Effect of Periodic Granulocyte Colony-Stimulating Factor Administration on Endothelial Progenitor Cells and Different Monocyte Subsets in Pediatric Patients with Muscular Dystrophies

Abstract

Muscular dystrophies (MD) are heterogeneous group of diseases characterized by progressive muscle dysfunction. There is a large body of evidence indicating that angiogenesis is impaired in muscles of MD patients. Therefore, induction of dystrophic muscle revascularization should become a novel approach aimed at diminishing the extent of myocyte damage. Recently, we and others demonstrated that administration of granulocyte colony-stimulating factor (G-CSF) resulted in clinical improvement of patients with neuromuscular disorders. To date, however, the exact mechanisms underlying these beneficial effects of G-CSF have not been fully understood. Here we used flow cytometry to quantitate numbers of CD34+ cells, endothelial progenitor cells, and different monocyte subsets in peripheral blood of pediatric MD patients treated with repetitive courses of G-CSF administration. We showed that repetitive cycles of G-CSF administration induced efficient mobilization of above-mentioned cells including cells with proangiogenic potential. These findings contribute to better understanding the beneficial clinical effects of G-CSF in pediatric MD patients.

Full text

Research Article
Effect of Periodic Granulocyte Colony-Stimulating
Factor Administration on Endothelial Progenitor
Cells and Different Monocyte Subsets in Pediatric
Patients with Muscular Dystrophies
Andrzej Eljaszewicz,1Dorota Sienkiewicz,2Kamil Grubczak,1,3
Bohena Okurowska-Zawada,2Grahyna Paszko-Patej,2Paula Miklasz,1Paulina Singh,1
Urszula Radzikowska,1Wojciech Kulak,2and Marcin Moniuszko1,4
1Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, 15-269 Bialystok, Poland
2Department of Pediatric Rehabilitation and Center of Early Support for Handicapped Children “Give a Chance”,
MedicalUniversityofBialystok,15-274Bialystok,Poland
3Department of Immunology, Medical University of Bialystok, 15-269 Bialystok, Poland
4Department of Allergology and Internal Medicine, Medical University of Bialystok, 15-276 Bialystok, Poland
Correspondence should be addressed to Marcin Moniuszko; marcin.moniuszko@umb.edu.pl
Received  May ; Accepted  July 
Academic Editor: Tao Wang
Copyright ©  Andrzej Eljaszewicz et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Muscular dystrophies (MD) are heterogeneous group of diseases characterized by progressive muscle dysfunction. ere is a
large body of evidence indicating that angiogenesis is impaired in muscles of MD patients. erefore, induction of dystrophic
muscle revascularization should become a novel approach aimed at diminishing the extent of myocyte damage. Recently, we and
others demonstrated that administration of granulocyte colony-stimulating factor (G-CSF) resulted in clinical improvement of
patients with neuromuscular disorders. To date, however, the exact mechanisms underlying these benecial eects of G-CSF have
not been fully understood. Here we used ow cytometry to quantitate numbers of CD+ cells, endothelial progenitor cells, and
dierent monocyte subsets in peripheral blood of pediatric MD patients treated with repetitive courses of G-CSF administration.
We showed that repetitive cycles of G-CSF administration induced ecient mobilization of above-mentioned cells including cells
with proangiogenic potential. ese ndings contribute to better understanding the benecial clinical eects of G-CSF in pediatric
MD patients.
1. Introduction
Muscular dystrophies (MD) are a heterogeneous group of
muscle diseases characterized by progressive muscle weak-
ness and wasting [, ]. Despite promising gene-based thera-
peutic approaches being tested in MD, there is no cure availa-
ble and thereby the need for developing novel therapies is still
warranted [–]. ere are at least two physiological mecha-
nisms for tissue regeneration: (a) cell renewal, the replace-
ment of damaged cells by newly generated cells delivered
from resident stem cells; (b) cell proliferation, the self-repair
of terminally dierentiated well-functioning cells. Moreover,
tissue regeneration requires angiogenesis for microvascular
network restoration and to provide nutrient and oxygen
delivery [, ]. It should be noted that progressive decline
in muscle strength is caused in part by impaired blood ow
in dystrophic muscles. ere is a substantial body of evi-
dence indicating that vascularity of muscles is signicantly
decreased in MD subjects [, –]. In addition, the process
of angiogenesis is impaired in the course of MD. erefore,
induction of dystrophic muscle revascularization should con-
tribute to diminishing the eect from functional ischemia
Hindawi Publishing Corporation
Stem Cells International
Volume 2016, Article ID 2650849, 9 pages
http://dx.doi.org/10.1155/2016/2650849

Stem Cells International
and decrease myocyte damage. Accordingly, the proper ther-
apy for skeletal muscle regeneration in MD needs to consider
both revascularization of the tissue and myober regenera-
tion. erefore, use of biological therapies is an interesting
approach in the treatment of muscular dystrophies [].
