Postischemic gene transfer of midkine, a neurotrophic factor ... - Nature

Gene Therapy (2005) 12, 487–493 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt

RESEARCH ARTICLE

Postischemic gene transfer of midkine, a neurotrophic factor, protects against focal brain ischemia J Takada1, H Ooboshi1, T Ago1, T Kitazono1, H Yao1, K Kadomatsu2, T Muramatsu2, S Ibayashi1 and M Iida1 1

Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan

2

Gene therapy may be a promising approach for treatment of brain ischemia. In this study, we examined the effect of postischemic gene transfer of midkine, a heparin-binding neurotrophic factor, using a focal brain ischemia model with the photothrombotic occlusion method. At 90 min after induction of brain ischemia in spontaneously hypertensive rats, a replication-deficient recombinant adenovirus encoding mouse midkine (AdMK, n ¼ 7) or a control vector encoding b-galactosidase (Adbgal, n ¼ 7) was injected into the lateral ventricle ipsilateral to ischemia. At 2 days after ischemia, we determined infarct volume by 2,3,5-triphenyltetrazolium chloride staining. There were no significant differences in

cerebral blood flow 1 h after ischemia between AdMK and Adbgal groups. Infarct volume of AdMK group was 51727 mm3, which was significantly smaller than that of Adbgal group (86727 mm3, Po0.05). TUNEL-positive and cleaved caspase-3-positive cells in the periischemic area of AdMK-treated rats were significantly fewer than those in Adbgal-treated rats, suggesting that the reduction of infarct volume by midkine was partly mediated by its antiapoptotic action. Thus, gene transfer of midkine to the ischemic brain may be effective in the treatment of brain ischemia. Gene Therapy (2005) 12, 487–493. doi:10.1038/sj.gt.3302434 Published online 10 February 2005

Keywords: midkine; brain ischemia; gene transfer; adenovirus; apoptosis

Introduction Midkine is a heparin-binding growth factor with rich basic amino acids and cysteine.1,2 Midkine has various biological activities, that is, neurite extension,3,4 survival of embryonal nerve cells,5–7 protection against constant light-induced retinal degeneration,8 and enhancement of fibrinolytic activity of endothelial cells.9 The expression of midkine is intensely induced in the surrounding ischemic zone but not in the necrotic lesion in the early stage of cerebral infarction,10 where apoptotic DNA fragmentation occurs.11 In addition, midkine inhibits apoptosis in cultured neurons12 and in hippocampal neurons after transient ischemia.13 These findings support that midkine may have neuroprotective actions on the cerebral ischemia. Gene therapy may be an attractive treatment of cerebrovascular disease.14,15 Previous experimental studies have demonstrated the efficacy of gene transfer for brain ischemia.16–18 Although recent studies have demonstrated that gene transfer of neurotrophic factors such as glial cell line-derived neurotrophic factor and hepatocyte growth factor inhibits ischemic cerebral damages,19,20 most of these gene were delivered before induction of ischemia, and the efficacy of postischemic Correspondence: Dr H Ooboshi, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan Received 28 January 2004; accepted 13 September 2004; published online 10 February 2005

gene therapy is still not clear. Therefore, we examined whether postischemic gene transfer of midkine had protective effects against cerebral ischemia using the rat middle cerebral artery occlusion model.

Results Overexpression of midkine in cerebrospinal fluid (CSF) We used a replication-deficient recombinant adenovirus with the cytomegalovirus immediate early promoter, carrying bacterial b-galactosidase (Adbgal) as control gene, or mouse midkine (AdMK). To determine the secreted midkine in CSF after transgene expression, Western blot analysis was performed with CSF 1 day after administration of AdMK vector into the lateral ventricle. With positive controls, the amount of the midkine in CSF from the rat treated with AdMK (n ¼ 3) was estimated as more than 1 ng per 15 ml, that is, more than 100 ng/ml (Figure 1). Adbgal (n ¼ 3) injection and control (n ¼ 3) treatment did not show detectable midkine in CSF. Moreover, pretreatment of heparin markedly augmented the amount of the secreted midkine in AdMK (n ¼ 2) group. Quantitative analysis of infarct volume To determine the effect of postischemic gene transfer of midkine, we compared the infarct volume after gene transfer between Adbgal and AdMK. The physiological variables before and after ischemia in Adbgal (n ¼ 7) and AdMK (n ¼ 7) groups are shown in Table 1. There were no significant differences in the physiological variables

