representative dot plots showing the proportion of cells in each phase of the cell cycle after in
vivo treatment with saline or DMOG for 18 days. Data in histograms are means SD from 5 different mice per group. Significance levels were calculated using a t test.
762 FORRISTAL et al BLOOD, 31 JANUARY 2013
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limited to LKS
CD48
HSC and LKS
CD48
HPC phenotypes,
because it was also observed in LKS
Flt3
CD34
HSCs and
LKS
Flt3
HPCs (supplemental Figure 2). Therefore, pharmaco
-
logic stabilization of the HIF-1 protein forces HSCs into quies-
cence in vivo. Interestingly, the effect of HIF-1 protein stabiliza-
tion was not restricted to HSCs but was extended to lineage-
restricted HPCs. Shorter DMOG treatments (6-12 days) did not
alter HSPC cycling or BrdU incorporation (data not shown).
We confirmed the effects of DMOG by treating mice with
FG-4497, which significantly decreased BrdU incorporation and
significantly increased the proportion of HSPCs in the G
0
phase
after only 6 days of treatment (Figure 3), likely because of the more
lasting effect of FG-4497 on HIF-1 protein stabilization.
To eliminate the possibility that this effect was not mediated by
EPO, the production of which by the kidneys is enhanced in
response to these PHD inhibitors,
27
we injected a parallel cohort of
mice with EPO for 18 days. Despite a strong increase in erythro-
cytes and hemoglobin concentration in the blood, EPO had no
effect on HSPC cycling (supplemental Figure 3).
PHD inhibition enhances myeloid potential
Because PHD inhibition slowed HSC cycling in vivo, we next
investigated whether the frequency and number of HSCs and HPCs
were altered by PHD inhibitors. We found a significant increase in
both the frequency (supplemental Figure 4A) and number of
Figure 3. FG-4497 decreases HSPC proliferation in vivo. A total of 20 mg/kg of FG-4497 (black bars) or saline (white bars) was administered daily into C57BL/6 mice for
6 days. BrdU was administered in the drinking water for the last 3 days of the experiment. (A) Percentage of BrdU
cells. Data are show as the means SD from 5 mice per
group. (C) Typical BrdU incorporation profiles for saline-treated (gray) and FG-4497–treated (black) mice. (B,D) Cell-cycle analysis on LKS
48
HPCs and LKS
48
HSCs
from mice treated with FG-4497 or saline. Representative dot plots showing the proportion of cells in each phase of the cell cycle after in vivo treatment with FG-4497 or saline.
Data are shown as the means SD from 5 different mice per treatment group. Significance levels were calculated using a t test.
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LKS
CD48
CD150
HSCs (P .02), lineage-restricted HPCs
(LKS
48
; P .0002), and myeloid progenitors (LKS
; P .02)
after a 6-day treatment with FG-4497 compared with saline (Figure
4A). In addition, we found a significant increase in the number of
total CFCs and CFU-GMs (P .007) per femur when mice were
treated with FG-4497 compared with saline (Figure 4B).
We next measured the effect of PHD inhibition on BM
reconstitution potential by performing long-term competitive re-
population assays using 2 10
5
BM cells from DMOG-treated
mice in competition with 2 10
5
BM cells from untreated
congenic CD45.1
mice. HIF-1 stabilization significantly in
-
creased the CD45.2
donor chimerism after transplantation (Figure
4D) with a 2.5-fold increase in repopulating units per femur at
week 16 compared with saline-treated donors (P .01; Figure 4E).
However, this increase in chimerism and RUs/femur was essen-
tially because of higher myeloid reconstitution with the BM from
DMOG-treated donors (Figure 4C).
