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Hypoxia-induced Apoptosis
Abstract
Brain ischemia is the underlying cause of neuron death during stroke and brain trauma.
In addition to necrosis, neural cells exposed to the condition of oxygen depletion during ischemia
can also undergo apoptosis, which significantly contributes to brain injury. Adrenomedullin
(AM), a multifunctional hormone, in combination with its enhancing binding protein, AMBP-1,
has been shown to effectively reduce tissue damage under hemorrhage and ischemia/reperfusion
in animal models. To evaluate a beneficial effect of AM/AMBP-1 administration in brain
ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human
neuroblastoma SH-SY5Y cells. After exposure to 1% O2 for 20 h, the neural cells were injury
with a reduction of the cellular ATP levels and an increase of lactate dehydrogenase released in
culture medium. Pre-administration of AM/AMBP-1 significantly reduced the hypoxia-induced
cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and
activation of caspase-3 in comparison to those cells exposed to hypoxia alone. AM/AMBP-1
prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after
hypoxia exposure. Correspondingly, treatment of forskolin, a stimulator of cAMP production,
also protected neural cells from hypoxia-induced injury. Inhibition of PKA, a downstream target
of cAMP, by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced
apoptosis. These results indicate that AM/AMBP-1 elevates cAMP levels, followed by
activating PKA activity, to protect neural cells from the injury caused by hypoxia. This study
suggests that AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from
brain ischemia.
Keywords: Adrenomedullin; Adrenomedullin binding protein-1; Neural cells; Hypoxia;
Apoptosis; cAMP; Protein kinase A
1. Introduction
Cerebral ischemia results from an insufficient supply of blood and oxygen to the brain
after injuries. It is the underlying cause of neuron death during stroke and brain trauma, two
leading perpetrators of death in the United States [1]. According to the National Center of Injury
Prevention and Control, about 1.4 million Americans sustain traumatic and ischemic brain
injuries annually. More than 10% of those injured are either severely disabled or die, and
patients surviving from this disease require massive sending on long-term rehabilitations such as
speech, occupational, and physical therapies [1]. So far, very limited treatments are available for
patients with stroke and brain trauma. Although tissue plasminogen activator has been approved
by FDA, it is only beneficial for a small number of patients [2]. Development of new
therapeutics is imperative in this field.
During the brain damage, two major processes that lead to cell death:
necrosis and apoptosis. Within the core of the ischemic area, where blood
flow is most severely restricted, necrotic cell death is dominate and occurs
within a few days after damage. In the periphery of the ischemic area,
where collateral blood flow can buffer the full effects of the damage, which
may start several days after transient ischemia, is mainly apoptosis [3,4]. In
principle, the apoptotic cascades during brain damage are reversible, which
can be the major target of therapeutic interventions [5-7].
Adrenomedullin (AM), a potent vasoactive peptide was discovered by Kitamura et al [8].
This peptide, consisting of 52 amino acids, acts as a circulating hormone to induce various
biological activities in a paracrine or autocrine manner [8]. AM is multifunctional in nature and
can regulate the proliferation, differentiation, and migration of a number of different cell types as
well as exert its regulatory abilities on blood pressure, water, and electrolyte balance [9]. A
specific AM binding protein (i.e., AMBP-1) has been identified in mammalian blood [10] and
further purified and characterized by Pio et al as human complement factor H [11]. AMBP-1 can
facilitate AM to confine in the interstitial space for the accession to AM receptors and modulate
AM biological activity. Moreover, AMBP-1 can prolong half-life of AM in the circulation by
protecting it from protease degradation (reviewed by [12]). Administration of AM combined
with AMBP-1 has been shown to have protective effects on tissue damage caused by low oxygen
and blood supply conditions, such as gut ischemia-reperfusion [13] and hemorrhagic shock [14]
in animal models. These studies prompt us to evaluate the effectiveness of AM/AMBP-1 on
protecting brain damage under ischemic condition.
Here, we used an in vitro cell model to study neuron injury caused by oxygen depletion.
Human neuroblastoma SH-SY5Y cells were treated with retinoic acid to differentiate into
neuron-like cell type and exposed to hypoxic condition at 1% O2. We first identified the injury
and apoptosis of differentiated SH-SY5Y cells after hypoxia exposure. We then examined the
effect of AM in combined with AMBP-1 on hypoxia-induced cell injury and apoptosis. We
further elucidated the signaling pathway mediated by AM/AMBP-1 in regulating hypoxiainduced apoptosis in differentiated SH-SY5Y cells.
