Experimental Hematology 2017;-:--

Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial

retention and associated ROS levels in the red blood cells of sickle cell disease
Ramasamy Jagadeeswarana,b, Benjamin A. Vazqueza, Muthusamy Thiruppathic, Balaji B. Ganeshd,
Vinzon Ibanezb,e, Shuaiying Cuif, James D. Engelg, Alan M. Diamondh, Robert E. Molokieb,e,
Joseph DeSimoneb,e, Donald Lavelleb,e, and Angela Riversa,b
a
Department of Pediatrics, University of Illinois at Chicago, Chicago, IL, USA; bJesse Brown Veterans Affairs Medical Center, Chicago, IL, USA;
c
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL, USA; dResearch Resources Center, University of
Illinois at Chicago, Chicago, IL, USA; eDepartment of Medicine, University of Illinois at Chicago, Chicago, IL, USA; fDepartment of Medicine,
Boston University School of Medicine, Boston, MA, USA; gDepartment of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI,
USA; hDepartment of Pathology, University of Illinois at Chicago, Chicago, IL, USA
(Received 3 January 2017; revised 6 February 2017; accepted 8 February 2017)

Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders
hemoglobin susceptible to polymerization when deoxygenated, affects millions of people
worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful
vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD
pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in
SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors elim-
inate their mitochondria during the terminal differentiation process. Strikingly, we observed
an increased percentage of RBCs retaining mitochondria in SCD patient blood samples
compared with healthy individuals. In addition, using an experimental SCD mouse model,
we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mito-
chondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing
agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan
and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the
SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1
showed increased expression of mitophagy genes. Our findings suggest that reduction of
mitochondria-retaining RBCs may provide a new therapeutic approach to preventing exces-
sive ROS in SCD. Copyright 2017 ISEH - International Society for Experimental Hema-
tology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Sickle cell disease (SCD) is an inherited blood disorder that
affects millions of people worldwide. The disease is caused
by a mutation of the b-globin gene that allows for the poly-
merization of sickle hemoglobin (HbS) when deoxygen-
ated. HbS polymerization is considered a main driving
force of hemolysis in SCD [1]. Increased expression levels
of fetal hemoglobin (HbF) results in decreased disease
severity and increased life expectancy due to the inhibition
of HbS polymerization. SCD manifestations start to appear
shortly after birth, when HbF levels start to decline and are
replaced by adult HbS. Because of this observation,
Offprint requests to: Angela Rivers, Department of Pediatrics, 840 South
Wood St., MC 856, University of Illinois, Chicago, IL 60612, USA;
E-mail: riversa@uic.edu
research over the past three decades has focused on the in-
duction of HbF expression as a possible therapeutic
approach for SCD. Currently, the only Food and Drug
Administration-approved disease-modifying drug available
is hydroxyurea, which increases HbF in approximately
60% of patients [2]. Other reported mechanisms of its ac-
tion include the reduction of reactive oxygen species
(ROS) through increased nitric oxide [3] and selenium-
dependent Gpx-1 activity [4].
Excessive ROS levels in SCD are directly associated
with hemolysis, vaso-occlusion, tissue damage, inflamma-
tory response, and pain crises [5]. Recent studies have
demonstrated that activation of Nrf2 ameliorates ROS-
mediated inflammation and tissue damage in SCD by
expression of antioxidant enzymes [6]. Although excessive

