Manuscript title
Combined gene therapy of vascular endothelial growth factor and apelin for a chronic
cerebral hypoperfusion model in rats

Authors
Masafumi Hiramatsu, MD1); Tomohito Hishikawa, MD1); Koji Tokunaga, MD2);
Hiroyasu Kidoya, PhD3); Shingo Nishihiro, MD1); Jun Haruma, MD1); Tomohisa
Shimizu, MD1); Yuji Takasugi, MD1); Yukei Shinji, MD1); Kenji Sugiu, MD1); Nobuyuki
Takakura, MD3); Isao Date, MD1)

1) Department of Neurological Surgery, Okayama University Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences
2) Department of Neurosurgery, Okayama City Hospital3) Department of Signal Transduction, Research Institute for Microbial Diseases,
Osaka University

Corresponding author: Masafumi Hiramatsu, MD
Department of Neurological Surgery, Okayama University Graduate School ofMedicine, Dentistry and Pharmaceutical Sciences
2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan

Fax: +81-86-227-0191
Tel: +81-86-235-7336
E-mail: mhiramatsu@okayama-u.ac.jp

Key words (3-6): “apelin”, “gene therapy”, “moyamoya disease”, “revascularization”,“vascular endothelial growth factor”
Running head (~65 characters): Gene therapy for moyamoya disease model
Sources of financial support: None
Sources of material support: From co-authors in the Department of Signal Transduction,
Research Institute for Microbial Diseases, Osaka University

Number of words: 4,182 words
Figures: 4
Tables: 0

Abstract
OBJECT: The aim of this study is to evaluate whether the combined gene therapy ofvascular endothelial growth factor (VEGF) and apelin in indirect vasoreconstructive
surgery enhances brain angiogenesis in a chronic cerebral hypoperfusion model in rats.
METHODS: A chronic cerebral hypoperfusion model induced by the permanent ligationof bilateral common carotid arteries (CCAs) in rats was employed in this study. Sevendays after the ligation of bilateral CCAs, encephalo-myo-synangiosis (EMS) andplasmid administration in the temporal muscle were performed. Rats were divided into
four groups by injected plasmids (i.e., LacZ group, VEGF group, apelin group, andVEGF/apelin group). Fourteen days after EMS, immunohistochemical analyses ofcortical vessels were performed. Seven days after EMS, protein assays of cortex andattached muscle were performed.
RESULTS: In the VEGF group and the VEGF/apelin group, the total number of bloodvessels in the cortex was significantly larger than that in the LacZ group (p < 0.05,
respectively). In the VEGF/apelin group, larger vessels were induced than in the othergroups (p < 0.05, respectively). Apelin protein was not detected in the cortex of anygroups. In the attached muscle apelin protein was detected only in the apelin group andthe VEGF/apelin group. Immunohistochemical analysis revealed that apelin and its

receptor APJ were expressed on the endothelial cells 7 days after the ligation of CCAs.
CONCLUSIONS: The combined gene therapy of VEGF and apelin with EMS in achronic cerebral hypoperfusion model in rats can enhance angiogenesis. This has
potential as a feasible treatment option for moyamoya disease in a clinical setting.

Background and Purpose
Moyamoya disease (MMD) is a chronic, progressive cerebrovascular diseasecharacterized by stenosis or occlusion of the bilateral supraclinoid internal cerebral
arteries and the development of an abnormal vascular network called moyamoya vessels
at the base of the brain that blocks cerebral flow.21 Indirect bypass surgeries such as
enchepalo-myo-synangiosis (EMS) are mostly performed for pediatric patients with
MMD. The critical issue in indirect bypass for MMD is the fact that the amount ofcollateral circulation by indirect bypass surgery is sometimes insufficient for most adultand some pediatric cases.20, 25, 27, 32, 39
To develop sufficient collateral circulation, we have investigated the effect of EMS
combined with vascular endothelial growth factor (VEGF) gene administration to thetemporal muscles in a chronic ischemia model in rats.15, 22 Adding the EMS surgery forbilateral common carotid arteries (CCAs) ligation, we have simulated the indirect
bypass surgery for MMD. The data demonstrated that EMS with administration ofplasmid human VEGF significantly increased angiogenesis in the cerebral cortex
compared to EMS without administration of the VEGF gene.15, 22 The over-expressionof VEGF can, however, introduce a risk of immature vessel formation that result inplasma leakage and angioma formation.4, 11, 24, 35