To date, experimental therapies mainly focused on Vas-
cular Endothelial Growth Factor- (VEGF-) related strategies.
It is well established that VEGF function as a potent promotor
of angiogenesis and promyogenic factor. In dystrophin de-
cient muscles VEGF was shown to promote myober regener-
ation and protect cells from apoptosis []. Moreover, VEGF
leads to an increased blood vessels permeability, induction
of endothelial progenitor cell (EPC) migration, and prolifer-
ation []. us, it is possible that, at least partially, VEGF-
relatedbenecialeectscouldbeattributedtoanincrease
in EPC numbers. On the other hand, VEGF administration
should be closely monitored due to carcinogenic properties
[, ]. us, it is tempting to hypothesize that therapeutic
strategies aimed at selective enhancement of EPC in muscular
dystrophies could provide an attractive alternative for VEGF
treatment.
Notably, there is a growing body of evidence that mono-
cytes/macrophages are also important players in muscle
regeneration. It should be noted that two distinct and func-
tionally dierent subpopulations of macrophages are present
in regenerating muscle tissue, namely, MI (classically acti-
vated) and MII (alternatively activated) macrophages. MI
macrophages are referred to as proinammatory cells and are
involved in immune activation, phagocytosis, and muscle cell
lysis. In contrast, MII macrophages are usually considered to
exert anti-inammatory properties as they have been shown
to regulate inammatory cell function and participate in
vascularization process. is subpopulation is able to support
muscle cell regeneration, by inducing satellite cell prolifera-
tionandtissuerevascularization[].However,inthecourse
of muscular dystrophy, myober degeneration leads to mus-
cleinvasionbybothMIandMIImacrophages.Similarto
tissue macrophages, activated blood monocytes may display
both anti-inammatory and proinammatory activities. Par-
tially, these dierential activities of monocytes are associated
with their distinct phenotypes delineated by dierential
expression of CD and CD. us, classical CD++CD−
monocytes exert mostly phagocytic activities while inter-
mediate CD++CD+ and nonclassical CD+CD++
monocytes play numerous immunomodulatory functions
[, ]. It should be emphasized that biological properties
of macrophages depend to a large extent on monocyte acti-
vation and maturation process that occurs at the periphery
[]. us the examination of distribution of peripheral
blood monocyte subsets allows for assessing the pattern
of monocyte-related immune responses. However, despite
potentialroledierentmonocytesubsetscouldplayinmuscle
regeneration, their dynamic changes in the course of MD and
MD-targeted therapies were not yet examined.
Recently, the members of our group demonstrated that
G-CSF administration brought benecial clinical eects in
pediatricpatientswithMD[].G-CSFisamemberofcolony
stimulating factors that regulate the growth and dieren-
tiation of granulocytes and was shown to induce skeletal
T : Clinical characteristics of studied patients.
Patient Gender Age
(years)
Typ e of
muscular
dystrophy
Functional status
Boy
 DMD Nonwalking
Boy
 DMD Walking
Boy
 DMD Walking
 Girl  FSHD Walking
Boy
 DMD Nonwalking
Boy
 BMD Walking
 Girl  MCMD Nonwalking
Boy
 DMD Walking
 Girl  FSHD Walking
 Boy DMDWalking
 Boy DMD Walking
BMD: Becker musculardystrophy; DMD: Duchenne muscular dystro-
phy; FSHD: Facioscapulohumeral muscular dystrophy; MCMD: Merosin-
negative congenital muscular dystrophy.
myocyte development and regeneration [, ]. It is used
routinely in clinical practice for the treatment of neutropenia
and in conditioning donors before stem cell transplantation
[, ].