2.5. Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
SH-SY5Y cells were grown on a chamber slide. After 20 h in hypoxia, cells were fixed
with 4% paraformaldehyde for 1 h at room temperature. Cells were washed with PBS
(phosphate buffer saline; pH 7.2) and permealized on ice for 2 min with 0.1% Triton X-100/0.1%
sodium citrate. After washing with PBS, cells were incubated with a TUNEL reaction mixture
from Roche Applied Science (Indianapolis, IN) at 37C for 1 h. The slides were sealed with
Vectashield mounting medium containing propidium iodide (PI) from Vector Labs (Burlingham,
CA). The green fluorescent, TUNEL-positive cells were counted under a fluorescent
microscope. Total cell number in the field was determined by PI staining.
manufacturer's instructions. The cAMP levels and PKA activity were normalized to protein
concentration.
3. Results
3.1. Effect of AM/AMBP-1 on cellular damage under hypoxia
To assess the cellular damage of SH-SY5Y cells under hypoxia (1% O2), we measured
the ATP levels in these cells. The correlation between ATP levels and cell death has been well
established in neural cells [17]. Oxygen depletion for 20 h resulted in a significant decrease of
ATP levels in SH-SY5Y cells by 32.6% in comparison to normoxia (Fig. 2). We then examined
the effect of AM/AMBP-1 on hypoxia-induced cell injury. It has been reported that an
administration of AM at 100 nM exert an optimal effect on inhibiting cytokine release in
macrophage stimulated with lipopolysaccharide [18]. Furthermore, AM co-administered with
AMBP-1 at ratio of 2:1 has better outcomes than AM treatment alone [18]. Therefore, we
selected two doses of AM/AMBP-1 (100/50 and 200/100 nM) to examine whether these
molecules could protect SH-SY5Y cells from hypoxia-induced cell injury. As shown in Fig. 2,
AM alone and AMBP-1alone at 100 nM and 50 nM, respectively, didnt change the ATP levels of
cells exposed to hypoxia. When AM was administered at 200 nM, there was a slight increase of
ATP levels in hypoxia-treated cells, but it didnt reach a statistical significance. However, when
AM co-administered with AMBP-1 at 100/50 nM, the ATP levels of hypoxia-treated cells had an
83.4% increase in comparison to hypoxia controls (Fig. 2). Similar results were also observed at
200/100 nM of AM/AMBP-1 (Fig. 2). Thus, the AM/AMBP-1 at 100/50 nM was applied to the
following experiments.
To confirm the protective effect of AM/AMBP-1 on hypoxia-induced injury, we analyzed
the release of LDH in the supernatant, another marker for measuring cell injury and death. LDH
activity was increased by 41.4% under the condition of hypoxia in comparison to the normoxia
controls (Fig. 3A). When adding 100/50 nM of AM/AMBP-1, the LDH activity of the hypoxia-
treated SH-SY5Y cells reduced to the level similar to that of the normoxia controls (Fig. 3A).
Correspondingly, the ATP levels of AM/AMBP-1 administered cells were significantly higher
than those of cells exposed to hypoxia alone (Fig. 3B).
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4. Discussion
In this study, we used human neuroblastoma SH-SY5Y cells differentiated to neuron-like
cell type, followed by hypoxia (1% O2) exposure, to simulate brain ischemia. We first
demonstrated that hypoxia caused damage of SH-SY5Y cells shown a reduction of cellular ATP
levels and an increase of LDH released into supernatant. By administration of AM/AMBP-1, the
cell injury induced by hypoxia was significantly alleviated. The cellular ATP levels and release
of LDH in the AM/AMBP-1-treated cells exposed to hypoxia were comparable to those in cells
grown in normal condition. Although we doubled amount of AM/AMBP-1 administration from
100/50 nM to 200/100 nM, we didnt observe further improvement of cell-injury, indicating that
AM/AMBP-1 at 100/50 nM was an optimal dose for treating SH-SY5Y cells to protect them
from hypoxia-induced injury. Furthermore, AM co-administered with AMBP-1 had a better
protective effect on hypoxia-induced injury than administration of AM alone in SH-SY5Y cells,
which is agreed with a previous study shown an enhancement of AM activity in combined with
AMBP-1 [18]. Administration of AMBP-1 alone had no protective effect on the cell injury
induced by hypoxia.
Although the small intestine is the major source of AM production under both
physiological and pathophysiological conditions [22], AM is also produced in the central nervous
system (CNS) at a relative low amount [23,24]. AM plays important roles in the maintenance of
homeostasis through central mechanisms [25]. The primary biosynthesis site of AMBP-1 is liver
[26]; however, AMBP-1 can be detected in the rat brain by immunohistochemical staining [27].
