Moyamoya disease

Background: Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates the adaptive response to hypoxia in mammalian cells. It consists of a regulatory subunit HIF-1α., which accumulates under hypoxic conditions, and a constitutively expressed subunit, HIF-1β. In this study, we investigated HIF-1α naked DNA-induced angiogenesis in a cerebral ischemic model in vivo.

Methods: We utilized a rat encephalo-myo-synangiosis (EMS) model and inoculated HIF-1α. DNA into the brain surface or the temporal muscle. We analysed whether HIF-1α induced angiogenic factors and collateral circulation.

Results: A histological section treated with HIF-1α DNA showed an increased expression of HIF-1α and VEGF with collateral circulation, in comparison with control DNA (p

Conclusion: These results suggest the feasibility of a novel approach for therapeutic collateral circulation of cerebral ischemia in which neovascularization may be achieved indirectly using a transcriptional regulatory strategy. [Neurol Res 2005; 27: 503-508]

Keywords: HIF-1α angiogenesis; ischemia; encephalo-myo-synangosis

INTRODUCTION

Moyamoya disease is characterized by progressive cerebrovascular occlusive disorder1. Its primary disease process is stenosis and/or occlusion of the intracranial arterial trunks, leading to the formation of extensive collateral circulation, including 'moyamoya vessels'. This disease causes ischemic symptoms or hemorrhage. To avoid unfavorable symptoms caused by ischemia, various anastomotic bypass surgical methods are used213. Indirect non-anastomotic bypass surgery for ischemia brain requires vascular-rich tissue, such as the superficial temporal artery and the temporal muscle on the brain surface to be perfused. This may cause vascular anastomosis between extracranial tissue and the brain to maintain blood flow to the ischemic tissue4. The mechanism is not completely known, but many cases receiving this procedure have had a favorable outcome. However, some cases have had clinical problems involving insufficient collateral blood supply, in spite of surgery5.

On the other hand, research into angiogenesis is progressing and some angiogenetic factors, such as vascular endothelial growth factor (VEGF), are being used for clinical trials6. Recently, hypoxia-inducible factor (HIF-1), which is induced under hypoxic conditions, was shown to induce VEGF gene expression7. This transcriptional factor is thought to play an important role in the pathogenesis of a number of conditions, including myocardial infarction, cerebrovascular disease, hypoxic lung disease and tumor vascularization8-11. Under hypoxic conditions, HIF-1 binds to the hypoxia responsive elements (HREs) located in either the 5' or the 3' flanking region of the gene. Approximately 30 genes are known to be activated by HIF-1, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), insulin-like growth factor 2, glycolytic enzymes and glucose transporter 1(12).

Since we do not have an animal model of moyamoya disease to evaluate the development of collateral vessels, we produced a temporal muscle procedure to stimulate the encephalo-myo-synangosis model of bypass. We show here that HIF-1α mediates an increase of VEGF on the brain surface and in connective tissue. In vivo, this still translates into increased collateral vascular formation. Therefore, HIF-1α gene transfer may provide a therapeutic avenue towards simulated indirect non-anastomotic bypass surgery.

MATERIALS AND METHODS

All experimental procedures were approved by the Care of Experimental Animals Committee of Oita University School of Medicine.

Animal models

Male Wistar rats weighing 220-270 g were anesthetized intraperitoneally with sodium pentobarbital (40 mg/kg) and intramuscular atropine sulfate (0.05 mg/kg). Initially, in the supine position, a midline linear skin incision was performed in the neck to expose the bilateral internal carotid arteries, which were ligated with 3-0 silk sutures. In the prone position, a midline linear skin incision was then performed on the sagittal suture. The right temporal muscle was dissected from the skull and the right fronto-temporo-parietal bone was extensively removed. The dura of this area was opened and the right temporal muscle was placed on the brain surface. This procedure simulates encephalomyo-synangiosis (EMS). At this time, plasmids were sprinkled onto brain surface or injected into the temporal muscle, and the skin was closed.

