Recombinant Milk Fat Globule–EGF Factor-8 Reduces
Oxidative Stress via Integrin β3/Nuclear Factor Erythroid
2–Related Factor 2/Heme Oxygenase Pathway in
Subarachnoid Hemorrhage Rats
Fei Liu, MD, PhD; Qin Hu, MD, PhD; Bo Li, MD, PhD; Anatol Manaenko, PhD;
Yujie Chen, MD; Junjia Tang, MD; Zongduo Guo, MD, PhD; Jiping Tang, MD;
John H. Zhang, MD, PhD
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Background and Purpose—Milk fat globule–EGF factor-8 (MFGE8) has been reported to be neuroprotective in ischemic
stroke. However, the effects of MFGE8 in early brain injury after subarachnoid hemorrhage (SAH) have not been investigated.
We investigated the role of MFGE8 in early brain injury and the potential mechanisms in antioxidation after SAH.
Methods—Two dosages (1 μg and 3.3 μg) of recombinant human MFGE8 were injected intracerebroventricularly at 1.5
hours after SAH. SAH grades, neurological scores, and brain water content were measured at 24 and 72 hours. For
mechanistic study, MFGE8 siRNA, integrin β3 siRNA, and heme oxygenase (HO) inhibitor SnPP IX were used for
intervention. The oxidative stress and expression of MFGE8, integrin β3, HO-1, extracellular signal-regulated kinase, and
nuclear factor erythroid 2–related factor 2 were measured by Western blots 24 hours after SAH.
Results—The expression of MFGE8 and HO-1 increased and peaked 24 hours after SAH. Administration of recombinant
human MFGE8 decreased brain water content and improved neurological functions both at 24 hours and at 72 hours
after SAH. Recombinant human MFGE8 reduced oxidative stress and enhanced the expression of extracellular signalregulated kinase, nuclear factor erythroid 2–related factor 2, and HO-1; and the effects were abolished by integrin β3
siRNA and HO inhibitor SnPP IX.
Conclusions—Recombinant MFGE8 attenuated oxidative stress that may be mediated by integrin β3/nuclear factor erythroid
2–related factor 2/HO pathway after SAH. Recombinant MFGE8 may serve as an alternative treatment to ameliorate
early brain injury for SAH patients. (Stroke. 2014;45:3691-3697.)
Key Words: heme oxygenase-1 ◼ MFGE8 protein, rat ◼ oxidative stress ◼ subarachnoid hemorrhage
A
neurysmal subarachnoid hemorrhage (SAH) is one of
the most life-threatening diseases with high mortality
and disability rates.1 Early brain injury has been reported as
the primary cause of mortality in SAH patients and has been
considered as a primary target for treatment.2–4 One of the
key factors involved in the pathogenesis of early brain injury
is oxidative stress,5 which is caused by free radicals, including excess production of reactive oxygen species and reactive nitrogen species. Therefore, an antioxidative strategy has
attracted attention in the treatment of SAH.
Milk fat globule–EGF factor-8 (MFGE8) is a multifunctional glycoprotein originally identified as part of the milk
fat globule membrane. MFGE8 seems to be instrumental
in cell-cell interactions and has been involved in diverse
physiological and pathophysiological functions, including
fertilization,6 angiogenesis,7 and phagocytosis of apoptotic
cells.8 More recent studies have shown that MFGE8 regulates
adaptive immune responses9 and inflammation.10 In the central nervous system, administration of recombinant human
MFGE8 (rhMFGE8) has neuroprotection against cerebral
ischemia through suppression of inflammation and apoptosis
after brain ischemia.10,11 rhMFGE8 also exhibits significant
antioxidative effects through increasing heme oxygenase-1
(HO-1) in the Alzheimer disease model.12 However, the
effects of MFGE8 in early brain injury after SAH have not
been investigated. In the present work, we addressed the
role of MFGE8 in SAH. We showed for the first time that
rhMFGE8 attenuated early brain injury through suppression of the oxidative stress involving integrin β3–dependent
pathway.
Received July 1, 2014; final revision received September 24, 2014; accepted September 30, 2014.