0301-472X/Copyright 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
http://dx.doi.org/10.1016/j.exphem.2017.02.003
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2 R. Jagadeeswaran et al./ Experimental Hematology 2017;-:--
ROS is linked to the hemolysis of red blood cells (RBCs) in
SCD and other hemolytic diseases [7], identifying the pri-
mary source and removal mechanisms of ROS are critical
steps toward mitigating the hemolytic processes in SCD.
ROS production in RBCs of patients with SCD is due, at
least in part, to factors such as HbS auto-oxidation, the Fen-
ton reaction, and low levels of antioxidants such as sele-
nium and GPx-1 [8] and is mediated via an increased
activity of NADPH oxidase [9,10]. Previous studies pro-
posed that the activation of NADPH oxidases is by up-
stream sources including mitochondrial ROS [11].
We therefore investigated whether SCD RBCs retain
mitochondria abnormally. The possibility of
mitochondria-derived ROS, particularly in RBCs, has
received no attention because, in normal physiological con-
ditions, the mitochondria are successfully eliminated during
terminal differentiation, particularly at the reticulocyte
maturation stage. Here, we show by flow cytometry anal-
ysis that RBCs obtained from SCD patients and in a mouse
model of SCD retain mitochondria. Second, we show that
RBCs with mitochondria are associated with high levels
of ROS accumulation. Finally, we demonstrate that two
preclinical candidate drugs, the LSD1 inhibitor, RN-1,
[12,13] and the mammalian target of rapamycin (mTOR)
inhibitor, sirolimus, [14] both reduce the levels of
mitochondria-retaining RBCs in an SCD mouse model.
Methods
Human samples
The study was approved by the institutional review board of the
University of Illinois at Chicago. Blood samples were collected
from SCD patients and control human subjects at the University
of Illinois Hospital & Health Sciences System Sickle Cell Center.
A total of six blood samples from patients with SCD disease and
six control individuals were collected in K2EDTA tubes and stored
at 4 C until use within 24 hours of collection.
Mouse model of SCD
SCD (HbSS) mice, also known as the Townes model mice (B6;
129-Hbatm1(HBA)TowHbbtm2 (HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J),
and wild-type (WT) (HbAA) mice (B6; 129-Hbatm1(HBA)Tow
Hbbtm3(HBG1,HBB)Tow) were purchased from The Jackson Labora-
tory (Bar Harbor, ME). HbSS mice carry human a, g, and bS globin
genes, which replace mouse globin genes and exhibit sickle cell
phenotypes similar to human SCD [15]. Heterozygous female
sickle cell trait (HbAS) mice were bred with male HbSS mice to
generate both sexes of SCD (HbSS) mice. HbAA mice were inter-
bred to produce age-matched HbAA control mice. All animal ex-
periments were performed under an approved protocol and
according to guidelines set by the Institutional Animal Care and
Use Committee at University of Illinois at Chicago. Peripheral
blood from the SCD (HbSS) and control (HbAA) mice was
collected from the tail vein or using retro-orbital bleeding methods.
Hematology parameters were analyzed using an automated
analyzer (DVIA 120 Hematology System, Bayer Corporation).
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Analysis of mitochondria and ROS by flow cytometry
Analysis of mitochondria and ROS levels was performed using
methods described previously [9,16]. RBCs were allowed to settle
by gravity and the overlying plasma and buffy coat were removed
by aspiration and then further washed twice with phosphate-
buffered saline (PBS) to remove plasma and white blood cells.
For the quantification of mitochondria in RBCs, hematocrit-
adjusted cells were stained with 250 nmol/L tetramethylrhodamine
methyl ester perchlorate (TMRM) (Invitrogen) in Dulbeccos modi-
fied Eagle medium (DMEM) for 30 minutes in a 37 C humidified
incubator. Cells were washed twice with 0.5% bovine serum albu-
min (Sigma-Aldrich) containing PBS and stained with allophyco-
cyanin (APC)-conjugated CD71 (BD Biosciences) antibody and
an RNA dye, thiazole orange (TO). Anti-mouse CD71 (transferrin
receptor) APC-conjugated antibody (Life Technologies) and
APC-mouse anti-human CD71 antibody (BD Pharmingen) were
used for mouse and human samples, respectively. For the simulta-
neous quantification of ROS levels and mitochondria-retaining
RBCs, cells were washed twice with PBS after the staining with
250 nmol/LTMRM (Life Technologies) and APC-conjugated trans-
ferrin receptor (CD71) antibody labeling and then incubated with
the ROS probe CM-H2DCFDA (Invitrogen) at 4 mmol/L for
30 min in a 37 C humidified incubator. Samples were analyzed
for fluorescence using fluorescein isothiocyanate (FITC), phycoer-
ythrin (PE), and APC channels on a BD LSRFortessa cell analyzer
using Kaluza analysis (Beckman Coulter) software.
Imaging flow cytometry
Image Stream X MKII Imaging flow cytometry (Amnis) was used
as described previously [17]. TO was excited with GFP laser and
emission collected in Channel 2 (480560 nm); TMRM was
excited with PE laser and emission collected in Channel 3 (595
660 nm). Bright-field images were collected in Channel 1. Images
were acquired using 60 magnification. A minimum of 30,000
100,000 positive events were collected. Individual TO and
TMRM fluorescent controls were collected for determining spec-
tral overlap (compensation) across image channels. Data were
analyzed using IDEAS version 6.0 software.
Immunofluorescence microscopy
Peripheral blood cells from SCD and controls were stained with
500 nmol/L MitoTracker Green FM (Invitrogen) in DMEM for
30 min 37 C, transferred to a glass-bottom dish (MatTek), and
viewed with a Carl Zeiss LSM 710 confocal microscope. Images
were captured and analyzed using ZEN software.
LSD1 and mTOR inhibition
The LSD1 inhibitor RN-1 (EMD Millipore) was delivered at doses
of 2.5 or 5 mg/kg by intraperitoneal injection to homozygous SCD
mice 5 days per week for 24 weeks as described previously [12].
For mTOR inhibition, sirolimus (rapamycin; Selleck Chemicals)
was given at a dose of 5 mg/kg by intraperitoneal injection to
SCD mice 5 days per week for 24 weeks and vehicle was admin-
istered to the control group.
RBC lifespan
N-hydroxysuccinimidebiotin was injected to WT controls and
SCD mice via the tail vein and w10 mL of blood was collected
at 24-hour intervals for 5 days. Survival of circulating, labeled
RBCs was measured by staining with TER-119 PE and streptavi-
din AF488 and analyzed for fluorescence in FITC and PE
 R. Jagadeeswaran et al./ Experimental Hematology 2017;-:-- 3