PHD inhibition accelerates blood recovery and protects HSCs
after severe irradiation
Because PHD inhibition slows HSPC cycling in vivo, we next
investigated whether this could protect HSPCs from severe sub-
lethal irradiation. Mice were treated with 400 mg/kg of DMOG or
saline for 18 days and then irradiated with 9.0 Gy (Figure 5A). Both
cohorts were leukopenic ( 1500 leukocytes/L) between days
7 and 14 after irradiation. However, DMOG-treated mice showed
significantly accelerated recovery (Figure 5B-F). Using the clinically
Figure 4. In vivo stabilization of HIF-1 increases the number of HSCs and HPCs in the BM. (A) Number of LKS
48-150
HSCs, LKS
48
lineage-restricted HPCs, and
LKS- myeloid progenitors in mouse BM after a 6-day treatment with saline or FG-4497 in vivo. Data are shown as the means SD from 6 mice per treatment group. (B) Number
of total CFCs and CFU-GEMM after a 6-day treatment with saline or FG-4497 in vivo. Data are shown as the means SD from 6 mice per treatment group. (C) Competitive
repopulation assay after treatment with DMOG or saline for 18 days. BM cells from 10 CD45.2
donor mice per treatment group were pooled within each treatment group.
A total of 200 000 CD45.2
BM cells from each treatment group were transplanted with 200 000 competitive whole BM cells from untreated congenic B6.SJL CD45.1
mice into
9 lethally irradiated CD45.1
recipients. CD45.2
donor contribution was measured in the blood 16 weeks after transplantation in total CD45
leukocytes, CD11b
myeloid
cells, B220
B cells, and CD3
T cells by flow cytometry (all recipients showed multilineage chimerism with over 0.5% donor CD45.2
contribution in each lineage). Each dot
represents an individual recipient; the bar represents the average. (D) Percentage of CD45.2
donor leukocytes in the blood at 8, 12, and 16 weeks after transplantation. Each
line represents an individual mouse (black lines are DMOG-treated donors; gray lines are saline-treated donors). (E) Number of RUs/femur from donor chimerism at 16 weeks
after transplantation. Data are shown as the means SD from 9 mice per group. Significance levels were calculated using a t test (A-B) or Mann-Whitney test (C,E).
764 FORRISTAL et al BLOOD, 31 JANUARY 2013
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relevant threshold of 10 granulocytes/L blood for severe neutrope-
nia, we found that all (12 of 12) saline-treated control mice became
severely neutropenic after irradiation, which was most severe at day 14
(Figure 5C). In contrast, only 1 of the 12 (8%) DMOG-pretreated mice
became severely neutropenic at any measured time point after irradia-
tion (P 10
4
by the Fisher exact statistic).
Saline-treated mice also became severely anemic ( 70 g/L of
hemoglobin and 4 10
6
erythrocytes/L) between days 14 and
22 after irradiation, whereas DMOG-treated mice showed only
moderate anemia (70-100 g/L of hemoglobin; Figure 5D-E).
Finally, saline-treated mice were thrombocytopenic ( 20 platelets/
L) between days 14 and 21, whereas DMOG-treated mice showed
significantly faster platelet recovery and shorter thrombocytopenia
(Figure 5F).
By day 50, mice from both groups had recovered to pre-
irradiation levels of RBCs, hemoglobin, granulocytes, and plate-
lets. To investigate whether DMOG pretreatment also promoted
long-term HSC survival or the ability to recover, BM was harvested
from these mice 50 days after irradiation. We found that DMOG
treatment induced a significant 2-fold increase in the frequencies
(supplemental Figure 4B) and numbers of phenotypic HSCs,
HPCs, and myeloid progenitors compared with control mice
(Figure 6A) and a significant 2-fold increase in the number of CFCs
and CFU-GMs per femur (Figure 6B).
This increase in the number of phenotypic HSCs after treatment
with a PHD inhibitor was confirmed in a repeat experiment in
which we performed long-term competitive repopulation assays
with BM cells. HIF-1 stabilization induced by the administration
of DMOG before irradiation increased long-term competitive
repopulation potential of the BM from irradiated mice (Figure 6D),
with a significant increase in myeloid and B-cell repopulation
potentials at 16 weeks after transplantation (Figure 6C). Indeed,
although only 2 of 9 recipients of irradiated CD45.2
BM from the
saline-treated group had multilineage chimerism (above 0.5% in
Figure 5. HIF-1 stabilization enhances blood recovery after severe sublethal irradiation. (A) Timeline of DMOG/saline administration, irradiation, and follow-up during
recovery. Time course of blood leukocytes (B), granulocytes (C; measured by flow cytometry on CD11b
Ly6-G
cells), erythrocytes (D), hemoglobin (E), and platelets (F) after
9.0 Gy of irradiation of C57BL/6 mice pretreated with saline or DMOG for 18 days. Dashed lines show levels of leukopenia, neutropenia, anemia, and thrombocytopenia. Data
are shown as the means SD from 12 different mice per treatment group. Significance levels were calculated using a t test.
