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[Frontiers in Bioscience 12, 712-724, January 1, 2007]
712
Neuroprotective Effect of Granulocyte-Colony Stimulating Factor
Ihsan Solaroglu
1
, Vikram Jadhav
1
, and John H. Zhang
1,2,3
1
Department of Physiology and Pharmacology,
2
Division of Neurosurgery,
3
Department of Anesthesiology, Loma Linda
University School of Medicine, Loma Linda, CA
TABLE OF CONTENTS
1. Abstract
2. Introduction
3. G-CSF and its receptor
3.1. G-CSF and G-CSFR in the brain
4. Clinical applications of G-CSF
4.1. G-CSF in general clinical use
4.2. G-CSF treatment in patients with cerebral injuries
5. Neuroprotective properties of G-CSF
5.1. G-CSF, inflammation, and apoptosis
5.2. G-CSF, hematopoietic stem cell mobilization, and brain repair
5.3. G-CSF and neurogenesis
6. Summary and Perspective
7. Acknowledgement
8. References
1. ABSTRACT
Granulocyte-colony stimulating factor (G-CSF) is
a growth factor which stimulates proliferation,
differentiation, and survival of hematopoietic progenitor
cells. G-CSF is being used extensively in clinical practice
to accelerate recovery of patients from neutropenia after
cytotoxic therapy. However, growing evidences have
suggested that G-CSF has important non-hematopoietic
functions in central nervous system. Recent studies have
shown the presence of G-CSF/G-CSF-receptor (G-CSFR)
system in the brain, and their roles in neuroprotection and
neural tissue repair as well as improvement in functional
recovery. The increased expression of G-CSF/G-CSFR on
neurons subjected to hypoxia provides evidence that G-
CSF may have an autrocrine protective signaling
mechanism in response to neural injury. G-CSF exerts
neuroprotective actions through the inhibition of apoptosis
and inflammation and the stimulation of neurogenesis.
Moreover, G-CSF has been shown to mobilize bone
marrow stem cells into the injured brain improving neural
plasticity. In this review, we summarize some of the recent
studies on G-CSF and the corresponding signal
transduction pathways regulated by G-CSF in
neuroprotection.
2. INTRODUCTION
Each year about 700,000 people experience a
new or recurrent stroke which is the leading cause of adult
disability and remains the third most common cause of
death in the United States (1). For the last two decades, a
great deal of attention has been paid to identify the
pathophysiological pathways leading to neural death in
stroke and to design neuroprotectant agents to modulate
these pathways. Although numerous agents have been
found to reduce infarct size and improve clinical outcome
in experimental models, the clinical use of these
neuroprotective agents has been hampered by toxicity of
the compounds. Recombinant tissue plasminogen activator
(rt-PA) remains the only approved agent for acute stroke
management in humans. However, its narrow therapeutic
window and potential side effects including increased risk
of intracerebral
hemorrhage and neurotoxicity limit the
efficacy of rt-PA (2-4).
In recent years, several independent research
groups have reported neuroprotective properties of growth
factors such as erythropoietin (EPO), insulin-like growth
factor, basic fibroblast growth factor, brain-derived
neurotrophic factor (BDNF), and granulocyte-colony
Neuroprotection by G-CSF
713
stimulating factor (G-CSF) in central nervous system
diseases such as stroke, neurotrauma, neuroinflammatory,
and neurodegenerative diseases using experimental animal
models (5-12). G-CSF, a member of growth factor family, not
only stimulates the development of committed progenitor cells
to neutrophils but also modulates the neutrophil functions and
their distribution in the body (13). Furthermore, like other
cytokines, it also has trophic effects on the different cell types
including neuronal cells (14). This could be of interest, because
neurotrophic factors are not only essential for the survival and
differentiation of normally developing neurons, but they also
play important roles in the protection and recovery of mature
neurons under pathologic conditions (15). Although there is
evidence from recent experimental studies that administration
of G-CSF is neuroprotective (16-20), the precise mechanisms
of the neuroprotective effect of G-CSF are not entirely
explored.
In this article, we review the biological properties
and clinical applications of G-CSF, and the possible role
and novel mechanisms of G-CSF as a potential therapeutic
agent for neuroprotection.