channels, respectively, on a BD LSRFortessa cell analyzer. Data
were analyzed using Kaluza analysis software. The percentage
of biotin-labeled cells on day 1 at 2 hours after biotin injection
was considered 100%. The percentage reduction of labeled
RBCs was calculated relative to the first day. The percentage
RBC survival was measured using circulating biotin-labeled
RBCs as described previously [13].
Gene expression analysis
Microarray analyses were performed on bone marrow cells from
SCD mice treated with RN-1 for 4 weeks. Data were processed
based on mouse gene ST 2.1 strips using the Affy Plus kit.
Mitophagy-related genes were selected from the Mouse Genome
Informatics international database (http://www.informatics.jax.
org) for mitophagy pathway gene expression analysis. For com-
parison between treated and control groups, probe sets were
selected with a twofold change or greater and an adjusted p value
of 0.05 or less.
Statistical analysis
Data are presented as mean 6 SEM and analyses were performed
using GraphPad Prism Software version 7.01. For comparison be-
tween means of two groups, a two-sided Students t test was used
to determine the degree of significance. Pearsons correlation anal-
ysis was used to test the relationship between two groups. Differ-
ences were considered significant when p # 0.05.
Results
Fluorescence-conjugated CD71 antibody and the RNA
dye TO were used to distinguish reticulocytes from
RBCs. In TO-negative and CD71-low RBCs, mitochondria
levels were measured by flow cytometric analysis using the
cell-permeable, cationic, red-orange fluorescent mitochon-
drial probe TMRM. We observed an increased percentage
of mitochondria-retaining RBCs in SCD (HbSS) patient
blood samples compared with normal HBAA controls (con-
trol: 0.46 6 0.1%, n 5 6; SCD [HbSS]: 10.09 6 2.1%,
n 5 6; p ! 0.001; Fig. 1).
Evaluation of mitochondria in RBCs of SCD mice and
control AA mice
Similar to humans, an increased percentage of
mitochondria-retaining RBCs was observed in SCD mice
compared with control mice (HbAA), as shown in the
confocal images (Fig. 2A) and by flow cytometry analysis
(Fig. 2B) (control [HbAA]: 0.29 6 0.18%; SCD [HbSS]:
16.68 6 1.9%, p ! 0.0001). Image Stream X MKII Imag-
ing flow cytometry (Amnis) was used for further confirma-
tion of the presence of mitochondria in RBCs of SCD. TO
was used to clearly distinguish RBCs from reticulocytes.
Representative images of TO-negative, TMRM-positive
RBCs (R6 fraction) reveal the presence of mitochondria
in the RBCs of SCD (Fig. 3).