Here we wished to assess the eects of repeated cycles
of G-CSF administration on mobilization of bone marrow
derived stem/progenitor cells (most specically endothelial
progenitor cells) and dierent monocyte subsets in pediatric
patients with MD. In parallel, we set out to analyze the eects
of G-CSF administration on angiopoietins that similarly to
EPC are involved in angiogenesis (e.g., via mobilization
of EPCs) or a marker that is associated with changes of
monocyte/macrophage phenotype, namely, soluble CD
(sCD).
2. Materials and Methods
2.1. Patients. Atotalofelevenmusculardystrophypatients
were enrolled in this study. Detailed clinical characteristics of
all patients are summarized in Table . Patients received their
current standard treatment which was supplemented only by
administration of lgrastim (Neupogen, Amgen) at the fol-
lowing doses:  𝜇g/kg of body weight/day for ve consecutive
days (course ). Such treatment course was repeated aer 
month (course ) and aer  months (course ).
2.2. Extracellular Staining and Flow Cytometry. Fresh EDTA-
anticoagulated whole blood samples were stained with a
panel (Table ) of mouse anti-human monoclonal antibodies,
according to stain-and-then-lyse-and-wash protocol. Briey,
 𝜇L(formonocytes)and𝜇L (for EPCs) of whole blood
were stained with monoclonal antibodies and incubated for
 min at room temperature, in the dark. ereaer, ery-
throcytes were lysed by adding  mL of FACS lysing solution
(BD), followed by min incubation in the dark. Cells were

Stem Cells International 
T:Monoclonalantibodiesusedforowcytometryanalysis.
Specicity Fluorochrome Origin Clone Supplier
CD PE Mouse M𝜑P Becton Dickinson
CD FITC Mouse B. Becton Dickinson
CD FITC Mouse  Becton Dickinson
CD PE Mouse HI Becton Dickinson
CD APC Mouse AC Miltenyi Biotec
CD PE Mouse  Becton Dickinson
washed twice with cold PBS (phosphate-buered saline) and
xed with CellFix (BD Biosciences). Fluorescence-minus-
one (FMO) controls were used for setting compensation
and to assure correct gating. Specimen acquisition was per-
formed using FACSCalibur ow cytometer (BD Biosciences).
Obtained data were analysed using FlowJo ver. .. soware
(Tree Star).
2.3. Cytokine Assay. Angiopoietin-, Angiopoietin-, and
sCD levels in EDTA-plasma samples from patients with
MD were quantied by means of commercially available
enzyme-linked immunosorbent assays (ELISA). To deter-
mine sCD plasma levels all samples were initially diluted
-fold with reagent diluent (% BSA (Sigma-Aldrich) in
PBS). Next, the specimens were assayed using sCD DuoSet
ELISA kit (R&D Systems), according to the manufacturer’s
instruction. In order to determine Ang- and Ang- levels
samples were directly assayed using Ang- DuoSet ELISA
kit and Ang- DuoSet ELISA kit (both from R&D Systems).
Finally, the protein levels in the specimens were calculated
from a reference curve generated by using reference stan-
dards. e samples were analyzed with automated light abso-
rbance reader (LEDETEC  system). Results were calculated
by MicroWin  soware.
2.4. Statistical Analysis. Statistical analysis was carried out
using GraphPad Prism  (GraphPad soware). Wilcoxon test
wasusedtocomparechangesinmonocytesandEPCsnum-
bers and plasma protein levels in single treatment course.
Kruskal-Wallis test with post hoc Dunn’s multiple compari-
son test was used to determine dierences between all treat-
ment courses. Spearman correlation coecient was used to
determine correlations between plasma protein levels and cell
subsets. e dierences were considered statistically signi-
cant at 𝑝 < 0.05. e results are presented as median (inter-
quartile range).
3. Results
First, we analyzed the eect of G-CSF treatment on hemato-
poietic stem/progenitor cells mobilization in children with
MD. We observed substantial increase in CD+ cell num-
bers aer course  (from  (–) to  (–
), Figure (a)), course  (from  (.–) to 
(–), Figure (b)), and course  (from  (–)
to  (–), Figure (c)) of G-CSF administration.