3. G-CSF AND ITS RECEPTOR
G-CSF is a glycoprotein with a molecular mass
of 19 kDa and is structurally characterized by four anti-
parallel alpha-helices (21). G-CSF is produced by a variety
of cells including bone marrow stromal cells, endothelial
cells, macrophages, fibroblasts, and astrocytes in response
to specific stimulation (13, 22, 23). Human G-CSF is
encoded by a single gene that is located on chromosome 17
q11-12 (24, 25).
G-CSF binds to its specific receptor and
stimulates the proliferation and differentiation of
neutrophilic progenitor cells. The G-CSF receptor (G-
CSFR) is a type I membrane protein and has a composite
structure consisting of an immunoglobulin-like domain, a
cytokine receptor-homologous domain and three
fibronectin type III domains in the extracellular region (26).
G-CSFR is expressed not only on a variety of
hematopoietic cells including neutrophils, and their
precursors, monocytes, platelets, lymphocytes, and
leukemia cells, but also on non-hematopoietic cells such as
endothelial cells, neurons and glial cells (20, 27-32).
G-CSFR activates a variety of intracellular
cascades, including the Janus
kinase (JAK)/signal
transducer and activator of transcription (STAT), the
Ras/mitogen-activated
protein kinase (MAPK), and
phosphatidylinositol 3-kinase (PI3-K)/protein kinase B
(PKB) (also known as Akt) (33-36). Activation of these
cascades subsequently activates their downstream
substrates and affects target genes which mediate
proliferation, differentiation, and survival of hematopoietic
cells (37). These signaling cascades are summarized in
Figure 1.
3.1. G-CSF and G-CSFR in the brain
G-CSF receptor and its ligand have been shown
to be expressed by neurons in a variety of brain regions
including pyramidal cells in cortical layers (particularly in
layers II and V), Purkinje cells in the cerebellum,
subventricular zone (SVZ) and cerebellar nuclei in rats. G-
CSF positive cells in CA3 region of the hippocampus,
subgranular zone and hilus of the dentate gyrus, entorhinal
cortex, and olfactory bulb have been identified (12).
Moreover, G-CSF receptor expression has shown in the
frontal cortex of human brain by postmortem studies (12).
G-CSF and its receptor are co-expressed in
neurons in the rodent central nervous system and are
upregulated in the ipsilateral forebrain hemisphere after 2
hours occlusion of the middle cerebral artery (MCAO) and
reperfusion (12). Similarly, Kleinschnitz et al. showed that
4 hours after permanent MCAO, G-CSF mRNA levels
massively increases compared to normal cortex and
decreases after 2 days to its control levels (38). The authors
pointed out that the increase in G-CSF mRNA expression is
not only within the ischemic lesions but also in the
nonischemic frontal cortex after photothrombosis model of
focal cerebral ischemia (38). Taken together, current
evidences suggest that G-CSF may have an autrocrine
protective signaling mechanism in response to neural
injury. Similar mechanisms have been suggested for other
growth factors, especially EPO which have been
extensively reviewed by others (39, 40).
4. CLINICAL APPLICATIONS OF G-CSF
4.1. G-CSF in general clinical use
G-CSF, specifically Filgrastim (r-metHuG-CSF),
a genetically engineered drug was approved by FDA on
February 21, 1991 to decrease the incidence of infection, as
manifested by febrile neutropenia, in patients with
nonmyeloid malignancies receiving myelosuppressive
anticancer drugs associated with a significant incidence of
severe neutropenia with fever. During the last decade, G-
CSF has found wide spread use in reducing the duration of
febrile neutropenia in cancer patients treated with
chemotherapy. G-CSF is also being used clinically to
facilitate hematopoietic recovery after bone marrow
transplantation, the mobilization of peripheral blood
progenitor cells in healthy donors and in the treatment of
severe congenital neutropenia (41-44).
The role of G-CSF treatment in variety of other
conditions is still matter of discussion. There is evidence
from clinical studies that administration of G-CSF is safe,
beneficial, and well
tolerated for the prevention or
treatment of infections in patients with
non-neutropenic
infections, patients undergoing surgery, patients with
human immunodeficiency virus, and patients with diabetic
foot infections (45-50). On the other hand, controlled trials
and systematic reviews have concluded that there is no
current evidence supporting the routine use of G-CSF in the
treatment of pneumonia, in patients undergoing surgery, for
treating or preventing neonatal infections, and in the
treatment of diabetic foot infections (51-54).