Evaluation of mitochondria in RBCs of SCD patients

and control subjects
One of the key steps in the final differentiation of RBCs in-
volves the elimination of mitochondria via an erythroid-
specific mitophagy pathway [18,19]. Previous studies using
mouse models with deletions in mitophagy genes indicated
reduced RBC survival and severe anemia due to defects in
mitochondrial clearance [2022].
To investigate whether mitochondria containing RBCs
exist in SCD, peripheral blood samples from six SCD pa-
tients and six controls were obtained (Table 1).

Table 1. Characteristics of human subjects and levels of mitochondria-

retaining RBCs

Human Study group Age Mitochondria-retaining

subjects (Genotype) (Years) Gender RBCs (%)

C1 Control (HbAA) 24 Female 0.52

C2 Control (HbAA) 24 Female 0.44
C3 Control (HbAA) 43 Female 0.20
C4 Control (HbAA) 45 Female 0.15
C5 Control (HbAA) 26 Male 0.74
C6 Control (HbAA) 28 Male 0.73
S1 SCD (HbSS) 24 Female 3.80
Figure 1. Mitochondria are retained in RBCs of SCD patients. (A)
S2 SCD (HbSS) 29 Female 10.55
S4 SCD (HbSS) 38 Female 17.08 Whisker plot showing the percentage of mitochondria-containing RBCs
S3 SCD (HbSS) 45 Female 15.30 in control (HbAA) and SCD (HbSS) mice. Center line in the plot repre-
S5 SCD (HbSS) 31 Male 5.90 sents the mean value. (B) Representative flow cytometry plots showing
the percentage of cells in TO (RNA)-negative RBCs that retain mitochon-
S6 SCD (HbSS) 31 Male 7.91
dria located in the upper- left quadrant in control and SCD mice.

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4 R. Jagadeeswaran et al./ Experimental Hematology 2017;-:--

Figure 2. Mitochondria are present in RBCs of humanized HbSS sickle mice and levels were associated with excessive ROS. RN-1 and sirolimus treatment
reduced the level of abnormal mitochondria-retaining RBCs and decreased ROS levels. (A) Peripheral blood cells obtained from WT and SCD mice were
stained with MitoTracker Green FM. (B) Peripheral blood cells obtained from WT and SCD mice or SCD mice treated with RN-1 (2.5 mg/kg), RN-1 (5 mg/
kg), or sirolimus (5 mg/kg) were stained with TMRM and TO or CD71-APC-conjugated antibody. RBCs were gated based on specific forward and side
scatter properties and analyzed by flow cytometry. The bar graph shows the mean percentage of peripheral mitochondria-retaining RBCs in WT versus
SCD versus SCD treated with RN-1 (5 mg/kg) or sirolimus (5 mg/kg). (C) Representative flow cytometry plots showing the percentage of mitochondria-
retaining RBCs located in the upper-left quadrant in WT, SCD, and SCD mice treated with RN-1 (5 mg/kg) or SCD mice treated with sirolimus (5 mg/
kg). (D) Peripheral blood from control (HbAA) and SCD (HbSS) mice were incubated with the ROS probe CM-H2DCFDA after staining with TMRM
and CD71-APC. The bar graph shows the mean percentage of RBCs with high ROS in WT versus SCD versus SCD treated with RN-1 (5 mg/kg) or sirolimus
(5 mg/kg). (E) Representative flow cytometry plots showing the percentage of cells in the RBC fraction with ROS located in the upper-left quadrant in WT,
SCD, and SCD mice treated with RN-1 (5 mg/kg) or sirolimus (5 mg/kg). (F) Pearson correlation plot showing the correlation between mitochondria and
ROS levels in RBC of control and treated SCD mice.