Under inflammation and ischemia/reperfusion injury in the gut, the expression of AMBP-1 is
down-regulated [13,28]. Administration of AM/AMBP-1 has been shown to prevent tissue
damage in animal models of intestinal and hepatic ischemia/reperfusion injury [13,29]. In view
of that, the observation of the protective effect of AM/AMBP-1 in cultured neural cells exposed
to hypoxia provides a rational strategy of using AM/AMBP-1 to treat brain damage caused by
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ischemia. There is a concern on crossing blood-brain barrier (BBB) for AM/AMBP-1 to reach
the therapeutic targets. It is well known that stroke can disrupt the BBB [30,31]. A previous
study has demonstrated that distribution of AM in the brain increases significantly in sepsis [32],
suggesting that brain permeability increases under disease conditions. In this scenario, we
expect that BBB permeability to human AM and AMBP-1 will be markedly enhanced after
ischemic stroke.
After demonstrating that AM/AMBP-1 has neuroprotective properties under hypoxic
conditions, we then elucidated the potential mechanism responsible for this novel protective
effect. It is widely accepted that hypoxia promotes cell apoptosis [33], we first tested whether
AM/AMBP-1 can decrease hypoxia-induced neural cell apoptosis. By using TUNEL assay and
detection of cleaved caspase-3 from Western blotting, we confirmed an induction of apoptosis in
SH-SY5Y cells under hypoxic condition of 1% O2. When AM/AMBP-1 was administered, the
number of apoptotic cells and levels of cleaved caspase-3 were significantly reduced in neural
cells exposed to hypoxia. The protective effect of AM on apoptosis has also been observed in
cultured rat endothelial cells under serum deprivation [34]. Apoptosis has pathological
consequences on immune and other cell function under various detrimental circulatory
conditions such as ischemia/reperfusion injury [35,36]. Moreover, inhibition of cell apoptosis
has proven to be beneficial in reducing inflammation [37]. Thus, an inhibition of neural cell
apoptosis not only can directly reduce brain injury but also can prevent the damage caused by
inflammation after ischemia.
AM and AMBP-1 are extracellular molecules. The next question is how intracellular
apoptotic cascade in SH-SY5Y cells can be regulated by AM/AMBP-1 during hypoxia. AM
mediates its activities via heterodimeric receptors that are composed of a seven transmembrane
calcitonin-receptor-like receptor (CRLR) [38] and a receptor activity modifying protein (RAMP)
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[39,40]. The RAMP family comprises three members (RAMP1, RAMP2 and RAMP3) [39,40].
AM receptors are widely distributed in most cell populations, including neurons [39,40]. In this
study, we demonstrated that treatment of AM receptor antagonist diminished the protective effect
of AM/AMBP-1 on hypoxia-induced injury, indicating that the interaction between AM and its
receptors was required for this protection. Several signaling pathways can be stimulated by AM
in various cell types [41] and the cAMP-PKA signaling pathway has been shown to mediate
many of AM effects, including cell proliferation and migration [42,43], cell protection [44], and
cell regeneration [45]. It has been demonstrated that activation of the cAMP-PKA pathway can
protect neuronal cells against apoptosis and improve survival [46,47]. Therefore, we examined
whether the cAMP-PKA pathway could mediate AM/AMBP-1 in regulating hypoxia-induced
apoptosis. We first demonstrated that cAMP levels were decreased in SH-SY5Y cells after
hypoxia and AM/AMBP-1 treatment could effectively prevent the reduction of cAMP levels.
Correspondingly, by adding forskolin, a stimulator of cAMP production, cell damage and
activation of caspase-3 induced by hypoxia were reduced in SH-SY5Y cells. To further identify
whether the protective effect by the elevated cAMP levels is PKA-dependent, we demonstrated
that down-regulation of PKA activity was prevented by administration of AM/AMBP-1 after
hypoxia. Furthermore, we inhibited the PKA activity by treating cells with KT5720, a
pharmacological inhibitor of PKA. KT5720 overturned the protective effect of AM/AMBP-1 on
hypoxia-induced injury and activated caspase-3 to the levels as same as hypoxia alone. These
mechanistic studies involved in the regulation of hypoxia-induced apoptosis by AM/AMBP-1 in
neural cells are summarized in Fig. 9. This proposed model indicates that the beneficial effect of
AM/AMBP-1 on protecting neural cells from hypoxia-induced cell injury is mediated by the
cAMP-PKA pathway.
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16
Figures
100m
Fig. 1. Differentiation of human neuroblastoma cells. SH-SY5Y cells were (A) untreated or (B)
differentiated through treatment with retinoic acid (10 M). The differentiated cells show typical
neuron morphology, such as dendrites and neuronal processes.
17
1200
# #
1000
800
* *
600
400
200
0
AM
AMBP-1
100
-
200
-
- 100
50 50
200 nM
100 nM
Hypoxia
Fig. 2. Effect of AM, AMBP-1, and AM/AMBP-1 on cellular ATP levels after hypoxia.