Plasmid preparation

HIF-1α cDNA was a kind gift from Dr C. A. Bradfield13 and was inserted into pcDNA3.1 (Invitrogen, Carlsbad, CA). 100 μg HIF-1a/pcDNA3.1 plasmid was used for the treatment group, while 100 µg pcDNA3.1 plasmid was used for the control group.

β-Galactosidase gene expression

To investigate the plasmid expression, pcDNA3.1 Lac Z plasmid was used. Rats underwent the above procedure and 100 µg Lac Z plasmids were inoculated. Two weeks later, the rats were anesthetized by an overdose of pentobarbital and perfused transcardially with cold 0.9% saline followed by 4% paraformaldehyde. The brains and temporal muscles were removed en bloc to produce a frozen section and X-Gal (Sigma, St Louis, MO) staining was performed14.

HIF-1α and VEGF immunohistochemistry

Two weeks after surgery, the rats were killed as above and perfused transcardially with cold 0.9% saline followed by 4% paraformaldehyde. The brains and temporal muscles were removed en bloc, and embedded in paraffin. Coronal sections (3 µm) were de-paraffinized. Immunohistochemistry for HIF-1α was performed with a monoclonal antibody to HIF-1α (Neomarkers Fremont, CA) and detected with a catalysed signal amplification system (DAKO lab kit; Dako, Inc., CA). Immunohistochemistry for VEGF was performed with a monoclonal antibody to VEGF (Oncogene, San Diego, CA) and detected with an LSAB2 kit/HRP (DAKO). Ten sections were investigated per rat and positive cells were counted in χ 200 power electromicroscopic fields for each section. Five rats were used for each group, so 50 fields were counted for each group.

Evaluation of collateral vessels

Two weeks after surgery, the rats were killed as above and perfused with an intracardiac infusion of a solution containing 4% neutral paraformaldehyde (PFA) and India ink in 0.9% sodium chloride. The brains and temporal muscles were removed en bloc and embedded in paraffin: only the middle third of the block was used. Twenty serial coronal sections (3 µm each) were made from the rostral and de-paraffinized. We defined the collateral vessels as vessels running from the connective tissue to the brain (as shown in Figure 4A) and counted them. Six rats were used for each group. The data were analysed by Student's f-test and presented as the mean ± SD. A value of p

RESULTS

We first tested the gene expression of β-galactosidase after inoculation with the LacZ-containing plasmid. Plasmids were sprinkled onto the brain surface (BS group) or injected into the temporal muscle (TM group). We compared the distribution of X-gal-stained sections in the BS and TM groups. A section of the BS group showed many blue positive cells in the connective tissue between the brain and temporal muscle. LacZpositive cells in the TM group were mainly located in the temporal muscle (Figure 7). The control group treated with plasmid without LacZ had no blue cells (data not shown). These results showed that the LacZcontaining plasmid worked 2 weeks after inoculation.

The number of HIF-1a positive cells was around 2-3-fold higher in HIF-1α-treated BS and TM groups than in the control plasmid-treated group (p

We identified an apparently new capillary network developed from the brain surface. As shown in Figure 4A, collateral vessels treated with HIF-Ia were longer and more angiogenic compared with the control. In comparison with the control plasmids sprinkled onto the brain surface, collateral vessels in HIF-1 α-treated rats (BS group) were significantly induced (p

DISCUSSION

Many modified techniques of cerebral revascularization have been developed for the treatment of moyamoya disease and other similar diseases. Direct revascularization via the superficial temporal artery and middle cerebral artery (STA-MCA) bypass, and indirect revascularization are the most frequently used techniques. The total level of neovascularization is thought to be mainly dependent on the extent of ischemia in the brain. However, the underlying mechanism is not completely understood. To avoid unfavorable outcomes caused by ischémie brain, we investigated the role of HIF-1α in the induction of neovascularization. VEGF is a well-known angiogenetic factor, and VEGF gene therapy for ischemic heart disease and chronic obstructive arteriosclerosis is clinically applied. However, there are some reports on incomplete angiogenesis and the problem of vascular permeability15. Moreover, Schwarz et al.16 showed that the injection of a high dose of VEGF plasmid to the ischemic heart caused complications of angioma formation. Increased systemic VEGF levels could cause unwanted side effects, including inappropriate angiogenesis at sites of vascular derangement or at sites where angiogenesis may have major adverse consequences; for example, in the retina, the synovium and occult tumors17,18.