4.2. G-CSF treatment in patients with cerebral injuries
The prophylactic administration of G-CSF
against severe infections in critically ill patients with
Neuroprotection by G-CSF
714
Figure 1. Figure shows the hematopoetic actions of Granulocyte-Colony Stimulating Factor (G-CSF). G-CSF plays an important
role in the survival, proliferation and differentiation of the hematopoetic cells. The figure also depicts the signaling pathways
suggested in the existing literature for the G-CSF mediated actions. G-CSFR activates Janus
kinase (JAK)/signal transducer and
activator of transcription (STAT), the Ras/mitogen-activated
protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-
K)/Akt pathways. Activation of JAK2/STAT3 leads
to increased expression of cellular inhibitor of apoptosis protein2 (cIAP2) in
human neutrophils resulting in their survival. Activation of STAT3 by G-CSF has also been linked to myeloid cell differentiation.
STAT5, however, seems to be involved in G-CSF-dependent cell
proliferation.
Neuroprotection by G-CSF
715
cerebral injuries is controversial. The primary outcome of
studies is a reduction in frequency of bacteremia and in
life-threatening infections in patients with cerebral injuries.
For the first time, Heard et al. conducted a randomized,
placebo-controlled, double-blind, multi-center, phase II
study to determine whether the use of prophylactic
recombinant human G-CSF reduces the frequency of
nosocomial infections in patients with either acute
traumatic brain injury or cerebral hemorrhage (55). The
primary efficacy end points of this study were the
frequency of nosocomial infections, increase in absolute
neutrophil count, and the safety of G-CSF. Secondary
efficacy end points included serum G-CSF concentrations,
28 day survival, and duration of hospitalization, antibiotic
use, intensive care unit stay, and mechanical ventilation.
Authors reported that G-CSF increases absolute neutrophil
counts and markedly reduces the frequency of bacteremia
in a dose-dependent fashion in critically ill and intubated
patients (55). They concluded that phase III trial is
warranted to confirm these results. However, the authors
did not report neurological outcomes and cerebral
physiological variables of the patients. Also, the study was
criticized by Whalen et al. because the systemic
administration of G-CSF to patients with traumatic brain
injury may carry potential risk of increased acute
inflammatory reactions in the injured brain due to excessive
accumulation of neutrophils (56). Whalen et al. have
suggested that it was premature to use G-CSF in humans
with TBI until animal studies have demonstrated G-CSF
effects on brain injury after trauma or stroke (56).
Combined therapy with high-dose barbiturates
and mild hypothermia has been used widely for patients
with severe head injuries to decrease their intracranial
pressure (ICP). However, both therapies alone may result in
a decrease in leukocytes and suppression of neutrophil
functions which increase susceptibility to infectious
complications (57, 58). Ishikawa et al. have used G-CSF to
ameliorate life-threatening infections in patients with
severe head injury who were treated with combined therapy
involving high-dose barbiturates and mild hypothermia
(59). Authors studied the daily changes in total leukocyte
count, leukocyte differentiation, C-reactive protein,
respiratory index (RI), and ICP. Patients treated with G-
CSF showed significant improvement in the RI which
reflects the recovery from pneumonia. Authors reported
that ICP was not elevated by G-CSF administration, and a
significant improvement was achieved in survival rate.
Moreover, G-CSF treatment resulted in decrease of blood
IL-6 concentrations of patients. Although previous studies
that have attempting to correlate IL-6 production
with
patient outcome following TBI have produced inconsistent
results, Arand et al. suggested that elevated IL-6 in the
serum
or CSF is associated with poor outcome (60).
Ishikawa et al. concluded that G-CSF treatment ameliorates
life-threatening infections in patients with severe head
injury who were treated with combined therapy involving
high-dose barbiturates and mild hypothermia without
causing lung injury or increasing brain swelling (59).
However, this was not a prospective study and the number
of patients was small to conclude whether the use of G-CSF
in brain injured patients is a safe prophylactic agent against
severe infections.
In summary, the therapeutic potential of
prophylactic G-CSF therapy in critical ill patients, and G-
CSF administration for the prevention or treatment of
infections in non-neutropenic patients are currently
unproven and further controlled trials will be necessary to
clarify these issues.
5. POTENTIAL NEUROPROTECTIVE
PROPERTIES OF G-CSF
There is growing number of studies about the
neuroprotective properties of growth factors in the last
decade. Although G-CSF has been used for many years in
clinical practice and is a well known growth factor, there
are only a few experimental studies exploring the
neuroprotective effects of G-CSF. These studies are
summarized in Table 1.