Notably, repetitive courses of G-CSF treatment did not aect
the eciency of CD+ cell mobilization in MD children
(𝑝 > 0.05).
Next, we evaluated the numbers of endothelial progeni-
tor cells (delineated by CD+CD+CD+ phenotype)
following repetitive courses of G-CSF treatment. We found
signicant increase in EPC numbers aer course  (from 
(–) to  (–), Figure (a)), course  (from . (.–
.) to . (–.), Figure (b)), and course  (from 
(.–.)– (.–), Figure (a)) of G-CSF administra-
tion. Again, no signicant dierences were observed in eec-
tiveness of EPC mobilization between courses (𝑝 > 0.05). In
parallel, we assessed the levels of two major angiopoietins,
Ang- and Ang-, during treatment with G-CSF and found
that none of them was aected by this therapy (Figure ).
Next we set out to investigate changes in absolute num-
bers of dierent monocyte subsets. We found that G-
CSF administration induced mobilization of CD++CD−,
CD++CD+, and CD+CD++ monocytes in all studied
individuals (Figure (a)). Moreover, repeated administration
ofG-CSFalsoresultedinanincreaseinthenumbers
of all three above-mentioned subpopulations (Figures (b)
and(c)).Interestingly,wedidnotobserveanystatistically
signicant changes of monocyte mobilization eectiveness
between courses of treatment (𝑝 > 0.05).
Next, we assessed the eects of G-CSF treatment on
sCD levels. We observed substantial increase in sCD
levels in all studied individuals undergoing initial treatment
(Figure (a)). Interestingly,  out of  (%) MD patients
presented with an increase in sCD levels aer course 
(𝑝 < 0.05, Figure (b)). Moreover,  out of  (%) MD
children showed an increase in sCD levels aer course  of
GM-CSF administration (𝑝 > 0.05, Figure (c)). Again, there
were no signicant dierences in eectiveness of treatment
response based on sCD plasma levels (𝑝 > 0.05).
Finally, we investigated whether plasma Ang-, Ang-,
andsCDlevelswerecorrelatedtonumbersofCD+cells,
EPCs, and monocytes subsets in peripheral blood. We did
not nd any signicant correlations among above-mentioned
parameters.
4. Discussion
G-CSF-induced mobilization of hematopoietic stem/progen-
itor cells is usually delayed, with peak levels achieved within
–days.Infact,inpresentstudyweobservedasubstantial
increase of CD+ cells, including hematopoietic stem cells
(HSCs) (as expected, 𝑝 = 0.015 for course ; 𝑝 = 0.007 for
course ; 𝑝 = 0.031 for course ; data not shown) and EPCs,
in all studied individuals. Interestingly from clinical point
of view, the growth rate of analyzed cell populations did not
dier between courses of treatment (at monthly intervals).
Similar to our study, de Kruijf et al. reported in mice model
that multiple cycles of recombinant human G-CSF admin-
istration (up to  cycles) did not lead to bone marrow
HSC pool depletion []. However, the long-term eects of
repetitive or chronic G-CSF treatment on hematopoiesis and
bone marrow steam/progenitor cells pool were not known.
e contribution of CD+ cells to muscle regeneration has

Stem Cells International
CD34+ cells events/200 𝜇L whole blood
6000
4000
2000
0
Before Aer
Course 1
p = 0.0156
(a)
CD34+ cells events/200 𝜇L whole blood
Before Aer
Course 2
5000
4000
3000
2000
1000
0
p = 0.0020
(b)
CD34+ cells events/200 𝜇L whole blood
Before Aer
Course 3
8000
6000
4000
2000
0
p = 0.0625
(c)
F : e summary of analyses of changes in CD+ cells numbers aer(a) course , (b) course , and (c) course  of G-CSF administration
in MD pediatric patients.