Recently, the activation of HIF-1α in the rat cerebral cortex was observed after ischemia7,19 (Kimba et al., unpublished data). We therefore utilized the HIF-1α gene to develop angiogenesis under almost physiological conditions. We sprinkled HIF-1α plasmids onto the brain surface (BS group) or injected into the temporal muscle (TM group). We compared the distribution of HIF-1α and VEGF between the BS and TM groups. A section of the BS group showed many positive cells in the connective tissue between the brain and temporal muscle. The TM group showed distribution mainly in the temporal muscle. HIF-1α sprinkled onto the brain surface induced significant collateral vessels. However, HIF-1α injected into the temporal muscle (TM group) was not statistically significant. These results suggested that the injection site may be vital to develop collateral vessels. The transcription factor, HIF-1, appears to be a universal molecular master switch, controlling cellular survival, glucose metabolism and transport, and metabolic adaptation20. One of the most relevant target genes of HIF-1 is the glycoprotein hormone, erythropoietein (EPO). Interestingly, it has been discovered that:

* EPO and its receptor EPO-R are expressed in the brain;

* the therapeutic administration of EPO can protect neurons against hypoxia-ischemia in vitro and in vivo21-25.

Further studies are required to investigate whether EPO is induced by the HIF-1α plasmid in the brain and acts as a paracrine mediator for neuroprotection in ischemic pro-conditioning. In addition to VEGF and EPO, HIF-1α may activate the expression of additional genes that promote angiogenesis26. It has been reported that the hypoxic induction of the expression of Flt-1, one of two VEGF receptors, is mediated by an HIF-1 binding site found upstream of the gene . HIF-1 may also upregulate the expression of the urokinase receptor to enhance cellular migration and invasion28. Furthermore, there may be additional, as yet uncharacterized, factors involved in angiogenesis that are regulated by HIF-1 and therefore possibly activated by HIF-1α29. Our data demonstrated that certain features of HIF-1α make it an attractive candidate for strategies of therapeutic angiogenesis in the ischemic brain.

ACKNOWLEDGMENTS

We thank Y. Kimba (Oita University) and E. A. Chiocca (Ohio State University) for our fruitful discussions, H. Wakimoto (Tokyo Medical and Dental University) for providing β-galactosidase cDNA, and Ms Yuko Sumita and Ms Yuka Hagimori for technical assistance.

REFERENCES

1 Suzuki J, Takaku A. Cerebrovascular 'moyamoya' disease. Disease showing abnormal net-like vessels in base of brain. Arch Neurol. 1969; 20: 288-299

2 Kobayashi H, Hayashi M, Manda Y, et al. EC-IC bypass for adult patients with moyamoya disease. Neurol Res. 1991; 13: 113-116

3 Isono M, Ishii K, Kamida T, et al. Long-term outcomes of pediatric moyamoya disease treated by encephalo-duro-arterio-synangiosis. Pediat Neurosurg 2002; 36: 14-21

4 Goda M, Isono M, Ishii K, et al. Long-term effects of indirect bypass surgery on collateral vessel formation in pediatric moyamoya disease. J Neurosurg 2004; 100: 1 56-1 62

5 Houkin K, Kuroda S, Ishikawa T, et al. Neovascularization (angiogenesis) after revascularization in moyamoya disease. Which technique is most useful for moyamoya disease? Acta Neurochir (Wien) 2000; 142: 269-276

6 Rajagopalan S, Mohler ER IIIrd, Lederman RJ, et al. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 2003; 108: 1933-1938