5.1. G-CSF, inflammation, and apoptosis
It is well established that the inflammation, in
response to brain injury, involves infiltration of neutrophils
and monocytes/macrophages into the injured brain
parenchyma and activation of resident brain cells which are
capable of generating inflammatory mediators (61, 62).
Although there is not a clear cause-effect relationship
between neutrophil recruitment and CNS pathogenesis
(63), it is well known that neutrophil activation results in
release of proteolytic enzymes and generation of free
oxygen radicals. Excessive generation of free oxygen
radicals causes DNA damage, lipid peroxidation and
inactivation of proteins and finally leads to severe tissue
injury (64-66). Free oxygen radicals also contribute to the
breakdown of the blood-brain barrier (BBB) and brain
edema (62, 67). It could be of interest because G-CSF
administration increases absolute neutrophil count in
peripheral blood which may result in acute inflammatory
reactions in the injured brain. In support of this, it has been
reported that in vivo chloroquine or colchicine treatment
that reduce the number of mononuclear phagocytes in
damaged brain, help to block reactive astrogliosis and
neovascularization, and slow the rate of debris clearance
from sites of traumatic injury (68). For the first time,
Whalen et al. evaluated the effect of G-CSF-induced
neutrophilia on BBB permeability and brain edema after
traumatic brain injury in a rat model (69). They have
demonstrated that subcutaneous administration of G-CSF
(25 µg/kg every 12 hrs for five doses, with the last dose
administrated immediately before cortical impact injury
(CCI)) resulted in increased systemic absolute neutrophil
count (ANC) at the time of controlled CCI which correlated
with BBB damage in the injured hemisphere 24 hours later.
However, no difference in brain edema and hemispheric
neutrophil accumulation were reported between control and
G-CSF treated rats. They have suggested
that the ability of
G-CSF-stimulated neutrophils to migrate
into injured tissue
may be impaired (69). Recently, Park et al. have reported
that intra-peritoneal G-CSF administration after
intracerebral hemorrhage reduces brain edema and
inflammation in a rat model (17). However, they also
Neuroprotection by G-CSF
716
Table 1. Previous experimental studies about neuroprotective effect of G-CSF treatment after CNS injuries
Species Model Results Author Year Ref.
Rat
Pretreatment
TBI
No increase in brain edema and posttraumatic brain neutrophil accumulation;
Increase in BBB damage;
Whalen M. J., et al. 2000 69
Mice EAE
Limitation of demyelination and inflammation;
Clinical score improvement;
Reduction of pro-inflammatory cytokines;
Zavala F., et al. 2002 74
Mice MCAO
Improving survival rate;
Reduction of infarct volume;
Six I., et al. 2003 18
Rat
MCAO
Cell Culture
Reduction of infarct volume;
Reduction of inflammation;
Protection against excitotoxicity;
Schabitz W. R., et
al.
2003 20
Mice TBI
Small functional effect on functional outcome;
No effect on histopathology or motor outcome;
Sheibani N., et al. 2004 19
Rat MCAO
Neurological improvement;
Reduction of infarct volume;
Improving neural plasticity and vascularization;
Shyu W. C., et al. 2004 16
Rat ICH
Neurological improvement;
Reduction of edema, inflammation and
perihematomal cell death;
Park H. K., et al. 2005 17
Mice MCAO
Neurological improvement;
Reduction of infarct volume;
Gibson C. L., et al. 2005 11
Rat
MCAO
CCCA/MCAO
Cell culture
Neurological improvement;
Reduction of infarct volume;
Induce neurogenesis;
Schneider A., et al. 2005 12
TBI; traumatic brain injury, EAE; experimental allergic encephalomyelitis, MCAo; middle cerebral artery occlusion,
ICH; intracerebral hemorrhage, CCCA/MCAo; combined common carotid artery/ middle cerebral artery occlusion, BBB; blood
brain barier, Ref: Reference
showed that G-CSF treatment also decreases the BBB
permeability after injury leading to contradictory results
(17). The neuroprotective effect of G-CSF in neurons was
suggested by Schabitz et al., by demonstrating that intra-
venous administration of G-CSF after focal cerebral
ischemia reduced the volume of infarction and the number
of infiltrated neutrophils into the ischemic hemisphere 24
hours later. Furthermore, neuroprotective effect of G-CSF
is further supported by the finding that G-CSF protects
cerebellar granule cells exposed to glutamate in vitro (20).