Gene transfer of midkine to ischemic brain J Takada et al

488 Cont Adβgal AdMK Adβgal AdMK

(%)

1ng 10ng 100ng

100

Adβgal

MK

AdMK heparin

CBF

80

Figure 1 Midkine secreted into CSF 24 h after gene transfer. In the left lane, midkine detected by Western blotting in each group is shown. In the right lane, 1, 10 and 100 ng midkine are indicated as measure. Cont represents control CSF from a rat without gene transfer; Adbgal, CSF from a rat injected with adenoviral vector encoding b-galactosidase (1 109 pfu); AdMK, CSF from a rat injected with adenoviral vector encoding midkine (1 109 pfu). Bar represents CSF after intraventricular injection of heparin (20 U/kg) 30 min before sampling.

60

40

20 Table 1

Physiological variables Adbgal (n ¼ 7)

AdMK (n ¼ 7)

0 At rest MABP (mmHg) Head temperature (1C) Rectal temperature (1C) pH PaCO2 (mmHg) PaO2 (mmHg) Hematocrit Blood sugar (mmol/l)

153710 36.070.1 37.170.1 7.4070.01 3773 123710 0.4470.01 7.170.7

15078 36.070.1 37.170.1 7.3970.02 3773 124712 0.4270.02 7.270.6

After ischemia (60 min) MABP (mmHg) Head temperature (1C) Rectal temperature (1C) pH PaCO2 (mmHg) PaO2 (mmHg) Hematocrit Blood sugar (mmol/l)

159720 36.070.1 37.170.0 7.4070.02 3775 12677 0.4470.02 7.570.7

14879 36.070.1 37.070.0 7.3970.01 3672 12679 0.4370.01 7.070.5

Values are mean7s.d. MABP ¼ mean arterial blood pressure.

between the two groups. In addition, distal middle cerebral artery (MCA) occlusion did not affect these values in each group. Cerebral blood flow (CBF) at the occluded side began to decrease within 10 min after vessel occlusion, and changes in CBF were not different between the two groups (Figure 2). Gene transfer of midkine attenuated cerebral ischemic damages, mainly at inner parts of parietal cortex, as compared with that of b-galactosidase (Figure 3a, b). The infarct area was reduced in all sections in AdMK group compared with Adbgal group, and the reduction was significant in the second and third portion of the brain slices (Po0.05 and o0.01, respectively, Figure 3c). Infarct volume was significantly smaller in AdMK group (51727 mm3, n ¼ 7) than that in Adbgal group (86727 mm3, n ¼ 7, Po0.05), and the reduction of infarct volume by the gene transfer of midkine was 41% (Figure 3d).

Histological study To determine the antiapoptotic action of midkine, we used terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end-labeling (TUNEL) staining Gene Therapy

0

10

30

60

(min)

Time After Onset of Ischemia Figure 2 Changes of CBF after onset of ischemia. There were no significant differences in CBF changes between Adbgal group (open circles, n ¼ 7) and AdMK group (closed circles, n ¼ 7).

and immunostaining for cleaved caspase-3. In Adbgal group, a large number of the TUNEL-positive cells at the periischemic area of cerebral cortex were observed 2 days after gene transfer (Figure 4a, b). In contrast, overexpression of midkine reduced the TUNEL-positive cells. The number of TUNEL-positive cells at the caudoputamen level in AdMK group was 1107169 cells/mm2 (n ¼ 7), which was significantly fewer than that in Adbgal group (5067269 cells/mm2, n ¼ 7; Po0.01). As TUNEL-positive cells contain necrotic cells, we also used the immunostaining for cleaved caspase-3, which was a specific product in the apoptotic cells (Figure 4c, d). The number of cleaved caspase-3-positive cells at the same area with TUNEL staining in AdMK group was 36727 cells/mm2 (n ¼ 7), which was also significantly fewer than that in Adbgal group (80748 cells/mm2, n ¼ 7, Po0.05).