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myeloid, B-, and T-lineages), multilineage chimerism increased to
7 of 9 recipients when irradiated BM from the DMOG-treated
group was transplanted (P .03 by Fisher exact test). Based on
chimerism 16 weeks after transplantation, the number of RUs/
femur in irradiated mice pretreated with DMOG was 89-fold higher
compared with irradiated mice pretreated with saline (Figure 6E).
Figure 6. HSPC radiation resistance increased by HIF-1 protein stabilization in vivo. Mice were treated with saline or DMOG for 18 days, irradiated with 9.0 Gy, and the
BM harvested 7 weeks later (see timeline in Figure 5A). Shown are the numbers of LKS
48
HSCs, LKS
48
lineage-restricted HPCs, and LKS
myeloid progenitors
(A) and total CFCs and CFU-GMs (B) in mouse BM 50 days after 9.0 Gy of irradiation. Data are shown as the means SD from 6 mice per group. (C) Competitive repopulation
assay at day 36 (week 5) after 9.0Gy irradiation following a pre-treatment with DMOG or saline for 18 days (see timeline in Figure 5A). BM cells from 10 CD45.2
donor mice per
treatment group were pooled within each treatment group. A total of 200 000 CD45.2
BM cells from each treatment group were transplanted with 200 000 competitive whole
BM cells from untreated congenic B6.SJL CD45.1
mice into 9 lethally irradiated CD45.1
recipients. (C) Percentages of CD45.2
donor contribution in total CD45
leukocytes, CD11b
myeloid cells, B220
B cells, and CD3
T cells 16 weeks after transplantation. Data are shown as the means SD from 9 mice per group. Each symbol
represents an individual recipient mouse, bars are averages, dotted lines represent the 0.5% CD45.2
threshold above which chimerism was considered to be positive.
(D) Percentage of CD45.2
donor leukocytes in the blood at 8, 12, and 16 weeks after transplantation. Each line represents an individual mouse (black lines are DMOG-treated
donors; gray lines are saline-treated donors). (E) Number of RUs/femur from donor chimerism at 16 weeks after transplantation. (F) Comparison of RUs/femur in mice treated
with DMOG or saline before (Figure 4C) and 50 days after (Figure 6C) 9.0 Gy of irradiation. Data are shown as the means SD from 9 mice per group. Significance levels were
calculated using a t test (A-B) or a Mann-Whitney test (C,E,F). NS indicates not significant.
766 FORRISTAL et al BLOOD, 31 JANUARY 2013
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Plots of RUs/femur in DMOG- and saline-pretreated mice before
and after irradiation showed that whereas the number of RUs/femur
was reduced 27-fold in the saline-treated group 50 days after
irradiation, irradiation did not decrease the number of RUs/femur
in DMOG-pretreated mice (Figure 6F). These results suggest that
pretreatment with DMOG enhanced HSPC survival to severe
sublethal irradiation, enabling faster recovery of the irradiation-
induced neutropenia, anemia, and thrombocytopenia and mainte-
nance of functional HSCs within the irradiated BM.
Discussion
This is the first report on the in vivo effects of pharmacologic
stabilization of the HIF-1 protein on hematopoiesis in wild-type
mice. Two structurally unrelated PHD inhibitors, DMOG and
FG-9947, efficiently stabilized the HIF-1 protein in the BM
independently of O
2
concentration. HIF-1 stabilization occurred
in all BM leukocytes, including HPCs and HSCs. This resulted in a
significantly higher proportion of HSCs and multipotent HPCs in
quiescence, leading to decreased HSPC proliferation. This effect
was not mediated by EPO, the production of which by the kidneys
is enhanced in response to these PHD inhibitors.