Peripherally derived leukocytes not only release
proteolytic enzymes and free oxygen radicals but also
produce and secrete cytokines including tumor necrosis-
alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) which
mediate BBB injury and enhance the migration of blood
leucocytes into the injured tissue by modulating the
expressions of various surface antigens (62, 70, 71). The
possible mechanisms of G-CSF treatment on the prevention
of BBB damage may be through its ability to decrease the
levels of proinflammatory cytokines. A
series of reports
have demonstrated that G-CSF displays immunoregulatory
properties through interaction with its receptor. It has been
shown that G-CSF treatment
protects rodents against
lipopolysaccharide (LPS)-induced lethal
toxicity by
suppressing systemic TNF-alpha
production in vivo (72).
Such reduced cytokine production as well as by an increase
in production of anti-inflammatory counterregulatory
molecules has also been shown in peripheral blood
leukocytes from G-CSF-treated
healthy volunteers (73).
Also, G-CSF reduces the T cell infiltration and
inflammation within the CNS in experimental
allergic
encephalomyelitis by reducing interferon-gamma (IFN-
gamma) and increasing IL-4 and TGF-beta1 levels, and
with a reduction of systemic and lymphocyte TNF-alpha
production (74). Nishiki et al. have suggested that G-CSF
inhibits LPS-induced TNF-alpha
production in human
monocytes
through selective activation of JAK2/STAT3
pathways (75).
G-CSF activates JAK2 in turn activates a
transcription
factor, STAT3, triggering a signaling
transduction cascade that leads
to increase in cellular
inhibitor of apoptosis protein2 (cIAP2) expression in
human neutrophils resulting in their survival (76) (Figure
1). Activation of STAT3 by G-CSF has also been linked to
myeloid cell differentiation
(77). However, STAT5 seems
to be involved in G-CSF-dependent cell
proliferation (78).
Also, G-CSF activation of JAK2-STAT3 pathway and the
resulting induction of antiapoptotic proteins and inhibition
of apoptotic death of cardiomyocytes in the infarcted hearts
have been recently reported by Harada et al. (79). Schabitz
et al. showed for the first time that G-CSF treatment results
in increased STAT3 expression in the penumbra of the
infarction after focal cerebral ischemia in rats and they
have suggested that upregulation of STAT3 in neurons may
mediate the antiapoptotic effects of G-CSF (20).
Antiapoptotic effect of G-CSF in neural tissue was also
pointed by Park et al. by demonstrating reduced number of
TUNEL positive neurons in the penumbra following G-
CSF treatment after intracerebral hemorrhage in rats (17).
More recently, G-CSF has been reported to
protect cortical neurons in vitro against camptothecin-
induced and NO-induced apoptosis by reducing caspase-3
and poly(ADP-ribose) polymerase (PARP) cleavage (12).
Authors showed that G-CSF exposure results in rapid
STAT3 phosphorylation by JAK2 kinase in neurons, which
was inhibited by AG 490, a specific inhibitor of JAK2. The
authors further reported that G-CSF leads to increase of
protein levels of the antiapoptotic STAT3 target Bcl-X
L
(Figure 2)
(12). However, determination of activation levels
of the extracellular signal-regulated kinase (ERK) family
showed that while ERK1/2 was transiently and weakly
Neuroprotection by G-CSF
717
Figure 2. Figure shows the signaling pathways implicated
in G-CSF mediated neuronal survival. Activation of the
extracellular signal-regulated kinase (ERK) family
enhances neuronal survival. PI3-K/Akt and STAT3
signaling
pathways when activated prevent apoptotic cell
death by inhibiting activation of caspases and increasing
antiapoptotic protein members such as Bcl-xL.
ctivated by G-CSF, ERK5 kinase was strongly activated in
cultured neurons from rat cortex (12). ERK5, also known
as big MAP kinase 1 (BMK1), is a MAPK member whose
biological role is largely
undefined. However, it has been
suggested that ERK5 activation enhances neuronal survival
(80, 81). Schneider et al. also showed the activation of PI3-
K/Phosphoinositide-dependent
kinase (PDK)/Akt signaling
pathway by G-CSF in cortical neurons in vitro. They
suggested that anti-apoptotic action of G-CSF on neurons
mediated at least partially by the PI3-K/Akt pathway (12)
(Figure 2). It is well established that Akt, a downstream
target of PI3-K, is a critical anti-apoptotic factor in
controlling the balance between survival and apoptosis in
multiple cell systems including neurons by several
mechanisms such as by phosphorylating Bcl-2-associated
death protein (BAD) and caspase-9, and inducing Bcl-2
expression (82-86). PDK has also been shown to
phosphorylate and activate Akt to promote cell survival
(87).