Differentiated SH-SY5Y cells were incubated in normoxia or hypoxia (1% O2) for 20 h, in the
presence of AM alone, AMBP-1 alone, or AM/AMBP-1 at the indicated concentration. ATP
levels in total cell lysates were determined. Data are presented as means SE (n=6). *P<0.05
vs. Normoxia (open bar); #P<0.05 vs. Hypoxia alone.
18
L DH (mU/mg prot)
100
80
60
40
20
0
1200
1000
800
600
400
200
0
Normoxia
Vehicle AM/AMBP-1
Hypoxia
Fig. 3. Effect of AM/AMBP-1 on LDH release after hypoxia. Differentiated SH-SY5Y cells
were incubated in normoxia or hypoxia (1% O2) for 20 h, in the presence of vehicle or
AM/AMBP-1 (100/50 nM). The release of LDH in the culture medium (A) and cellular ATP
levels in cell lysate (B) were measured. Data are presented as means SE (n=4-6). *P<0.05 vs.
Normoxia; #P<0.05 vs. Hypoxia+Vehicle.
19
Normoxia
Hypoxia
Hypoxia+AM/AMBP-1
100 m
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Cleaved Caspase 3/
-Actin
0.15
-Cleaved Caspase 3
--Actin
0.12
0.09
0.06
0.03
0.00
Normoxia
Vehicle AM/AMBP-1
Hypoxia
Fig. 4. Effect of AM/AMBP-1 on formation of apoptotic cells after hypoxia. Differentiated SHSY5Y cells were incubated in normoxia or hypoxia (1% O2) for 20 h, in the presence of vehicle
or AM/AMBP-1 (100/50 nM). Apoptotic cells were identified by TUNEL assay with green
fluorescence labeling. (A) A representative filed of cells under fluorescence microscope. (B)
The apoptotic rate was determined by the number of TUNEL-positive cells divided by the total
number of cells in 6 fields. Data are presented as means SE (n=6-8). (C) Total cell lysates
20
were subjected to Western blot analysis. Representative blots against cleaved caspase 3 and actin are shown. Blots were scanned and quantified with the densitometry. Band intensity of
cleaved caspase-3 was normalized to the corresponding band intensity of -actin. Data are
presented as means SE (n=4). *P<0.05 vs. Normoxia; #P<0.05 vs. Hypoxia+Vehicle.
300
250
200
150
100
50
0
Normoxia
Vehicle AM/AMBP-1
Hypoxia
Fig. 5. Effect of AM/AMBP-1 on levels of intracellular cAMP after hypoxia. Differentiated SHSY5Y cells were incubated in normoxia or hypoxia (1% O2) for 20 h, in the presence of vehicle
or AM/AMBP-1 (100/50 nM). cAMP levels in total cell lysates were determined. Data are
presented as means SE (n=8-10). *P<0.05 vs. Normoxia; #P<0.05 vs. Hypoxia+Vehicle.
21
1000
A
#
800
600
400
200
0
-Cleaved Caspase 3
0.10
--Actin
0.08
0.06
0.04
0.02
0.00
Normoxia
Fig. 6. Effect of forskolin on cellular ATP levels and cleavage of caspase-3 after hypoxia.
Differentiated SH-SY5Y cells were incubated in normoxia or hypoxia (1% O2) for 20 h, in the
presence of vehicle or forskolin (1 M). (A) ATP levels were determined in total cell lysate. (B)
The cleaved caspase-3 was determined by Western blotting as described in Fig. 4. Data are
presented as means SE (n=4-10). *P<0.05 vs. Normoxia; #P<0.05 vs. Hypoxia+Vehicle.
22
Relative PK A activity
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Normoxia
Vehicle AM/AMBP-1
Hypoxia
Fig. 7. Effect of AM/AMBP-1 on PKA activity after hypoxia. Differentiated SH-SY5Y cells
were incubated in normoxia or hypoxia (1% O2) for 20 h, in the presence of vehicle or
AM/AMBP-1 (100/50 nM). PKA activity in total cell lysates was determined and normalized to
protein concentration. The PKA activity of the normoxia was considered to be 1. Data are
presented as means SE (n=6-8). #P<0.05 vs. Hypoxia+Vehicle.
23
-Cleaved Caspase 3
--Actin
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Normoxia
Fig. 8. Effect of PKA inhibitor on cleavage of caspase-3 after hypoxia. Differentiated SHSY5Y cells were incubated in normoxia or hypoxia (1% O2) for 20 h, in the presence of vehicle
or AM/AMBP-1 (100/50 nM) plus KT5720 (1 M). The cleaved caspase-3 was determined by
Western blotting as described in Fig. 4. Data are presented as means SE (n=4). *P<0.05 vs.
Normoxia.
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AMBP1
AM
Hypoxi
a
CRL
R
AC
RAMP
cAMP AT
P
Forskoli
n
KT572
0
PKA
Caspase-3
Neural
Cell
Apoptos
is
25
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