27
In vivo
treatment with PHD inhibitors also increased myeloid reconstitu-
tion of transplanted BM cells. Finally, pharmacologic stabilization
of HIF-1 protein before severe 9.0-Gy sublethal irradiation
enhanced recovery from irradiation-induced leucopenia, anemia,
and thrombocytopenia, resulting in an 89-fold higher recovery of
RUs/femur.
Our results show that treatment with DMOG in steady-state
conditions enhanced myeloid reconstitution of the BM in a
long-term competitive repopulation transplantation assay and in-
creased both the numbers and frequencies of phenotypic myeloid
progenitors and HSPCs. However, because we did not perform
transplantations with serial dilutions of BM cells, we cannot
confirm whether the number of actual reconstituting HSCs was
increased by DMOG treatment alone despite the increase in
frequency and number of phenotypic LKS
CD48
CD150
cells in
the BM of DMOG-treated mice. Nevertheless, DMOG pretreat-
ment before 9.0 Gy of irradiation very clearly protected HSCs from
irradiation, with multilineage reconstitution potential of the irradi-
ated BM increasing to levels comparable to multilineage reconstitu-
tion of naïve, nonirradiated BM. This result clearly suggests that
DMOG pretreatment protects long-term reconstituting HSCs from
severe sublethal irradiation.
Because PHD inhibitors stabilized the HIF-1 protein in all BM
cells in vivo, including HSPCs, our data do not distinguish whether
their effect on HSPC cycling is HSPC intrinsic or if it is indirectly
mediated by the microenvironment. However, conditional deletion
of the Hif1a gene in hematopoietic cells
18
or the effect of hypoxia
and PHD inhibitors on cultures of sorted HSPCs
28
suggest that the
in vivo effect of PHD inhibitors may be intrinsic to HSPCs. Indeed,
sorted LKS
Flt3
CD34
HSCs cultured in hypoxic conditions
accumulate in phase G
0
because of the accumulation of the
cyclin-dependent kinase inhibitors p21
cip
, p27
Kip1
, and p57
Kip2
in
response to HIF-1 protein stabilization
28
and reduced mitochon
-
drial oxidative glycolysis.
29
This is also consistent with the
decreased proportion of LKS
CD34
HSCs in phase G
0
observed
in vivo after inducible deletion of the Hif1a gene in hematopoietic
cells and, conversely, the increased proportion of HSCs in G
0
after
conditional deletion of the Vhl gene, which promotes the protea-
somal degradation of HIF- proteins when O
2
2%.
18
The
HSC-autonomous mechanisms by which hypoxia and HIF-1
increase HSPC quiescence may involve reduction of mitochondrial
activity, metabolic switch from mitochondrial oxidative glycolysis
to anaerobic glycolysis, reduced neogenesis of amino acids and
nucleotides from products of the tricarboxylic cycle, and inhibition
of the Akt/mTOR/S6 phosphorylation pathways.
29
Interestingly, the effect of stabilized HIF-1 protein on cycling
and proliferation was not restricted to HSCs, but was extended to
lineage-restricted LKS
CD48
HPCs and, to a lesser extent, to
myeloid progenitors. This is consistent with our observation that
pretreatment with DMOG lessens the impact of severe sublethal
irradiation on leukocytes, erythrocytes, and platelets and increases
the survival of HSPCs in the BM. Indeed, cells in phase G
0
are
more radiation resistant, with radiation sensitivity peaking in late
phase G
2
/M.
30
These data are also consistent with reports that
malignant cells cultured in hypoxic conditions become more
resistant to radiation.
31
In our experiments, stabilization of the
HIF-1 protein in vivo was sufficient to increase HSPC radiation
resistance. How hypoxia increases radioresistance is not fully
understood. Enhanced hypoxia may reduce the production of
deleterious reactive oxygen species that could further damage
DNA during irradiation.
31
Conversely, hypoxia has been reported
to activate the p53-dependent growth arrest and survival pathway
by attenuating p53 phosphorylation in a human colorectal carci-
noma cell line.
32
Whether such a mechanism takes place in HSPCs
in response to hypoxia or PHD inhibitors remains to be determined.