There is strong evidence from previous studies
that G-CSF exerts antiapoptotic effect on many
hematopoietic cell lines by triggering various signal
pathways. G-CSF inhibits spontaneous cytochrome c
release and mitochondria dependent apoptosis of
myelodysplastic syndrome hematopoietic progenitors (88).
It has been reported that G-CSF-induced survival of
neutrophils
associates with inhibition of cleavage of Bid,
Bid/Bax translocation
to the mitochondria, and prevention
of subsequent release of
the proapoptotic mitochondrial
constituents. Moreover, G-CSF blocks both processing of
the initiator
caspase-8 and caspase-9 and the executioner
caspase-3 and their
specific enzymatic activities in
apoptotic neutrophils (89). Furthermore, G-CSF inhibits the
processing of proIL-1beta and the release of mature IL-
1beta in LPS-stimulated whole blood via the inhibition of
caspase-1 which is also known as interleukin-converting
enzyme (90). It has also been shown that mice deficient in
interleukin-1 converting enzyme are resistant to neonatal
hypoxic-ischemic brain damage (91). These mechanisms
suggest probable mechanisms for the anti-apoptotic effects
of G-CSF treatment on hematopoietic cell lines. However,
it needs to be studied whether G-CSF maintains viability of
neuronal cells by similar mechanisms. The precise
mechanism by which G-CSF exerts anti-apoptotic function
in the neural tissue in vivo is not well understood.
5.2. G-CSF, hematopoietic stem cell mobilization, and
brain repair
There is a growing interest to restore brain
function after stroke by transplanting stem cells. Various
stem cells especially porcine fetal cells, neural stem cells
(NSCs), and bone marrow stem cells (BMCs) currently are
under investigation by various independent research groups
(92-95).
Recent studies have focused on the potential
plasticity of bone marrow stem cells (BMCs) after stroke.
There are two populations of bone marrow stem cells:
hematopoietic stem cells (HSCs), which can differentiate
into every type of mature blood cells; and mesenchymal
stem cells (MSCs), which can differentiate into adipose,
muscle, bone, cartilage, endothelium, hepatocyte, glia, and
neuron under appropriate stimuli in vitro and in vivo (96-
104). However, some controversy still exists about
differentiation of BMCs into neural tissues (105-107).
In previous studies, it has been reported that after
direct intrastriatal, intracarotid, and intravenous delivery,
MSCs migrate to the ischemic area, survive and
differentiate into neuronal and glial cell types, and improve
functional recovery after experimental stroke (92-94). The
migration of MSCs is supported by evidence that Y
chromosome-positive
neuron-like cells are seen in
postmortem brain samples from females who
had received
bone marrow transplants from male
donors in humans (95).
Although various studies attributed the beneficial
results of BMC treatment after stroke to possible neuronal
plasticity, the precise mechanisms of the functional benefits
still remains unclear. Several studies have showed
functional benefits may result either from enhanced
neurogenesis or from a favorable impact of stem cells or
their released cytokines which stimulate angiogenesis and
reduce cell death. It has been shown that treatment of
stroke with human MSCs enhances angiogenesis in the
host
brain which is mediated by increase
in levels of endogenous
rat vascular endothelial growth factor (VEGF) and VEGF-
Neuroprotection by G-CSF
718
receptor 2 (108). Other studies have shown reduction of
apoptosis, increase in BDNF and nerve growth factor in the
penumbral zone of the lesion, and the proliferation of
endogenous cells in the subventricular zone after
administration of human MSCs in rats (109). Moreover, it
has been suggested that intrastriatal transplantation of
mouse MSCs restores cerebral blood flow and BBB to
normal levels, elevates expression levels of neurotrophic
factors including activin A, the glial cell line-derived
neurotrophic factor, and transforming growth factor-beta 1
and 2 (110). So, these mechanisms of recovery may also be
due to the release of trophic factors into the damaged area
rather than neuronal differentiation and implant integration
to the injured ischemic site.