Apelin has been identified as the endogenous ligand of the orphan G protein-coupled
receptor APJ that is expressed in the cardiovascular and central nervous systems.29 The
apelin-APJ system is involved in a wide range of physiological activities, such as heart
contractility and blood pressure regulation6, appetite and drinking behavior,23
neuroprotection,30 and angiogenesis.7, 14, 17, 18, 26 It has been reported that apelin together
with VEGF effectively induced functional vessels that are larger than those with VEGF
alone in the hind limb ischemia model.18
In this report, we evaluated whether the combined gene therapy of VEGF and apelin
in indirect vasoreconstructive surgery enhances brain angiogenesis in a chronic cerebral
hypoperfusion model in rats.

Materials and Methods
Animals and Surgical Procedures
All animal procedures in this study were specifically approved by the Institutional
Animal Care and Use Committee of Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences (approval number: OKU-2013158).
Adult male Wistar rats (9-11 weeks old, weighing 250-350 g) were used for theexperiments. Under general anesthesia with 2.0% halothane in a mixture of 40% oxygen

and 60% nitrous oxide gas the common carotid arteries (CCAs) were carefully separated
from the sympathetic and vagal nerves using a ventrocervical incision. Bilateral CCAs
were ligated with 3-0 silk sutures. The body temperature in the rats was maintained
close to 37°C throughout the procedure and by using a heating pad. Sham operations
involved skin incision and exteriorization of bilateral CCAs without CCA ligation. An
interval of 7 days was allowed for postoperative recovery (Figure 1A).
Seven days after the bilateral CCAs ligation, the rats underwent EMS surgery (Figure
1B). The period between CCAs ligation and the EMS surgery is short, 7 days. This
period was recruited because bilateral CCAs ligation reduces CBF to 35-50% of thecontrol level, and CBF start to recover at 1 week. We thought that delayed EMS surgery
after the beginning of CBF recover could be a negative effect for angiogenesis. In past
literatures from other institutions, this period was recruited to develop similar model,
too. Under the general anesthesia with the intraperitoneal administration ofpentobarbital sodium (45mg/kg), the rats were placed in a stereotactic apparatus withthe top of the skull positioned horizontally. After the midline linear incision, the righttemporal muscle was detached from the temporal bone. Craniotomy was then performedin the temporo-parietal region using a dental drill. The dura mater was carefully opened
and removed with no disruption of the brain surface (Figure 1C). The exposed brainsurface was covered with the muscle flap (Figure 1D). Plasmid injection in the temporalmuscle was performed using GenomOne-Neo transfection reagent (Ishihara Sangyo)
according to the manufacturer’s protocol. Rats were divided into four groups by injected
plasmids (i.e., LacZ group, VEGF group, apelin group, and VEGF/apelin group).
Quantity of plasmid was 25µg in each group. In our previous report, we simply injected
50µg of VEGF plasmid into the temporal muscle, and found significant increase ofcapillary density.22 Moreover, we performed optimal dose analysis that demonstrated
the maximal angiogenic effect occurred with a 100µg dose of VEGF plasmid.15 In
comparison study of transfection efficacy between naked plasmid and method using
GenomOne-Neo transfection reagent, transfection efficacy of this method was morethan four times than injection of naked plasmid.38