Effect of RN-1 and sirolimus on mitochondria-retaining
RBCs, ROS levels, and the RBC lifespan in a sickle cell
mouse model
We investigated the currently used preclinical drugs RN-1, an
LSD1 inhibitor, and sirolimus, an mTOR inhibitor, in the SCD
mouse model. Previous studies have reported that RN-1
increased the lifespan of RBC in a SCD mouse model with
less than 3% induction of HbF as measured by high-
performance liquid chromatography [13]. Cui et al. hypothe-
sized that increased mouse embryonic globin chains promoted
longer RBC survival [13]. Here, we explored the reduction of
mitochondria-retaining RBCs as an additional mechanism for
the longer RBC survival. We observed that SCD mice treated
with either RN-1 (5 mg/kg) or sirolimus (5 mg/kg) showed a
significant decrease in mitochondria-retaining RBCs
compared with untreated SCD vehicle controls (vehicle con-
trol: 16.68% 6 1.9%; RN-1: 4.96% 6 1.0%, p ! 0.0005; si-
rolimus: 6.4% 6 1.8%, p ! 0.002) (Fig. 2B and C). We also
observed a significant reduction of ROS in RBCs coupled with
a decrease of mitochondria-retaining RBCs after in vivo treat-
ment with RN-1 (vehicle control: 16.8% 6 4.5%, RN-1:
2.4% 6 0.9%, p 5 0.04). Sirolimus treatment showed a
similar pattern, but this was not significant (sirolimus:
7.2% 6 1.4%, p 5 0.11; Fig. 2D and E). Mitochondrial levels
were correlated with ROS in RBCs (Pearson r 5 0.82, n 5 13,
p ! 0.001; Fig. 2F).
Further analysis was performed to determine whether the
decreased mitochondrial-retaining RBCs and ROS induced
by either RN-1 or sirolimus were related to increased RBC
lifespan. Both RN-1 and sirolimus increased the RBC

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 R. Jagadeeswaran et al./ Experimental Hematology 2017;-:-- 5

Figure 3. Mitochondria present in RBCs of SCD mice analyzed by imaging flow cytometry. Representative images of cells (R3, R4, R5, R6) obtained by
Image Stream X flow cytometer (Amnis). Gating strategy was used similar to regular flow cytometry detection of mitochondria-retaining RBCs methodology,
as described in the Methods. The top left panel (R6) shows TO-negative mitochondria (TMRM-positive)-retaining RBCs. The bottom left panel (R3) shows
TO-negative (TMRM-negative) RBCs that effectively eliminated mitochondria during terminal differentiation. The top right panel (R5) shows TO-positive
mitochondria-retaining RBC precursors (reticulocytes). The bottom right panel (R4) shows TO-positive mitochondria-negative RBC precursors (reticulo-
cytes). BF 5 bright field.

lifespan significantly (vehicle control: 40% 6 2.6%; RN-1 RBCs in SCD patients is mainly due to excessive ROS-
[2.5 mg/kg]: 69.9% 6 2.6%, p ! 0.01; RN-1 [5 mg/kg]: mediated hemolysis, as distinct from HbS-mediated sickle
72.3% 6 1.1%, p ! 0.01; sirolimus [5 mg/kg]: cell formation [9]. There is a direct association of
67.5% 6 6.1%, p ! 0.05; Fig. 4).

Effect of RN-1 treatment on mitophagy gene expression

Gene expression analysis of bone marrow cells obtained
from SCD mice treated with RN-1 showed significant in-
duction (Otwofold) in the expression of major mitophagy
genes including the erythroid-specific mitophagy gene
Atg7 (Fig. 5A and B) compared with untreated SCD
mice. The core autophagy gene Atg7 is a key molecular
effector in mitochondrial autophagy in mammalian hemato-
poietic cells and its loss in erythroid cells leads to the
incomplete removal of mitochondria and severe anemia
in vivo [20].

Discussion
Although the significance of ROS in SCD has been known
for years [10,23], the presence of mitochondria-retaining
RBCs in SCD and their role in ROS generation has not
been recognized previously. Normal RBCs survive in circu-
lation for approximately 90130 days [24], whereas RBCs
in SCD patients have a significantly shorter lifespan
(approximately 2040 days) [25]. The shorter lifespan of
Figure 4. RN-1 and sirolimus treatment increase RBC survival. The per-
centage of RBC survival was calculated by measuring the circulating
biotin-labeled RBCs by staining with TER-119 PE and streptavidin
AF488. The percentage reduction of labeled RBC was calculated relative
to the first day. n 5 23 of each type of mouse per group. Shown is the
RBC survival curve of WT and SCD with PBS, RN-1 (2.5 mg/kg), RN-
1 (5 mg/kg), or sirolimus (5 mg/kg) for 4 weeks. Each data point represents
the mean 6 SEM.