Recently, Bang et al. have examined the
feasibility, efficacy, and safety of cell therapy using
culture-expanded autologous MSCs in patients with
ischemic stroke (111). They have reported that intravenous
infusion of 1x10
8
autologous MSCs to patients, more than 1
month after the onset of stroke symptoms, improves
outcome (111). However, they stress the need of future
double-blind studies with larger cohorts to reach a
definitive conclusion regarding the efficacy of MSC
therapy (111).
It is well known that administration of G-CSF
mobilizes HSCs from bone
marrow into peripheral blood
(13). G-CSF has been shown to result in significant
decrease in infarct volume and enhance survival rate after
focal cerebral ischemia in mice, and these benefits have
been suggested to be mediated by mobilization of
autologous HSCs into circulation and their contribution to
brain repair (18). Moreover, Shyu et al. showed that G-CSF
treatment increases BrdU
+
cells coexpressing the neuronal
phenotypes of Neu-N
+
and microtubule-associated protein-
2 (MAP-2)
+
cells as well as the glial phenotype of GFAP
+
cells in the ischemic cortical areas of G-CSF-treated rats
after focal cerebral ischemia in rats (16). They have
suggested that G-CSF treatment enhances translocation of
HSCs into ischemic brain, and significantly improves
lesion repair by improving neuronal plasticity and
vascularization (16). Interestingly, they have reported that
neutralization of CXC chemokine receptor 4 (CXCR4) by
its specific antibody results in a slight reduction in infarct
volume in G-CSF-treated rats (16).
The chemokine receptor CXCR4 is expressed in
cells of both the immune and the central nervous systems
and can mediate migration of resting leukocytes and
hematopoietic progenitors in response to its ligand, stromal
cell-derived factor-1 (SDF-1). SDF-1, the strong
chemoattractant was also shown
to be crucial for cerebellar
development (112, 113). Moreover, it has been suggested
that SDF-1/CXCR4 system may control cerebral infiltration
of CXCR4-carrying leukocytes
during cerebral ischemia,
and
contribute to ischemia-induced neuronal plasticity
(114). SDF-1 also play pivotal role in the
migration of
NSCs toward the ischemic core and penumbra, and
enhances proliferation,
promotes chain migration and
activates intracellular
molecular pathways mediating
engagement (115). Both microglial cells and astrocytes in
humans can express chemokine receptors including CCR4,
CCR5, CCR6, CXCR2, and CXCR4 and activation of the
cells with TNF-alpha and IFN-gamma changes the
expression levels of these chemokine receptors (116, 117).
CXCR4-mediated signaling in astroglioma
cells also
provides pathway for these cells to express chemokines
involved in angiogenesis and inflammation (118). Taken
together these data, a better understanding
of this complex
dynamic and its relationships with G-CSF may permit us to
understand precise mechanisms of G-CSF related
functional recovery after stroke.
5.3. G-CSF and neurogenesis
The subgranular zone of dentate gyrus (DG) in
the hippocampus and subventricular zone (SVZ) of the
lateral ventricle are the main areas of the adult brain that
undergo neurogenesis and gliogenesis (119-121). The SVZ
in the forebrain is the largest source of neural stem cells
(NSCs) and progenitor cells which can differentiate into
neurons, astrocytes, and oligodendrocytes (120, 122). There
is accumulating evidence indicating that this
neurogenerative capacity of the adult CNS could open
novel therapeutic approaches to neurodegenerative diseases
based on the use of NSC transplantation. Previous in vivo
experiments showed that transplanted human NSCs
survive, migrate, and differentiate in host brain and
promote functional recovery after intracerebral hemorrhage
and cerebral ischemia (123-125). Endogenous NSCs
sustain neurogenesis and gliogenesis in response to several
different injuries including ischemic and traumatic brain
injury (126-128).
It has been reported that outcome from ischemia
appears to be worse when neurogenesis is inhibited by
irradiation in gerbils (129). However, augmentation of this
self-repair phenomenon by exogenous agents can further
enhance neurogenesis and
might also have therapeutic
applications after CNS disorders. Enhanced neurogenesis
with growth factors such as EPO or heparin-binding
epidermal growth factor-like growth factor (HB-EGF) have
been reported to decrease infarct size and improve
neurological functions after stroke in rats (130, 131).