Immunohistochemical Analysis
Bilateral CCAs ligation and sham rats were euthanized with an overdose ofpentobarbital (100mg/kg) 1 week after surgery (Figure 1A), and EMS model rats wereeuthanized 2 weeks after EMS (Figure 1B). They were perfused transcardially with 200
ml of cold phosphate-buffered saline (PBS) and 100 ml of 4% paraformaldehyde (PFA)in PBS. The brain and transfected temporal muscle were removed and post-fixed in thesame fixative overnight at 4°C, and subsequently stored in 30% sucrose in PBS until
completely submerged. Frozen coronal sections (17 μm thick) were cut from eachspecimen on a cryostat. The sections were thaw mounted on slides. Slides from thebilateral CCA ligation or sham operation without EMS surgery were evaluated withimmunohistochemical analysis of endothelial cells (ECs) and apelin/APJ protein. Slides
from the bilateral CCA ligation with EMS surgery 2 weeks after were evaluated withimmunohistochemical analysis of ECs. The number per group was 8 in each group.
Sections which include cortical surface were photographed at 10x magnification. The
number of all vessels per field, percentage of vessel area per field and number of large
vessels (>10 μm) per field were calculated in each photograph from the images using
ImageJ.
For the immunohistochemical staining of ECs, after several rinses in PBS, slides
were incubated in 10% fetal bovine serum in PBS for 1 h. Then, the slides were washedand incubated with an affinity-purified mouse monoclonal anti-endothelial cell antibody(RECA-1) with 1% fetal bovine serum for 2 h at room temperature. The slides were
washed and incubated for 1 h with a Cy3 anti-mouse IgG antibody at 1:200 dilution atroom temperature.
For the immunohistochemical staining of APJ and ECs, after several rinses in PBS,

slides were incubated in 10% fetal bovine serum in PBS for 1 h. Then, the slides were
washed and incubated with RECA-1 and an affinity-purified rabbit polyclonal anti-APJ
antibody with 1% fetal bovine serum for 2 h at room temperature. The slides were
washed and incubated for 1 h with a Cy3 anti-mouse IgG antibody at 1:200 dilution andAlexa fulor anti-rabbit IgG antibody at 1:200 at room temperature.
For the immunohistochemical staining of apelin and ECs, after several rinses in PBS,
slides were incubated in 10% normal goat serum in PBS for 1 h. Then, the slides were
washed and incubated with an affinity-purified rabbit polyclonal anti-von Willebrandfactor antibody and an affinity-purified mouse monoclonal anti-apelin antibody with 1%
normal goat serum for 2 h at room temperature. The slides were washed and incubatedfor 1 h with an FITC anti-rabbit IgG antibody at a 1:300 dilution and a Cy3 anti-mouse
IgG antibody at 1:300 dilution at room temperature.

Enzyme-linked Immunosorbent Assay (ELISA) Analyses
For protein assay, the CCAO and EMS models were quickly harvested after the
decapitation of animals anesthetized with an overdose of pentobarbital (100 mg/kg, i.p.)
1 week after EMS surgery. The number per group was 2 in each group. Their brains andmuscles were sliced with a thickness of 2mm. The brain tissue of the cortex waspunched out using a biopsy punch (3 mm hole, Kai Corporation and Kai Industries Co.,
Ltd., Japan). Brain and muscle tissues were then homogenized in T-PER (Pierce,
Rockford, IL) and centrifuged at 10,000 G for 10 min at 4°C, and the supernatant was
obtained. Induced VEGF and apelin levels of the brain and muscle of the CCAO andEMS models were measured using human VEGF ELISA and apelin-12 ELISA assay
kits.

Statistical Analyses
The number of vessels and ELISA were evaluated statistically using single analysis ofvariance (ANOVA), with subsequent post hoc Tukey-Kramer Fisher’s protected leastsignificance difference (PLSD) test. Statistical significance was preset at p < 0.05.

Results
At the capillary level, the number of blood vessels in the VEGF and the VEGF/apelingroups was significantly higher than that in the LacZ group (p < 0.05, respectively)(Figure 2A, 2B). Percentage of vessel area per field in the VEGF/apelin group was
significantly higher than that in the LacZ group (p < 0.05)(Figure 2C). Moreover, thenumber of large vessels in the VEGF/apelin group was significantly higher compared tothat in the LacZ, VEGF, and apelin groups (p < 0.05, respectively) (Figure 2DC).
The protein levels of VEGF and apelin in the attached muscle and the cortex 1 week
after EMS were evaluated in all four groups. The protein levels of VEGF in the attachedmuscle in the VEGF and the VEGF/apelin groups tended to be higher than in the othergroups but single ANOVA showed no significant difference (p = 0.095). VEGF proteinin the cortex was not detected in any of the groups (Figure 3A). Apelin protein in theattached muscle was detected only in the apelin group and the VEGF/apelin groups.
Apelin protein in the cortex was not detected in any of the groups (Figure 3B).
Immunohistochemical staining of the brain with monoclonal anti-apelin antibody andpolyclonal anti-APJ antibody 7 days after occlusion of the bilateral CCAs revealed thatECs stained by RECA-1 or vWF antibody in the cortex express apelin or APJ (Figure
4).