From the Department of Physiology and Pharmacology (F.L., Q.H., B.L., A.M., Y.C., Junjia Tang, Z.G., Jiping Tang, J.H.Z.) and Department of
Neurosurgery (J.H.Z.), Loma Linda University School of Medicine, Loma Linda, CA; and Department of Neurosurgery, the Third Xiangya Hospital,
Central South University, Changsha, Hunan, China (F.L.).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
114.006635/-/DC1.
Correspondence to John H. Zhang, MD, PhD, Department of Neurosurgery and Department of Physiology and Pharmacology, Loma Linda University
School of Medicine, 11041 Campus St, Risley Hall, Room 219, Loma Linda, CA 92354. E-mail [email protected]
© 2014 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.114.006635
3691
3692 Stroke December 2014
A
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B
Figure 2. Spatial expression of milk fat globule–EGF factor-8
(MFGE8) after subarachnoid hemorrhage (SAH). MFGE8 is
seldom expressed in brain in sham animals, and increased in
microglials, but not in neurons and astrocytes at 24 hours after
SAH. Scale bar=30 μm in (A) and 100 μm in (B). GFAP indicates
glial fibrillary acidic protein; HO-1, heme oxygenase-1; and
NeuN, neuronal nuclei.
SAH Model and Experimental Protocol
Figure 1. Time course of subarachnoid hemorrhage (SAH) grade,
neurological scores, and expression of milk fat globule–EGF factor-8 (MFGE8) and heme oxygenase-1 (HO-1) after SAH surgery. A,
SAH grades were decreased with time from 6 hours to day 7 after
SAH. B, Animals showed severely neurological deficits from 6 hours
after SAH, improved as time passed, and showed no significant difference with sham at day 7. C and D, The temporal expression of
MFGE8 and HO-1 after SAH. Both of them increased after SAH and
peaked at 24 hours. n=6 for each group. *P<0.05 vs sham.
Methods
All experiments were approved by the Institutional Animal Care and
Use Committee of Loma Linda University.
Two hundred ten male (280–320 g) Sprague-Dawley rats
(Indianapolis, IN) were used. The endovascular perforation model
of SAH in rats was performed as reported previously.13 Briefly, with
3% isoflurane anesthesia, a sharpened 4-0 monofilament nylon suture
was inserted rostrally into the right internal carotid artery from the
external carotid artery stump and perforated the bifurcation of the anterior and middle cerebral arteries. Sham-operated rats underwent the
same procedures, except the suture was withdrawn without puncture.
Two dosages (1 μg and 3.3 μg) of rhMFGE8 were injected intracerebroventricularly at 1.5 hours after SAH. SAH grades, neurological scores, and brain water content were measured at 24 and 72 hours.
To study mechanisms, MFGE8 small interfering RNA (siRNA), integrin β3 siRNA, and HO inhibitor SnPP IX were used for intervention.
The oxidative stress and expression of MFGE8, integrin β3, HO-1,
extracellular signal-regulated kinase (ERK), and nuclear factor erythroid 2–related factor 2 (Nrf2) were measured by Western blots at
24 hours after SAH.
Intracerebroventricular Drug Administration
Intracerebroventricular drug administration was performed as previously described.14 Briefly, rats were placed in a stereotaxic apparatus under 2.5% isoflurane anesthesia. The needle of a 10-μL
Hamilton syringe (Microliter 701; Hamilton Company, Reno, NV)
was inserted through a burr hole into the right lateral ventricles at
the following coordinates relative to bregma: 1.5 mm posterior, 1.0
mm lateral, and 3.2 mm below the horizontal plane of the skull.
rhMFGE8 (Sigma) and SnPP IX (Santa Cruz Biotechnology, 3.3
μg) were injected, respectively, at 1.5 hours after SAH induction
by a pump at a rate of 0.5 μL/min, respectively. MFGE8 siRNA,
Liu et al MFGE8 and Oxidative Stress 3693
integrin β3 siRNA, and scrambled siRNA (500 pmol/3 μL, Santa
Cruz Biotechnology) were injected at 2 days before SAH induction
by a pump at a rate of 0.5 μL/min.