Discussion In this study, we examined the effect of gene transfer of a neurotrophic factor, midkine, on ischemic brain damages using the thrombotic distal MCA occlusion model in rats. The major new finding of the present study was that overexpression of midkine by postischemic gene transfer markedly reduced cerebral infarct volume. Basic fibroblast growth factor is known to increase in neurons and astrocytes of the cerebral cortex transiently at the early stage (within 14 days) of permanent brain ischemia,21 and expression of brain-derived neurotrophic factor also increases transiently in the periischemic cerebral cortex a few hours after brain ischemia.22 The expression of midkine is also seen in the surrounding ischemic zone in the early stage of cerebral infarction.10 Previous studies demonstrated that application of several factors such as neurotrophic factors, antiapoptotic materials and antiinflammatory cytokines, reduced


489

a

b

c (mm2) 20

d ∗

100

Infarct Volume

Infarct Area

AdMK

+

15

§

(mm3 ) 150

Adβgal

10

50 5

0

0 1

2

3 4 Number of Slice

5

6

Adβgal

AdMK

Figure 3 Brain infarction after gene transfer. Brain sections were stained with TTC (2,3,5-triphenyltetrazolium chloride) 2 days after cerebral ischemia in rats injected with an adenoviral vector encoding b-galactosidase (a) or midkine (b) (1 109 pfu) after induction of ischemia. Bar indicates 5 mm. Infarct area (c) and volume (d) were determined by brain sections stained with TTC. Adbgal, rats (n ¼ 7) injected with adenoviral vector encoding b-galactosidase (1 109 pfu); AdMK, rats (n ¼ 7) injected with adenoviral vector encoding midkine (1 109 pfu). Values are mean7s.d. nPo0.01 and wPo0.05 by repeated measures ANOVA followed by post hoc Fisher’s protected least significant difference test. yPo0.05 by unpaired t-test.

neuronal damages following ischemia and cerebral infarct volume.23–26 Moreover, neuroprotective effects using gene transfer approaches were also demonstrated in some pretreatment studies.16,27,28 However, the studies that showed effective reductions in infarct size by postischemic gene transfer are limited.19 Therefore, the present study would provide useful information for the clinical application of the gene therapy for brain infarction. In a previous study,29 expression of LacZ gene, a reporter gene, in the ischemic brain was seen by 4–6 h after injection of herpes simplex virus vector using X-gal staining. In another report,30 transgene expression of LacZ was detected in rat pancreatic acinar cells 6 h after administration of adenoviral vector. We also detected the transgene expression in the ischemic brain 3 h after injection of adenovirus vector encoding LacZ by X-Gal

staining (unpublished data). As the reduction of infarct volume was achieved by the gene transfer 90 min after induction of ischemia, we assumed that midkine produced into the lateral ventricles by gene transfer started to work in the ischemic brain within 4–5 h after brain ischemia. Previous studies showed that midkine worked at the concentration of 10–100 ng/ml.9,12,31,32 In the present study, the concentration of midkine secreted into CSF 1 day after gene transfer was estimated as more than 100 ng/ml, suggesting that the amount of secreted midkine was high enough for the neuroprotective effect at the early phase. Our results in TUNEL staining and immunostaining for cleaved caspase-3 suggested that one of the mechanisms of midkine for attenuation of ischemic damages was its antiapoptotic action. In a study with cultured neurons, midkine inhibited caspase-dependent apoptosis Gene Therapy


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by suppressing the activation of caspase-3 via the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.12 Another study on the cell survival by midkine33 demonstrated that midkine bound to an LDL receptor-related protein, a transmembrane protein, followed by binding to nucleolin, a nucleocytoplasmic shuttle protein, after internalization,

suggesting that nuclear translocation of the midkine complex would be one of the key mechanism for cytoprotective action of midkine. These mechanisms may lead to the attenuation of ischemic damages. Moreover, midkine is reported to increase expression of urokinase-type plasminogen activator and decrease plasminogen activator inhibitor-1 expression in endo-