been well documented [–]. However, CD+ population
is not uniform as it is composed of dierent subpopulations
of progenitor/stem cells of which EPCs constitute a crucial
subset involved in development of new vessels. Notably,
we demonstrated here that G-CSF treatment of patients
with pediatric MD increased EPC numbers in peripheral
blood.isndingcanbeofimportanceintreatmentofMD
characterized by impaired vasculature. EPCs were shown to
migrate in response to angiogenic growth factors, including
angiopoietins to the site of ischemic tissue where they dier-
entiate into mature endothelial cells (ECs). ereaer, ECs
proliferatetosupportandformnewvessels.Furthermore,low
dose CD+VEGFR+ cell transplantation hinders apoptotic
cell death and reduces brosis in the ischemic muscles. ese
cells support ischemic muscle regeneration, improve the
clinical outcome, and accelerate the hemodynamic recovery
rate []. We showed here that repetitive use of G-CSF could
contribute to improved “endothelization” of dystrophic mus-
cles via ecient mobilization of EPCs. Given these promising
data, further mechanistic studies dening in detail the role of
EPCs in muscle regeneration in humans are still warranted.
In addition, further studies in MD patients focused on
measurements of possible triggering factors for progenitor
cells such as stromal cell-derived factor- (SDF-) or sphingo-
sine--phosphate (SP) would be of potential clinical benet.
Similarly, given the signicant eects of G-CSF on progenitor
cells, more detailed experiments addressing the eects of
G-CSF administration on mobilization of stem cells subsets
such as mesenchymal stem cells (MSCs) or very small
embryonic-like stem cells (VSELs) in MD patients would be
of signicant interest.
We reported here substantial increase of all absolute
monocyte subset numbers following G-CSF administration.
Similarly, G-CSF was found to increase monocyte numbers
in mice []. Moreover, Capoccia et al. showed that G-CSF-
mobilized monocytes stimulated angiogenesis at sites of
ischemia []. is study did not describe mechanism of
monocyte-related angiogenesis; however, it can be hypothe-
sized that this action was dependent on increased numbers
of these monocyte subsets with proangiogenic potential,

Stem Cells International 
400
300
200
100
0
p = 0.0156
CD34+CD133+CD309+ cells events/200 𝜇L
whole blood
Before Aer
Course 1
(a)
200
150
100
50
0
p = 0.0059
CD34+CD133+CD309+ cells events/200 𝜇L
whole blood
Before Aer
Course 2
(b)
500
400
300
200
100
0
p = 0.0625
CD34+CD133+CD309+ cells events/200 𝜇L
whole blood
Before Aer
Course 3
(c)
F : e summary of analyses of EPC numbers (expressing CD+CD+CD+ phenotype) in MD pediatric individuals aer
(a) course , (b) course , and (c) course  of G-CSF administration.
p = 0.8750
p = 1.0000
Angiopoietin-1
Angiopoietin-2
15000
10000
5000
0
4000
3000
2000
1000
0
Before Aer
Before Aer
Course 1
Angiopoietin-1 (pg/mL)
Angiopoietin-2 (pg/mL)
(a)
p = 0.6875
p = 0.4375
Angiopoietin-1 (pg/mL)
Angiopoietin-2 (pg/mL)
15000
10000
5000
0
4000
3000
2000
1000
0
Before Aer
Before Aer
Course 2
(b)
p = 0.4375
p = 0.3125
10000
8000
6000
4000
2000
0
4000
3000
2000
1000
0
Before Aer
Before Aer
Course 3
Angiopoietin-1 (pg/mL)
Angiopoietin-2 (pg/mL)
(c)
F : Time course changes in Ang- (upper row) and Ang- (bottom row) plasma levels in pediatric patients with MD aer (a) course ,
(b) course , and (c) course  of G-CSF administration.