Discussion
Indirect bypass surgery for moyamoya disease
Direct and/or indirect bypass surgery is often performed in patients with MMD as asurgical treatment. Although direct bypass surgeries such as superficial temporalartery-middle cerebral artery anastomosis are frequently performed in adult andpediatric patients, direct bypass surgeries are sometimes difficult, especially in young
children. Due to its easy and simple manner, indirect bypass surgery for MMD is acommonly used procedure to increase cerebral blood flow. Although it has beenreported that indirect bypass surgeries are effective for pediatric and young adultpatients with MMD, direct bypass surgery is the main treatment option for most adultpatients with MMD.27 The most important issue related to indirect bypass surgery for
MMD is the fact that the amount of collateral circulation by surgery is sometimes
insufficient for most adult and some pediatric cases.20, 25, 27, 32, 39 An endogenous
angiogenic factor may be involved in the development of collateral circulation. Park et
al. demonstrated that the genotype of the VEGF allele was related to better collateral
vessel formation after bypass surgery in patients with MMD.33 These data indicate thatthe addition of an exogenous angiogenic factor to indirect bypass surgery could enhance
the level of collateral vessels.

Gene therapy for a chronic cerebral hypoperfusion model
In this report, we confirmed that, in the VEGF group and the VEGF/apelin group, thetotal number of blood vessels in the cortex was significantly larger than that in the LacZ
group. Moreover, in the VEGF/apelin group, larger vessels were induced than in theother groups. In the hypoperfusion mode indirect bypass surgery without angiogenic
factors could increase the number of vessels, and additional angiogenic factors could
lead to a further increase.1, 2, 12, 15, 19, 22, 31 Hechet et al. showed that EMS with VEGF
expressing myoblasts for mice with unilateral ICA ligation improved not only the vessel
density of the cortex but also the cerebrovascular reserve capacity.12 Similar reports
focused on vessel caliber size were, however, limited. Ohmori et al. reported that theencephalogaleosynangiosis (EGS) with granulocyte-colony stimulating factor (G-CSF)
for hypoperfusion rats increased the number of smaller vessels.31 We thought that theincrease in the number of larger vessels was important factor in development of mature
angiogenesis. Kidoya et al. reported that the apelin/APJ system spatially and temporally
modulates caliber size enlargement during embryogenesis.17 Some reports suggested
that tissue hypoxia induced apelin expression on ECs.7, 14, 17, 18 The mechanism ofenlarged blood vessels by the apelin/APJ system was described as follows.36 VEGF
induces APJ expression on sprouted ECs, and angiopoietin-1 (Ang-1) induces apelin
expression on ECs. Apelin induces the assembly of ECs and the proliferation of ECs
with VEGF. Finally, APJ disappears on angiogenesis-inactive ECs and caliber size
regulation finishes.
Newly formed vessels promoted by the over-expression of VEGF can be immatureand are at risk of tissue edema11 or hemangioma.35 Some papers reported combined
gene therapy, such as VEGF and Ang-1, for ischemic hind limb models, myocardial
infarction models, or acute cerebral ischemia models, and VEGF and apelin forischemic hind limb models.3, 5, 18, 34, 40, 41 The merits of the combined gene therapy of
VEGF and apelin were reported to be the increase in vessel density and the maturationof newly developed vessels, including larger vessel formation and low permeability.36
Some studies reported that VEGF-mediated permeability occurred through thedisorganization of endothelial junction proteins, such as VE-cadherin.10, 16 It wasreported that apelin inhibited the down-modulation of VE-cadherin by VEGF, resulting
in the suppression of hyperpermeability.18 Although we reported the development oflarger vessels, we could not confirm the suppression of hyperpermeability in the
VEGF/apelin group. When we apply gene therapy to the indirect bypass of MMD, thedevelopment of mature newly formed vessels due to bypass surgery may lead to a
reduction in adverse effects and an increase in cerebral blood flow.
Secreted proteins were detected only in muscles. This was also described in ourprevious report.22 We thought that the injection of VEGF and apelin plasmids to themuscle increased angiogenesis not only in the muscle but also in the muscle-cortex
interface. Then, the transpial vessel sprouted to the cortex. Similar models have reportedthis mechanism.12, 28 In particular, Nakamura et al. showed the mechanism ofrevascularization in an experimental model after the EMS of pigs.28 During cerebral
ischemia, the infiltration of inflammatory cells between the temporal muscle and thearachnoid membrane developed angiogenesis and led to revascularization between theexternal cerebral artery and the cerebral cortex artery.