Figure 4 Histochemical staining after gene transfer. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling)positive cells (a, b) and cleaved caspase-3-positive cells (c, d) were determined in periischemic area at the caudoputamen level 2 days after cerebral ischemia. (a, c) The section of a rat injected with adenoviral vector encoding b-galactosidase (1 109 pfu). (b, d) The section of a rat injected with adenoviral vector encoding midkine (1 109 pfu). Arrowheads indicate cleaved caspase-3-positive cells. Bars indicate 50 mm. (e) The square indicates the area shown by (a–d). Gene Therapy


thelial cells.9 Enhanced fibrinolytic activity induced by midkine may also contribute to the reduction of infarct volume. However, further studies are required to elucidate the precise mechanisms. In conclusion, we demonstrated that postischemic gene transfer of midkine, a neurotrophic factor, produced an enough amount of midkine in the CSF and reduced cerebral infarct volume. The attenuation of ischemic damages by midkine was partly attributable to antiapoptotic actions.

Materials and methods Adenovirus preparation We constructed AdMK with the use of a cDNA for mouse midkine as described previously.34,35 The identity of the vector was confirmed by DNA sequencing, and secreted midkine into the culture medium were verified by Western blotting. Viral titers were determined by a plaque assay using human embryonic kidney 293 cells. After purification, the virus was suspended in phosphate-buffered saline (PBS) with 3% sucrose, and was kept at –801C until used for the experiment. Animals and surgical procedure All animal procedures were approved by the Animal Care and Use Review Committee at Kyushu University (12-053-0). Male spontaneously hypertensive rats (SHRs; age: 7–8 months; weight: 352–450 g) were used for the present study. Surgical procedures used in this study were performed as previously described.36 Rats were anesthetized with halothane (3% for induction, and 1.5% during the surgical preparation with a face mask, 0.75% after intubation and 0.5% for maintenance). The right femoral artery and vein were cannulated for monitoring mean arterial blood pressure or sampling blood, and for the injection of agents, respectively. The rats were endotracheally intubated, and mechanically ventilated after intravenous injection of pancuronium bromide (an initial dose of 0.3 mg followed by 0.1 mg every 30 min). Rectal and head temperature was maintained at 37 and 361C, respectively, by means of a heating pad and a warming lamp. Rats were mounted on a stereotaxic head holder, and burr holes were drilled above the right parietal and temporal cortices. CBF was measured by laser Doppler flowmetry (ALF21, ADVANCE Co., Ltd, Tokyo, Japan) at 4.0 mm lateral and 1.5 mm posterior to the bregma on the ischemic side. Brain ischemia and injection of adenoviral vector Brain ischemia was produced by photochemical occlusion of the distal MCA, as described previously.37 A krypton laser (wavelength ¼ 568 nm; Innova 301, Coherent Inc., Palo Alto, CA, USA) was used to irradiate the distal MCA at a power of 20 mW. The photosensitizing dye rose bengal (15 mg/ml in 0.9% saline; Wako Pure Chemical, Osaka, Japan) was administered intravenously to a body dose of 20 mg/kg over 90 s simultaneously with 4 min of laser irradiation. At 90 min after the distal MCA occlusion, 30 ml of viral suspension (3 1010 plaque forming units/ml) was injected into the lateral ventricle on the ischemic side. The burr holes were then covered with bone wax, and the scalp was sutured. The rats were

carefully weaned from the respirator, then were returned to their home cages and housed for 2 days.