Stem Cells International
p = 0.0286
p = 0.1250
p = 0.1250
2500
2000
1500
1000
500
0
400
300
200
100
0
400
300
200
100
0
CD14++CD16−
CD14++CD16+
CD14+CD16+
Absolute CD14++CD16−
Absolute CD14++CD16+
Absolute CD14+CD16++
Before Aer
Before Aer
Before Aer
Course 1
count
count count
(a)
p = 0.0078
p = 0.0078
p = 0.0234
2500
2000
1500
1000
500
0
1000
800
600
400
200
0
600
400
200
150
100
50
0
Absolute CD14++CD16−Absolute CD14++CD16+
Absolute CD14+CD16++
Before Aer
Before Aer
Before Aer
Course 2
count
count
count
(b)
p = 0.1250
p = 0.2500
p = 0.1250
2000
1500
1000
500
0
2000
1500
1000
500
0
200
150
100
50
0
Absolute CD14++CD16−
Absolute CD14++CD16+
Absolute CD14+CD16++
Before Aer
Before Aer
Before Aer
Course 3
count
count count
(c)
F : Eect of (a) course , (b) course , and (c) course  of G-CSF administration on absolute numbers of CD++CD−(upper row),
CD++CD+ (middle row), and CD+CD++ (bottom row) monocytes in pediatric patients with MD.
namely, those bearing high levels of Tie, receptor for angio-
poietins. ese monocytes are referred to as Tie express-
ing monocytes (TEMs). Tie receptor is also present on
HSCs and EPCs indicating that these cells constitute tar-
get populations for angiopoietin-mediated actions [–].
Angiopoietin- (Ang-) and Angiopoietin- (Ang-) are the
best known and are characterized of the four, so far discov-
ered, angiopoietins. Angiopoietin- is the principal activator
of Tie; additionally, it stimulates the migration of endothelial
cells in vitro and promotes satellite cell self-renewal [].
In contrast, Ang- is its natural inhibitor, blocking Ang-
-dependent phosphorylation of Tie receptor, which is
reected by destabilization of blood vessels and constitutes
the initial stage of neovascularization [, ]. It should be
noted that TEMs in the vast majority express CD; therefore
they fall into both intermediate and nonclassical monocytes
[, ]. Here we found that G-CSF treatment increased both
above-mentioned subpopulations; however, it did not aect
Ang- and Ang- plasma levels. us we can hypothesize that
G-CSF treatment increased monocyte numbers with proan-
giogenic potential in an angiopoietin-independent manner.
However, further studies are warranted to explore whether
such increase in both subpopulations of CD-expressing
monocytes could directly contribute to improved muscle
regeneration in MD.
Quite surprisingly, we found here that G-CSF treatment
tended to increase sCD levels. As surface CD can be
shed from monocytes to become soluble CD, one could
hypothesize that enhanced levels of sCD following G-CSF
therapy could result from enhanced levels of CD bearing
monocytes (mostly classical and intermediate ones, see []).
Interestingly, sCD has been considered as a surrogate mar-
ker of on-going monocyte-related inammation []. us, it
needs to be further examined whether G-CSF administration
could be linked to enhancement of inammation. How-
ever,ontheotherhandelevatedsCDlevelscouldhave
originated from alternatively activated macrophages (MII)
known to have derived most frequently from intermediate
monocytes. Previously, we have shown that intermediate
monocytes expressed highest levels of CD []. us, G-
CSF-induced enhancement of intermediate monocytes could
have resulted in subsequent increase of MII macrophages
known to exert benecial eects on muscle regeneration.
Nevertheless, potential use of sCD as a putative marker
of enhanced muscle repair related to accumulation of MII
macrophagesneedstobeclariedinfurtherstudies.

Stem Cells International 
p = 0.1250
600
400
200
0
sCD163 (𝜇g/mL)
Before Aer
Course 1
(a)
p = 0.0313
600
400
200
0
sCD163 (𝜇g/mL)
Before Aer
Course 2
(b)
p = 0.1250
600
400
200
0
800
sCD163 (𝜇g/mL)
Before Aer
Course 3
(c)
F : Time course changes in sCD plasma levels aer (a) course , (b) course , and (c) course  of G-CSF administration in MD
pediatric patients.
5. Conclusion
Insummary,toourknowledge,thisistherstreportshowing
that repetitive G-CSF treatment can induce ecient mobili-
zation of cells with proangiogenic potential, namely, EPC and
putative proangiogenic monocytes. ese ndings could help
better understand the benecial clinical eects of repetitive
G-CSF administration in MD pediatric patients. Neverthe-
less, the clinical safety of such treatment in this group of
patients needs to be carefully addressed in further follow-up
studies.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
is study was supported by grants from Medical University
of Bialystok and funds from Leading National Scientic Cen-
ter in Bialystok of Medical University of Bialystok.
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