7 Marti HJ, Bernaudin M, Bellail A, et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol 2000; 156: 965-976

8 Lee SH, Wolf PL, Escudero R, et al. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med 2000; 342: 626-633

9 Sharp FR, LuA, Tang Y, et al. Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab 2000; 20: 1011-1032

10 Haddad JJ. Antioxidant and prooxidant mechanisms in the regulation of redox(y)-sensitive transcription factors. Cell Signal 2002; 14: 879-897

11 Sondergaard KL, Hilton DA, Penney M, et al. Expression of hypoxia-inducible factor 1alpha in tumours of patients with glioblastoma. Neuropathol Appl Neurobiol 2002; 28: 210-217

12 Semenza GL. Hypoxia-inducible factor 1: control of oxygen homeostasis in health and disease. Pediat Res 2001; 49: 614-617

13 Hogenesch JB, Cu YZ, Jain S, et al. The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA 1998; 95: 5474-5479

14 Abe T, Wakimoto H, Bookstein R, et al. Intra-arterial delivery of p53-containing adenoviral vector into experimental brain tumors. Cancer Gene Ther 2002; 9: 228-235

15 Schoch HJ, Fischer S, Marti HH. Hypoxia-induced vascular endothelial growth factor expression causes vascular leakage in the brain. Brain 2002; 125: 2549-2557

16 Schwarz ER, Speakman MT, Patterson M, et al. Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor (VEGF) in a myocardial infarction model in the rat-angiogenesis and angioma formation. J Am Coll Cardiol 2000; 35: 1323-1330

17 Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; 267: 1093110934

18 Su H, Arakawa-Hoyt J, Kan YW. Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model. Proc Natl Acad Sci USA 2002; 99: 9480-9485

19 Chavez JC, LaManna JC. Activation of hypoxia-inducible factor-1 in the rat cerebral cortex after transient global ischemia: potential role of insulin-like growth factor-1. J Neurosci 2002; 22: 8922-8931

20 Bruick RK, McKnight SL. Transcription. Oxygen sensing gets a second wind. Science 2002; 295(5556): 807-808

21 Prass K, Scharff A, Ruscher K, et al. Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin. 5frofe2003; 34:1981 -1986

22 Bernaudin M, Marti HH, Roussel S, et al. A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J Cereb Blood Flow Metab 1999; 19: 643-651

23 Sakanaka M, Wen TC, Matsuda S, et al. In vivo evidence that erythropoietin protects neurons from ischemixc damage. Proc Natl Acad Sci USA 1 998; 95: 4635-4640

24 Brines ML, Chezzi P, Keenan S, et al. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc Natl Acad Sci USA 2000; 97: 10526-10531

25 Ruscher K, Freyer D, Karsch M, et al. Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model. J Neurosci 2002; 22: 1029110301

26 Vincent KA, Feron O, Kelly RA. Harnessing the response to tissue hypoxia: HIF-1 alpha and therapeutic angiogenesis. Trends Cardiovasc Med 2002; 12: 362-367

27 Gerber HP, Condorelli F, Park J, et al. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia. J Biol Chem 1997; 272: 23659-23667

28 Krishnamachary B, Berg-Dixon S, Kelly B, et al. Regulation of colon carcinoma cell invasion by hypoxia-inducible factor 1. Cancer Res 2003; 63: 1138-1143

29 Vincent KA, Shyu KG, Luo Y, et al. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1alpha/VP16 hybrid transcription factor. Circulation 2000; 102: 2255-2261

Takeshi Matsuda, Tatsuya Abe, Jian Liang Wu, Minoru Fujiki and Hidenori Kobayashi

Department of Neurosurgery, Oita University School of Medicine, Hasama-machi, ldalgaoka 7-7, Oita, 879-5593, Japan

Correspondence and reprint requests to: Tatsuya Abe, Department of Neurosurgery, Oita University School of Medicine, Hasama-machi, Idaigaoka 1-1, Oita, 879-5593, Japan. [abet@med.oita-u.ac.jp] Accepted for publication December 2004.

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