SAH Grade
applied in the dark for 1 hour at 21°C. For negative controls, the primary antibodies were omitted and the same staining procedures were
performed. The sections were visualized with a fluorescence microscope, and the photomicrographs were saved and merged with Image
Pro Plus software (Olympus, Melville, NY).
The severity of SAH was blindly evaluated using the SAH grading scale
at the time of euthanasia, as previously reported.15 Rats with mild SAH
(SAH grades ≤7 at 24 hours and SAH grades ≤5 at day 7) were excluded
from the study.16 A total of 16 animals were excluded as a result of mild
SAH (SAH, 2; SAH+vehicle, 3; SAH+10 μg/kg rhMFGE8, 2; SAH+3
μg/kg rhMFGE8, 1; SAH+MFEG8 siRNA, 2; SAH+Scramble siRNA,
2; SAH+rhMFGE8+Scramble+siRNA, 1; SAH+rhMFGE8+SnPP IX,
1; SAH+ rhMFGE8+integrin β3 siRNA, 2).
The brain samples were collected at 24 hours after SAH. Western
blotting was performed as described previously.18 Primary antibodies
used were MFGE8 (Santa Cruz Biotechnology), HO-1, Nrf2 (Santa
Cruz Biotechnology), 4-hydroxynonenal (4-HNE, Abcam Biotech
Company), Nitrotyrosine (Santa Cruz Biotechnology), integrin β3,
ERK, and phosphorylated ERK (p-ERK).
Neurological Score
Statistical Analysis
Neurological scores were evaluated at 1 hour before euthanization
by a blinded observer according to the 21-point scoring system described by Garcia et al,17 with modifications.
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Brain Water Content
Brains were removed at 24 or 72 hours after surgery and separated
into left hemisphere, right hemisphere, cerebellum, and brain stem.
Each part was weighed immediately after removal (wet weight) and
once more after drying in 105°C for 72 hours. The percentage of water content was calculated as (wet weight−dry weight)/wet weight.13
Immunofluorescence Staining
Double-fluorescence labeling was performed as previously described.13 Sections were incubated overnight at 4°C with goat
anti-MFGE8 (Santa Cruz Biotechnology), rabbit anti-ionized calcium-binding adaptor molecule 1 (Abcam), goat antiglial fibrillary
acidic protein (Santa Cruz Biotechnology), and mouse anti-neuronal
nuclei (EMD Millipore) primary antibodies. The expression of HO1was detected by fluorescence labeling with goat anti-HO-1 (Santa
Cruz Biotechnology). Appropriate fluorescence dye–conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) were
Western Blot
Data are expressed as a mean±SEM. All other data were analyzed
by 1-way analysis of variance followed by the Tukey post hoc test.
P value of <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism for Windows.
Results
The Expression of MFGE8 and HO-1 Increased and
Peaked at 24 Hours After SAH
After SAH, there were clear blood clots around basal cistern,
and the SAH grades decreased gradually as time passed. There
were significant differences of SAH grades between sham and
SAH animals up to day 7 (Figure 1A). Rather, the neurological deficits relived gradually and the animals showed no obvious difference on the neurological scores at 7 days after SAH
(Figure 1B).
Western blots showed that MFGE8 was seldom expressed
in sham animals and increased from 6 hours after SAH. The
increase in MFGE8 reached peak at 24 hours lasting to day 7
Figure 3. Recombinant human milk
fat globule-EGF factor-8 (rhMFGE8)
decreased brain water content and
improved neurological functions at 24
and 72 hours after subarachnoid hemorrhage (SAH). Both low dosage and high
dosage decreased brain edema at 24
hours after SAH (A), but only high dosage
improved the neurobehavioral deficits
(B). High dosage of rhMFGE8 decreased
brain water content (C) and improved
neurological functions (D) at 72 hours
after SAH. n=6 for each group. *P<0.05
vs sham; #P<0.05 vs SAH+PBS.
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(Figure 1C). The protein expression of HO-1 also increased
after SAH and peaked at 24 hours, and almost back to baseline
at 7 days after SAH (Figure 1D).