Study limitations
This study has some limitations. First, the observational period of immunohistochemical
analyses and protein assay were was short. Our previous studies and similar studies
from other institutions reported results from evaluations of 2 to 4 weeks.1, 12, 15, 19, 22, 31 In
general, angiogenesis developed several months after indirect bypass surgery in patientswith MMD. Future studies need to analyze collateral formation and protein assay in thismodel over a longer period.
Second, we could not conduct behavioral assessments in our model. Cognitive
impairment can occur in patients with MMD.9, 13 Assessment of the correlation between
the development of collateral formation and the change in cognitive function in thismodel is desirable.
Third, we did not conduct measurement of vessel dysfunction, such as breakdown of

brain blood barrier, vessel leakiness, tight junction protein assessment, brain edema.
Fourth, we could not conduct blood flow measurements. In the past literatures, after
bilateral CCAs ligation of rat, the greatest reduction in cerebral blood flow to 35-50% of
the control level.8, 37 In the future, we need blood flow measurement to show the effectof enhanced angiogenesis.
In the present study, we performed only vessel number analysis in short period,
therefore, further analyses as described above are needed before the clinical application.

Conclusions
The combined gene therapy of VEGF and apelin with EMS in a chronic cerebral
hypoperfusion model in rats can enhance mature angiogenesis. This could potentially bea feasible treatment option for MMD in a clinical setting.

Acknowledgments
The authors thank Dr. H. Kidoya for supplying VEGF, apelin and LacZ plasmids, andDr. H. Michiue, Ms. A. Ueda, Dr. T. Oohashi Ms. A. Maehara, Ms. M. Arao, and Ms. N.
Uemori for technical assistance.

Disclosure
The authors report no conflicts of interest related to the materials or methods used inthis study or the findings specified in this paper.

Figure legends

Figure 1. Time course of the experimental design and indirect bypass surgery(A) Scheme showing the experimental design for the carotid artery occlusion model(B) Scheme showing the experimental design for the indirect bypass surgery model(C) Exposed brain after the craniotomy in the right temporo-parietal region(D) Fascia attached (*) to the remaining parietal bone and brain covered with thetemporal muscle (**)
CCAO: common carotid artery occlusion, EMS: encephalo-myo-synangiosis, IF:
immuno-fluorescence

Figure 2. Analysis of angiogenesis of indirect bypass surgery model(A) RECA-1 staining of the cortex in the four groups of the indirect bypass surgery
model(B) Quantitative evaluation of the number of vessels (*p<0.05)(C) Percentage of vessel area per field (*p<0.05)(D) Quantitative evaluation of the number of large vessels (more than 10 μm) (*p<0.05)

Figure 3. Results of ELISA analysis for human VEGF and apelin(A) Human VEGF level in the cortex and attached muscle 1 week after indirect bypass
surgery and administration of plasmids.
(B) Apelin level in the cortex and attached muscle 1 week after indirect bypass surgery
and administration of plasmids

Figure 4. Immunohistochemical analysis of the cortex from the carotid artery occlusion
model (CCAO) and sham-operated (sham)(A) Immunohistochemical staining using anti-apelin antibody(B) Immunohistochemical staining using anti-APJ antibody
CCAO: common carotid artery occlusion, vWF: anti-von Willebrand Factor antibody,
RECA-1: anti-endothelial cell antibody

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