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

OBJECT: The aim of this study is to evaluate whether the combined gene therapy of vascular 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 ligation of bilateral common carotid arteries (CCAs) in rats was employed in this study. Seven days after the ligation of bilateral CCAs, encephalo-myo-synangiosis (EMS) and plasmid administration in the temporal muscle were performed. Rats were divided into four groups by injected plasmids (i.e., LacZ group, VEGF group, apelin group, and VEGF/apelin group). Fourteen days after EMS, immunohistochemical analyses of cortical vessels were performed. Seven days after EMS, protein assays of cortex and attached muscle were performed. RESULTS: In the VEGF group and the VEGF/apelin group, the total number of blood vessels 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 other groups (p < 0.05, respectively). Apelin protein was not detected in the cortex of any groups. In the attached muscle apelin protein was detected only in the apelin group and the 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 a chronic 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 disease characterized 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 of collateral circulation by indirect bypass surgery is sometimes insufficient for most adult and 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 the temporal muscles in a chronic ischemia model in rats.15, 22 Adding the EMS surgery for bilateral common carotid arteries (CCAs) ligation, we have simulated the indirect bypass surgery for MMD. The data demonstrated that EMS with administration of plasmid human VEGF significantly increased angiogenesis in the cerebral cortex compared to EMS without administration of the VEGF gene.15, 22 The over-expression of VEGF can, however, introduce a risk of immature vessel formation that result in plasma 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 the experiments. 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 the control 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 of pentobarbital sodium (45mg/kg), the rats were placed in a stereotactic apparatus with the top of the skull positioned horizontally. After the midline linear incision, the right temporal muscle was detached from the temporal bone. Craniotomy was then performed in 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 temporal muscle 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 of capillary 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 more than four times than injection of naked plasmid.38 Immunohistochemical Analysis Bilateral CCAs ligation and sham rats were euthanized with an overdose of pentobarbital (100mg/kg) 1 week after surgery (Figure 1A), and EMS model rats were euthanized 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 each specimen on a cryostat. The sections were thaw mounted on slides. Slides from the bilateral CCA ligation or sham operation without EMS surgery were evaluated with immunohistochemical analysis of endothelial cells (ECs) and apelin/APJ protein. Slides from the bilateral CCA ligation with EMS surgery 2 weeks after were evaluated with immunohistochemical 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 washed and 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 at room 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 and Alexa 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 Willebrand factor 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 incubated for 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 and muscles 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) an