Double immunostaining of MFGE8 with ionized calciumbinding adaptor molecule 1 (marker for microglia), glial fibrillary acidic protein (marker for astrocyte), and neuronal nuclei
(marker for neuron) showed that MFGE8 is intensely expressed
in the brain after SAH and highly colocalized with ionized
calcium-binding adaptor molecule 1 at 24 hours (Figure 2).
MFGE8 is not colocalized with glial fibrillary acidic protein
and neuronal nuclei after SAH (Figure 2A). In brain tissue, the
expression of HO-1 was low in shamed animals and remarkably increased at 24 hours after SAH (Figure 2B).
Knockdown Integrin β3 and Inhibition of HO
Abolished the Beneficial Effects of rhMFGE8 at 24
Hours After SAH
Administration of rhMFGE8 Decreased Brain
Water Content and Improved Neurological
Functions After SAH
Administration of rhMFGE8 significantly increased the
protein level of MFGE8 in the brain, and MFGE8 siRNA
effectively decreased it at 24 hours after SAH (Figure 5A).
Consistent with MFGE8, the protein level of HO-1 was
intensely increased by rhMFGE8 administration (Figure 5B).
After SAH, oxidative stress had been generated and Western
blots revealed a significant increase in 4-HNE (Figure 5C)
and nitrotyrosine (Figure 5D). Administration of rhMFGE8
suppressed the expression of 4-HNE and nitrotyrosine and
MFGE8 siRNA prevented the effects of rhMFGE8 (Figure 5).
HO inhibitor SnPP IX did not change the protein level of
HO-1 (Figure IA in the online-only Data Supplement) but
increased the expression of 4-HNE and nitrotyrosine in rhMFGE8-treated animals (Figure IB and IC in the online-only
Data Supplement).
Two dosages of rhMFGE8 (1 μg and 3.3 μg) were administrated intracerebroventricularly 1.5 hours after SAH. Brain
water content and neurological scores were measured. The
data showed that both high dosage and low dosage decreased
the brain water content at 24 hours (Figure 3A and 3C), but
only the high dosage improved the neurological deficits at 72
hours after SAH (Figure 3B and 3D). The results indicated
that the high dosage is more effective, so we chose the high
dosage for the following mechanism studies.
To investigate the potential mechanisms of recombinant
MFGE8, we administrated integrin β3 siRNA or HO inhibitor
SnPP IX with rhMFGE8 treatment. Both integrin β3 siRNA
and SnPP IX significantly increased brain water content
(Figure 4A; P<0.05 versus SAH+rhMFGE8) and decreased
the neurological scores at 24 hours after SAH (Figure 4B;
P<0.05 versus SAH+rhMFGE8).
Administration of rhMFGE8 Enhanced the
Expression of HO-1 and Decreased Oxidative Stress
at 24 Hours After SAH
MFGE8 Decreased HO-1 Dependent on Integrin β3/
ERK/Nrf2 Pathway
At 24 hours after SAH, administration of rhMFGE8 has no
significant effects on the protein level of its receptor integrin
β3 (Figure 6A). Integrinβ3 siRNA knocked down the receptor efficiently (Figure 6A). rhMFGE8 treatment increased
p-ERK, Nrf2, and HO-1 compared with vehicle group
(Figures 6B–6D). Integrin β3 siRNA remarkably abolished
the up-regulation of p-ERK, Nrf2, and HO-1 after rhMFGE8
administration (Figure 6B–6D).
Discussion
Figure 4. The protective effects of recombinant human milk fat
globule-EGF factor-8 (rhMFGE8) depend on integrin β3 and heme
­oxygenase-1 (HO-1). Silencing MFGE8 or its receptor integrin β3
by siRNA or blocking the activity of HO-1 by its inhibitor SnPP
IX abolished the effects of rhMFGE8 on brain edema (A) and
neurobehavioral deficits (B). n=6 for each group. #P<0.05 vs subarachnoid hemorrhage (SAH)+PBS, &P<0.05 vs SAH+rhMFGE8.