Hypoxia and HIF-1 may also affect directly the activity of the
ataxia telangiectasia mutated (ATM) pathway, which plays a
critical role in DNA repair in HSCs during aging
33
or after ionizing
radiation.
34-35
Indeed, HIF-1 is rapidly degraded even in hypoxic
conditions in cells with defective ATM kinase,
36
suggesting that
ATM is another critical regulator of HIF-1 protein stability.
Reciprocally, hypoxia activates ATM protein kinase by DNA
damage–independent mechanisms.
37-38
Hypoxia-activated ATM
kinase serine-phosphorylates HIF-1 protein, which stabilizes
HIF-1
37-38
and suppresses the catalytic activity of mammalian
target of rapamycin complex-1 (mTORC1).
37-38
Although it re
-
mains to be demonstrated, it may be that PDH inhibitors also
activate ATM kinase via HIF-1 stabilization and subsequently
attenuate p53 phosphorylation and mTORC1 activity in HSPCs
(Figure 7). This would enhance DNA repair and reduce prolifera-
tion by inhibiting activities of the mTORC1 targets S6 ribosomal
protein kinase and eukaryotic initiation factor 4E.
Finally, because Kit ligand expressed by endothelial cells in
vascular niches is critical to HSC survival and maintenance in the
BM,
39
our results must also be interpreted in the context of the
vascular niche. The perfusion of vascular HSC niches is likely to be
heterogeneous. BM sinusoids are flaccid without basal lamina and
sinusoidal blood flow is rhythmic, with a filling period of
1-2 minutes (during which blood flow slows and stops) followed by
a shorter emptying phase.
40
Consequently, corpuscular blood
velocity is low (0 to 0.2 mm/s) in these sinusoids, whereas it is
0.5-1.0 mm/s in capillaries feeding sinusoids in nutrients and
oxygen and 1.5 mm/s in more distant arterioles.
40
Therefore,
sinusoids more distant from arterioles could be more poorly
perfused than sinusoids more proximal to arterioles. Consequently,
hypoxia (which is inversely proportional to perfusion) and HIF-1
stabilization could be heterogeneous among BM vascular niches. In
this scenario, pharmacologic HIF-1 stabilization in HSCs located
in more perfused vascular niches could explain the increased
proportion of quiescent and radioresistant HSCs.
HIF-1 STABILIZATION INCREASES HSC QUIESCENCE 767BLOOD, 31 JANUARY 2013
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In conclusion, our data demonstrate that PHD and HIF-1 play
a critical role in regulating HSPC quiescence in vivo and that levels
of the HIF-1 protein can be pharmacologically manipulated with
PHD inhibitors to increase HSPC quiescence in vivo. We conclude
that the administration of PHD inhibitors may represent pharmaco-
logic tools to reduce HSPC proliferation and increase HSC
radiation resistance in vivo.
Acknowledgments
This work was supported by project grant 604303 from the
National Health and Medical Research Council of Australia. I.G.W.
is supported by a Career Development Fellowship from the
National Health and Medical Research Council (488817 and
APP1033736). During the course of this study, J.P.L. was sup-
ported by a Senior Research Fellowship from the Cancer Council
of Queensland.
Authorship
Contribution: C.E.F. planned and performed the experiments,
interpreted the results, and wrote and edited the manuscript; I.G.W.
planned and performed the experiments, interpreted the results, and
edited the manuscript; B.N. and V.B. performed the experiments
and analyzed the data; G.W. helped design the experiments with
FG-4497, provided background information, and edited the manu-
script; and J.-P.L. conceived and coordinated the study, planned and
performed the experiments, interpreted the results, and wrote and
edited the manuscript.
Conflict-of-interest disclosure: G.W. is an employee of and
owns equity in FibroGen Inc, which owns the commercial rights to
FG-4497. FibroGen did not provide any funding for this work. The
remaining authors declare no competing financial interests.
Correspondence: Jean-Pierre Levesque, Mater Medical Re-
search Institute, Level 4, 37 Kent Street, Woolloongabba 4102,
Australia; e-mail: jplevesque@mmri.mater.org.au.