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Measurement of infarct volume Rats were anesthetized with pentobarbital (50 mg/kg intraperitoneally) and decapitated 2 days after induction of ischemia. The brains were quickly removed and cooled in ice-cold saline for 10 min. After cutting into 2-mm thick coronal sections in a cutting block, the brain slices were then immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC, Wako Pure Chemical, Osaka, Japan) at 371C for 30 min in the dark. After the rinse with saline, the sections were incubated with 4% formaldehyde (Wako Pure Chemical, Osaka, Japan) for a day. Then the posterior surface of each section was photographed, and the infarct area in the right cerebral cortex, which was indicated by the lack of staining, was determined using NIH Image software (version 1.6.2). Infarct volume was calculated as sum of the infarct area multiplied by thickness of the slice. Immunohistochemical assay of periischemic cortical region The brain section at the caudoputamen level was fixed by 4% formaldehyde, embedded in paraffin, and cut into 5-mm thickness of slices. To detect cleavage of genomic DNA, the slice was stained with TUNEL using a kit (In Situ Cell Death Detection Kit POD, Roche, Mannheim, Germany). The slice was counterstained with hematoxylin. Another slice at the same level with TUNEL staining was used for immunostaining for cleaved caspase-3. The immunostaining was performed by avidin–biotin–peroxidase method using a kit (Histofine SAB-PO(R) Kit, Nichirei, Tokyo, Japan). In brief, after deparaffinization, the slices were incubated in PBS containing 0.0002% proteinase K for antigen unmasking. In order to block endogenous peroxidase, the slices were incubated for 30 min with methanol containing 0.3% hydrogen peroxide, followed by blocking with PBS containing 5% bovine serum in skim milk for 30 min at room temperature. The slices were then incubated with the rabbit monoclonal antibody against cleaved caspase-3 (Cell Signaling Technology, Inc., Beverly, MA, USA) diluted in PBS containing 5% bovine serum overnight at 41C. The sections were then washed with PBS containing 0.1% p-(1,1,3,3-tetramethylbutyl)phenol ethoxylate (Triton X-100, Polysciences Inc., Warrington, PA, USA), and incubated for 30 min with the biotinylated anti-rabbit polyclonal antibody, followed by incubation with avidin– biotin–horseradish peroxidase complex. Staining was developed with 3,30 -diaminobenzidine tetrahydrochloride (Histofine DAB Substrate Kit, Nichirei, Tokyo, Japan), and lightly counterstained with hematoxylin. The number of positively stained cells was counted at 3 areas of 0.15 mm2 each in the periischemic parietal cortical region (3 mm lateral to the midline at caudoputamen level, Figure 4e) by light microscopy. The total number of positive cells in three areas was expressed as cells/mm2. Detection of midkine in CSF Other sets of SHRs were injected with either AdMK, Adbgal, or none into the lateral ventricle under pentobarbital anesthesia (50 mg/kg i.p.), and 24 h later, CSF Gene Therapy


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was collected from the cisterna magna of rat under pentobarbital anesthesia. A part of the transfected rats (AdMK or Adbgal) were injected with heparin (20 U/kg) into the lateral ventricle 30 min before CSF was collected. CSF was centrifuged at 3000 r.p.m for 5 min at 41C to remove cell components and the supernatant was used for Western blotting to verify the secretion of midkine from the transfected tissue. As the sequence of mouse midkine was highly homologous to that of human,38 our rabbit antibody to human midkine was crossreactive to mouse midkine,39 and we used purified human midkine (a generous gift from Dr Ikematsu, Meiji Cell Technology Center, Meiji Milk Products Co., Ltd, Japan) as positive control. The supernatant (15 ml) was electrophoresed on a 12.5%-SDS polyacrylamide gel, and transblotted onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), which was blocked with BlockAce (Dainippon Pharmaceutical, Osaka, Japan) at 41C overnight. The membrane was probed with the rabbit anti-mouse midkine antibody (dilution, 1:200) in Trisbuffered saline (TBS) containing 10% BlockAce for 45 min at 371C. After washing three times with TBS containing 0.05% Tween-20 (TBST) and once with TBS, the membrane was incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG (1:4000, Promega, Madison, WI, USA) in TBS containing 10% BlockAce for 30 min at 371C. Protein bands were then visualized by revealing the alkaline phosphatase activity according to the manufacturer’s directions.

Statistical analysis Data were presented as mean7s.d. Differences in the data were analyzed by unpaired t-test, repeated measures ANOVA, and nonparametric Mann–Whitney U-test appropriately. P-values less than 0.05 were considered to be statistically significant.

Acknowledgements This work was supported in part by the research grant in aid from the Ministry of Health and Welfare Comprehensive Research on Aging and Health (H11-008), Japan and from the Ministry of Education, Science and Culture (14570604), Japan (HO). We would like to thank the University of Iowa Gene Transfer Vector Core, especially Maria Scheel, Kate Lamsey and Beverly L Davidson, for viral vector preparations.

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