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6 R. Jagadeeswaran et al./ Experimental Hematology 2017;-:--

Figure 5. Mitophagy gene expression profiling of bone marrow cells from SCD mice treated with RN-1. (A) Heat map of 186 mitophagy-related genes that
are differentially expressed (RN-1 treated vs. control) as determined by microarray analysis. Red denotes upregulation (fold changes). (B) Bar graph showing
greater than twofold changes in upregulated mitophagy gene expression of bone marrow cells from SCD mice treated with RN-1 compared with vehicle-
treated controls.

intravascular hemolysis with major clinical events in SCD.
In SCD, oxidative damage in erythrocytes can result in
early erythrocyte destruction and contribute to severe ane-
mia and disease pathogenesis [26,27]. Our current findings
establish that mitochondria-retaining RBCs in SCD are
associated with excessive ROS levels in RBCs. These find-
ings indicate that the mitochondria-retaining RBCs are
Figure 6. Schematic representation of the role of mitochondria-retaining
RBCs in SCD pathogenesis. Mitochondria-retaining RBCs in SCD
contribute excessive ROS, which destroy RBCs, leading to SCD pathogen-
esis. Inhibiting LSD1 by RN-1 or inhibiting mTOR by sirolimus reduces
hemolysis by reducing mitochondria-retaining RBCs and associated ROS
levels.
likely to be a major source of excessive ROS. Further
detailed human studies should be performed to determine
whether mitochondria-retaining RBCs are a new biomarker
for disease severity because they are most likely an up-
stream event of hemolysis.
In this study, pharmacological reduction of
mitochondria-retaining RBCs was achieved partially by
both sirolimus and RN-1. The inhibition of mTOR by siro-
limus has been shown to promote autophagy of mitochon-
dria in human cell hybrid lines carrying pathological
mitochondrial DNA mutations [28]. mTOR inhibition has
also been shown recently to improve anemia and reduce or-
gan damage in a murine model of SCD [14]. In baboons
(Papio anubis), chromatin immunoprecipitation analysis
showed that RN-1 increased the levels of activating chro-
matin marks (H3K4Me2, H3K4Me3, and H3K9ac) associ-
ated with g-globin, but not -globin, which suggests a
specific effect of the drug on g-globin expression [29].
RN-1 induction of Atg7 and other key mitophagy genes in-
dicates that RN-1mediates mitochondrial clearance in SCD
via epigenetic upregulation of pro-mitophagy genes. Previ-
ous mouse models with specific deletions in mitophagy
genes showed a reduced survival of RBCs due to mitochon-
dria retention [2022]. Therefore, future studies should
investigate whether RBC precursors of SCD patients have
mitophagy defects. Future studies should also determine
whether RN-1s effect on ROS reduction is due to epige-
netic regulation in erythroid-lineage-specific precursors.
In summary, the results presented here are the first to iden-
tify a role for abnormal mitochondria-retaining RBCs in SCD
as an upstream target for ROS generation and the

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 R. Jagadeeswaran et al./ Experimental Hematology 2017;-:--
complications of SCD. These findings indicate that the re-
tained mitochondria in RBCs of SCD are likely to be a major
contributor of excessive ROS accumulation and may play a
role in hemolysis-associated SCD complications. We also
present new evidence that mitochondria-retaining RBCs
contribute to oxidative stress in SCD erythrocytes. Our find-
ings indicate that the LSD1 inhibitor, RN-1, and the mTOR
inhibitor, sirolimus, can potentially improve SCD RBC sur-
vival by eliminating a key intracellular source of ROS (Fig.
6). Therefore, drugs targeting mitochondrial ROS may war-
rant inclusion in a multimodal approach to SCD therapy.
Acknowledgments
We thank Dr. Brian Hall (Amnis, Millipore-Sigma) for the Image
Stream X MKII imaging flow cytometry analysis.
This work was supported by National Institutes of Health
Grants R03HL135453, K01HL103172, and U01 HL117658.
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