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The inhibition of alpha-ketoglutarate (α-KG)-dependent dioxygenases is thought to contribute to isocitrate dehydrogenase (IDH) mutation-derived malignancy. Herein, we aim to thoroughly investigate the expression pattern and prognostic significance of genes encoding α-KG-dependent enzymes for lower-grade glioma (LGG) patients. In this retrospective study, a total of 775 LGG patients were enrolled. The generalized linear model, least absolute shrinkage and selection operator Cox regression, and nomogram were applied to identify the enzyme-based signature. With the use of gene set enrichment analysis and Gene Ontology, the probable molecular abnormalities underlying high-risk patients were investigated. By comprehensively analyzing mRNA data, we observed that 41 genes were differentially expressed between IDHMUT and IDHWT LGG patients. A risk signature comprising 10 genes, which could divide samples into high- and low-risk groups of distinct prognoses, was developed and independently validated. This enzyme-based signature was indicative of a more malignant phenotype. The nomogram model incorporating the risk signature, molecular biomarkers, and clinicopathological parameters proved the incremental utility of the α-KG-dependent signature by achieving a more accurate prediction impact. Our study demonstrates that the α-KG-dependent enzyme-encoding genes were differentially expressed in relation to the IDH phenotype and may serve as a promising indicator for clinical outcomes of LGG patients.
... The authors also found that DMOG-mediated radioprotection was achieved mainly through inhibition of PHD2 and upregulation of HIF-2 [21]. Forristal CE et al. reported similar results, that is, DMOG played radioprotective effects on the hematopoietic system through the PHD-HIF pathway [36]. Cummins EP et al. demonstrated that the upregulation of HIF with a PHD inhibitors could promote NF-κB translocation by activating IKK-β [37], while Greten FR et al. demonstrated that the activation of NF-κB in intestinal epithelial cells could inhibit their apoptosis [38]. ...
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Background Severe ionizing radiation (IR)-induced intestinal injury associates with high mortality, which is a worldwide problem requiring urgent attention. In recent years, studies have found that the PHD-HIF signaling pathway may play key roles in IR-induced intestinal injury, and we found that FG-4592, the PHD inhibitor, has significant radioprotective effects on IR-induced intestinal injury. Methods In the presence or absence of FG-4592 treatment, the survival time, pathology, cell viability, cell apoptosis, and organoids of mice after irradiation were compared, and the mechanism was verified after transcriptome sequencing. The data were analyzed using SPSS ver. 19 software. Results Our results show that FG-4592 had significant radioprotective effects on the intestine. FG-4592 improved the survival of irradiated mice, inhibited the radiation damage of intestinal tissue, promoted the regeneration of intestinal crypts after IR and reduced the apoptosis of intestinal crypt cells. Through organoid experiments, it is found that FG-4592 promoted the proliferation and differentiation of intestinal stem cells (ISCs). Moreover, the results of RNA sequencing and Western blot showed that FG-4592 significantly upregulated the TLR4 signaling pathway, and FG-4592 had no radioprotection on TLR4 KO mice, suggesting that FG-4592 may play protective role against IR by targeting TLR4. Conclusion Our work proves that FG-4592 may promote the proliferation and regeneration of ISCs through the targeted regulation of the TLR4 signaling pathway and ultimately play radioprotective roles in IR-induced injury. These results enrich the molecular mechanism of FG-4592 in protecting cells from IR-induced injury and provide new methods for the radioprotection of intestine.
... However, the role of VEGF signaling in the proliferation of hematopoietic stem cells and hematopoiesis is understudied. In line with our findings, a few other studies have shown that inhibition of Vegfr1 diminished HSC cell cycling and lineage differentiation after bone marrow suppression (81), and pharmacological stabilization of HIF-1a increases HSC quiescence (82). Our study demonstrates that the proliferation of HSPC and leukocytosis under hypoxic conditions is mediated by VEGFr1 although the data do not rule out the contribution of VEGFr2 in this process. ...
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... A l'inverse, une augmentation de la quiescence, via l'inhibition de CDK4/6, ou via la stabilisation de HIF1a, favorise la survie des CSH après une TBI (Forristal et al., 2013;Johnson et al., 2010). ...