Early brain injury, which occurs within 72 hours after cerebral aneurysm rupture, has been considered a new target for
improving the outcome after SAH. Recent studies reported
that oxidative stress was involved in the pathogenesis of early
brain injury and antioxidative treatment can be one of the
therapeutic candidates for early brain injury after experimental SAH or in a clinical setting.5 In this study, our goals were
to test first whether rhMFGE8 administration suppressed oxidative stress and improved the outcome after SAH; and second, to investigate the potential mechanisms of rhMFGE8 in
antioxidation. The results showed that MFGE8 was increased
in the early stage of SAH, and its expression was well consistent with the expression of HO-1, which is an essential cellular defense molecular of oxidative stress. Administration of
rhMFGE8 significantly decreased brain edema and improved
neurological deficits. rhMFGE8 up-regulated the expression
Liu et al MFGE8 and Oxidative Stress 3695
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Figure 5. Recombinant human milk fat globule-EGF factor-8 (rhMFGE8) modulated the expression of heme oxygenase-1 (HO-1) and oxidative stress. Administration of rhMFGE8 increased the cellular level of MFGE8 (A) and HO-1 (B) and attenuated lipid peroxidation (C) and
protein nitrification (D) at 24 hours after subarachnoid hemorrhage (SAH). Silencing MFGE8 by siRNA reversed the results. n=6 for each
group. #P<0.05 vs SAH+PBS.
of p-ERK, Nrf2, and HO-1, reduced lipid peroxidation and
protein nitrification after SAH; and knockdown of its receptor
integrin β3 by siRNA and inhibition of HO by SnPP IX abolished the antioxidation effects of rhMFGE8. These data indicated that rhMFGE8 might be an alternative therapy for early
brain injury after SAH by alleviating oxidative stress through
integrin β3/ERK/HO pathway.
MFGE8 plays important roles in several biological processes, including apoptotic cell clearance,19 angiogenesis,7
and anti-inflammation.10 Recent studies showed MFGE8mediated potential therapeutic benefits in several models of
central nervous system diseases. Li et al12 showed that MFGE8
was released from microglia and increased microglial neuroprotective activity against oligomeric amyloid β–induced neuronal cell death. This neuroprotection was mediated through
integrin receptor and activation of Nrf2/HO-1 pathway. In
permanent middle cerebral artery occlusion rats, endogenous
brain MFGE8 levels were decreased after cerebral ischemia,
and exogenous rhMFGE8 was reported to reduce the infarct
size and improve neurological function through suppression
of inflammation and apoptosis.11 In agreement with this study,
MFGE8 knockout mice had significantly larger infarct size
and showed a marked increase in the expression of proinflammatory mediators interleukin-1β and tumor necrosis factor-α
in the ischemic brain, which might be dependent on its receptor integrin β3.10 These results clearly indicated that endogenous MFGE8 is required for the neuroprotection against
neurodegenerative disease and ischemic cerebral damage. In
our study, we found that the level of MFGE8 was increased in
SAH rats and highly expressed in microglia. Administration
of rhMFGE8 improved the neurological deficits and reduced
brain edema via suppressing oxidative stress, whereas knockdown MFGE8 by siRNA exacerbated the outcomes and intensified oxidative stress. Our results were consistent with the
previous studies to support the beneficial effects of MFGE8 in
animal stroke models.
Oxidative stress became emerged as a key player in the
development and progression of many pathological conditions including stroke. HO-1 is an essential cellular defense
system against oxidant-induced injury during inflammatory
processes.20 HO-1 offers protection by catalyzing the first
and rate-limiting step in the oxidative degradation of heme
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Figure 6. Recombinant human milk fat
globule–EGF factor-8 (rhMFGE8) reduced
oxidative stress dependent on integrin
β3/extracellular signal-regulated kinase
(ERK)/heme oxygenase-1 (HO-1) pathway. Administration of rhMFGE8 has no
effects on protein level of integrin β3 (A),
increased the expression of phosphorylated ERK (p-ERK) (B), nuclear factor
erythroid 2–related factor 2 (Nrf2) (C), and
HO-1 (D). Silencing integrin β3 by siRNA
removed the effects of rhMFGE8. n=6 for
each group. #P<0.05 vs subarachnoid
hemorrhage (SAH)+PBS, &P<0.05 vs
SAH+rhMFGE8.