More recently, G-CSF has been shown to have
functional role in differentiation of adult rat NSCs both in
vitro and in vivo (12). Analysis of G-CSF responsiveness in
the cultured NSCs from the rat SVZ or hippocampal region
indicates that G-CSF exposure results mainly in increase in
the population of cells expressing mature neural markers
such as beta-III-tubulin, neuron-specific enolase (NSE),
and MAP-2 without elevating the number of immature stem
cells (12). Schneider et al. found that peripheral infusion of
G-CSF enhances recruitment of progenitor cells from the
lateral ventricular wall into the ischemic area of the
neocortex in rat (12). Moreover, authors reported that G-
CSF increases hippocampal neurogenesis not only in
ischemic animals, but also in the intact, non-ischemic rat.
Based on this evidence, they argued that G-CSF may
enhance structural repair and function even in healthy
subjects or at long intervals after stroke (12).
Neuroprotection by G-CSF
719
The intracellular mechanisms that regulate
neurogenesis both in normal and ischemic conditions
remain unclear. Increased cyclic guanosine monophosphate
(cGMP), a molecular messenger involved in regulation of
cellular proliferation, has been reported to enhance
neurogenesis (132). Recently, Wang et al. showed that PI3-
K/Akt pathway mediates cGMP enhanced proliferation of
adult progenitor cells derived from the SVZ of the rat
(133). Moreover, it has been suggested that stroke-
enhanced neuroblast migration is independent of cell
proliferation and survival, and PI3-K/Akt signal
transduction pathway mediates neuroblast migration after
stroke (134). The activation of PI3-K/Akt signaling
pathway by G-CSF has been shown in cortical neurons in
vitro (12). The anti-apoptotic effect of G-CSF on neurons is
dependent on activation of PI3-K/Akt signaling
pathway. It
can be further hypothesized that G-CSF may also augment
neurogenesis and neuroblast migration by activation of PI3-
K/Akt signaling
pathway.
It has been shown that CXCR4 is expressed in rat
and human neural progenitor cells (135). The
CXCR4/SDF-1 system is important in mediating specific
migration of neural progenitor cells to the site of ischemic
damaged neurons (115, 136). As discussed previously,
neutralization of CXCR4 by its specific antibody has been
reported to reduce neuroprotective effect of G-CSF in rats
after stroke (16). Thus, it can be hypothesized that
CXCR4/SDF-1 system may play important role in G-CSF
induced neurogenesis. Additional studies however will be
necessary to clarify this issue.
It is well known that the regulation of adult
neurogenesis and the maintenance of stem cell renewal are
modulated by various regulatory mechanisms including
growth factors. Although the ability of G-CSF to stimulate
the neurogenesis shows a lot of promise, additional studies
are required for determining the molecular basis of this
effect.
6. SUMMARY AND PERSPECTIVE
An increasing amount of data suggests a
neuroprotective role for G-CSF. The molecular
mechanisms of neuroprotective effect of G-CSF especially
under in vivo settings still remain unclear. However, the
neuroprotective property of G-CSF may be due, at least in
part, to the mobilization of BMCs to the injured brain.
However, the precise mechanism of BMCs-mediated brain
repair and/or neuroprotection after stroke is still under
investigation. Furthermore, G-CSF has anti-apoptotic, anti-
inflammatory, immunomodulatory, and neurogenesis-
inducing effects which provide other potential mechanisms
that may be responsible for neuroprotection and long term
benefits.
Neuroprotective effects of G-CSF however,
should be tested in multiple animal models in different
species with different time points, different doses and
application routes to confirm results. Also, the use of G-
CSF in conjunction with rt-PA should be tested for safety
before starting clinical trials in humans.
7. ACKNOWLEDGEMENT
This study was partially supported by grants from
NIH NS45694, HD43120, and NS43338 to JHZ.
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Cereb Blood Flow Metab doi:10.1038/sj.jcbfm.9600172
Key Words: Angiogenesis, Apoptosis, Cytokine,
Granulocyte-colony stimulating factor, Growth factor,
Inflammation, Neurogenesis, Neuroprotection, Plasticity,
Stem cell, Stroke, Review
Send correspondence to: John H. Zhang M.D., Ph.D.,
Division of Neurosurgery, Loma Linda University Medical
Center, 11234 Anderson Street, Room 2562B, Loma Linda,
California 92354, Tel: 909-558-4723, Fax: 909-558-0119,
E-mail: johnzhang3910@yahoo.com
http://www.bioscience.org/current/vol12.htm