Thesis
Avec plus d’un patient sur deux qui en bénéficie, la radiothérapie est l’une des méthodes les plus utilisées dans le traitement des cancers. Malgré son efficacité dans l’éradication des tumeurs, le principal inconvénient de cette technique est la toxicité qu’elle peut entrainer sur les tissus sains environnants. Au niveau de la moelle osseuse, qui contient les cellules souches hématopoïétiques (CSH), cette toxicité peut être très délétère. En effet, les CSH sont responsables de l’hématopoïèse tout au long de la vie d’un individu, d’où l’importance de les préserver. Ces cellules sont localisées dans un microenvironnement cellulaire, appelé niche hématopoïétique, jouant un rôle majeur dans leur protection et le maintien de leur intégrité. Au sein de cette niche, les macrophages résidents de la moelle osseuse, caractérisés par l’expression du marqueur de surface CD169, ont montré un rôle à la fois dans le maintien des CSH dans leur niche, mais aussi dans la protection des cellules vis-à-vis d’un stress oxydatif. Dans ce contexte, mon projet de thèse avait pour but de définir le rôle de ces macrophages résidents de la moelle osseuse dans la réponse des CSH à une irradiation corps entier (TBI) de 2 Gy, dose couramment utilisée en radiothérapie fractionnée. Afin de répondre à cette question, j’ai utilisé deux modèles de souris déplétées en macrophages CD169+ (Mϕ CD169+) : un modèle pharmacologique (clodronate-liposomes) et un modèle génétique (souris CD169DTR/+). Dans ces deux modèles, j’ai montré qu’une déplétion des Mϕ CD169+ avant l’irradiation entraine à court-terme une diminution moins importante du réservoir de CSH, accompagnée d’une diminution de leur apoptose et d’une absence d’espèces réactives de l’oxygène (ROS) généralement induites par l’irradiation. A plus long-terme, l’absence de Mϕ CD169+ permet un rétablissement complet d’un réservoir fonctionnel de CSH après irradiation. L’ensemble de ces résultats démontre que la présence des macrophages résidents lors d’une irradiation a un rôle délétère en diminuant la réserve de CSH au sein de la moelle osseuse. Afin de mettre en évidence le ou les mécanismes provoquant cet effet délétère, je me suis intéressée à la réponse directe des Mϕ CD169+ à l’irradiation, en ciblant particulièrement les phénomènes liés au stress oxydatif. J’ai observé que la TBI entraine une augmentation de la proportion de Mϕ CD169+ qui produisent de l’oxyde nitrique (NO), une des caractéristiques de la réponse de type pro-inflammatoire des Mϕ. Cette augmentation est corrélée à une augmentation des CSH ayant des peroxynitrites, oxydants extrêmement cytotoxiques issus de la réaction entre les NO et les ROS. L’utilisation de modulateurs négatifs ou positifs des NO a montré qu’après une TBI de 2 Gy, la diminution de la production de NO par les Mϕ CD169+ permet de limiter l’apoptose des CSH et de restaurer leur nombre, alors que l’augmentation de NO dans l’environnement médullaire entraîne une diminution de leur nombre. Cette étude démontre que l’irradiation accroit la production de NO par les macrophages résidents de la moelle, entrainant un effet nocif sur la réserve de CSH. Ces nouvelles données identifient le macrophage résident comme un candidat potentiel pour moduler les effets de l’irradiation sur les CSH.
... Fujimoto and his colleagues (Fujimoto et al., 2019) measured increasing microvessel density and quantified less epithelial apoptosis in roxadustat-treated K-ras; Trp53; Pdx-1-Cre(KPC) mice, a widely used transgenic pancreatic ductal adenocarcinoma model, which suggested that roxadustat is an effective remedy in improving the survival rate and maintaining intestinal function during radiation therapy. In addition, FG-4497 also functions to increase hematopoietic stem cell (HSC) quiescence and enhance HSC survival in the bone marrow of irradiated mice, as measured in long-term competitive repopulation assays, which efficiently alleviates marrow suppression (Forristal et al., 2013). ...
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