(Fe-protoporphyrin-IX) to carbon monoxide, ferrous iron
(Fe2+), and biliverdin.21 Studies have revealed that HO-1
played an important role in neuroprotection in ischemic
stroke.22,23 In this study, we found that rhMFGE8 decreased
the expressions of 4-HNE and nitrotyrosine, which are markers of lipid peroxidation (4-HNE) and protein nitrification
(nitrotyrosine), when compared with the vehicle group. We
further observed that the levels of HO-1 in ischemic brain tissue were up-regulated by rhMFGE8 after treatment, and HO
inhibitor SnPP IX removed the antioxidation effects of rhMFGE8. Therefore, we conceptualize that rhMFGE8 improved
the outcomes after SAH, possibly by suppressing oxidative
stress via increasing HO-1.
How did rhMFGE8 decrease the expression of HO-1 and
reduce oxidative stress after SAH? It is well-known that
integrin is a putative receptor of MFGE8 in various cell
types.10,24–27 In vascular smooth muscle cells, Wang et al26
demonstrated that MFGE8 facilitation of the cell cycle was
mediated by integrins/ERK signaling. In diabetic mice,
MFGE8 was found to coordinate fatty acid uptake through
integrin avβ3 and integrin avβ5–dependent phosphorylation
of Akt by phosphatidylinositide-3 kinase and mammalian
target of rapamycin complex 2.27 Recently, Deroide et al10
showed that MFGE8 attenuated inflammation in ischemic
cerebral injury through integrin β3–dependent inhibition of
NLRP3 inflammasome. However, the precise mechanisms
underlying its role in antioxidation after SAH are poorly
understood. Increasing evidence suggests that HO-1 is upregulated through the Nrf2 cascade.28,29 In the Alzheimer disease model, MFGE8 was released from microglia, activated
Nrf2/HO-1 pathway through integrin receptor, and suppressed oxidative stress.12 In our experiment, we found that
the expression of HO-1 was coincident with that of MFGE8
after SAH. Treatment with rhMFGE8 significantly increased
the expression of p-ERK, Nrf2, and HO-1 at 24 hours after
SAH and reduced oxidative stress; knockdown integrin β3
by siRNA deteriorated the neurological scores, increased
brain edema, and decreased the expression of p-ERK, Nrf2,
and HO-1. With the relevant cumulative findings, herein we
concluded that MFGE8 improved the outcome of SAH via
antioxidation, which may depend on integrin β3/ERK/HO
pathway.
In conclusion, our results demonstrate, for the first time,
that MFGE8 signaling is linked to modulation of oxidative
stress after SAH. Administration of rhMFGE8 improved neurological deficits and attenuated brain edema through decreasing oxidative stress; silencing MFGE8 or its receptor integrin
β3, or blocking the activity of HO-1 abolished the protective
effects of rhMFGE8. MFGE8-attenuated oxidative stress may
depend on integrin β3/ERK/HO pathway. Thus, targeting
MFGE8 could be a novel strategy to decrease oxidative stress
and ameliorate early brain injury after SAH.
Liu et al MFGE8 and Oxidative Stress 3697
Sources of Funding
This study was supported partially by a grant from National Institutes
of Health NS081740 and NS082184 to Dr Zhang.
Disclosures
None.
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Stroke. 2014;45:3691-3697; originally published online October 23, 2014;
doi: 10.1161/STROKEAHA.114.006635
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ONLINE SUPPLEMENT
Supplementary Figures
B
A
C
Supplementary figureⅠ. HO-­‐1 inhibitor SnPP IX has no effects on the expression of HO-­‐1 (A), and increased the expression of 4-­‐HNE (B) and nitrotyrosine (C) in rhMFGE8 treated animals. n=6 for each group. *p<0.05 vs. Sham; #p<0.05 vs. SAH+vehicle.