The Role of VEGF and KDR Polymorphisms in Moyamoya Disease and Collateral Revascularization

We conducted a case-control study to investigate whether vascular endothelial growth factor (VEGF −2578, −1154, −634, and 936) and kinase insert domain containing receptor (KDR −604, 1192, and 1719) polymorphisms are associated with moyamoya disease. Korean patients with moyamoya disease (n = 107, mean age, 20.9±15.9 years; 66.4% female) and 243 healthy control subjects (mean age, 23.0±16.1 years; 56.8% female) were included. The subjects were divided into pediatric and adult groups. Among the 64 surgical patients, we evaluated collateral vessel formation after 2 years and divided patients into good (collateral grade A) or poor (collateral grade B and C) groups. The frequencies and distributions of four VEGF (−2578, −1154, −634, and 936) and KDR (−604, 1192, and 1719) polymorphisms were assessed from patients with moyamoya disease and compared to the control group. No differences were observed in VEGF −2578, −1154, −634, and 936 or KDR −604, 1192, and 1719 polymorphisms between the control group and moyamoya disease group. However, we found the −634CC genotype occurred less frequently in the pediatric moyamoya group (p = 0.040) whereas the KDR −604C/1192A/1719T haplotype increased the risk of pediatric moyamoya (p = 0.024). Patients with the CC genotype of VEGF −634 had better collateral vessel formation after surgery. Our results suggest that the VEGF −634G allele is associated with pediatric moyamoya disease and poor collateral vessel formation.


Introduction
Although many studies have investigated the etiology of moyamoya disease, unsatisfactory progress has been made. Moyamoya disease etiology may be idiopathic, environmental, or genetic. The search for genetic loci linked to moyamoya disease has uncovered associations with chromosomes 3, 6, 7, 8, and 17 and the HLA haplotype [1][2][3][4][5][6][7][8], but relevant genes have not been identified [9]. Recent linkage analyses from East Asian families with moyamoya disease demonstrated a linkage of 17q25.3 with the disease at a locus that is 21480 bp from the transcription site of the Raptor gene. However, an allele of this gene was not detected in samples from Caucasian patients with this disease [10].
Vascular endothelial growth factor (VEGF) is involved in vasculogenesis and vascular permeability in various intracranial lesions [11]. In ischemic disease, cerebral angiogenesis is caused by the release of VEGF [12,13]. VEGF affects vasculogenesis, endothelial cell proliferation and migration, vascular permeability, and stromal degradation through the activation of proteolytic enzymes that are involved in angiogenesis [14,15]. VEGF binds its receptor tyrosine kinases, VEGF receptor-1 and VEGF receptor-2 (also known as kinase insert domain containing receptor, or KDR) but KDR is the key receptor mediating angiogenesis [16] and is essential for endothelial cell survival and integrity [17].
Although excess VEGF in moyamoya disease has been demonstrated [11,18] and the association is convincing, the specific role for VEGF remains unclear. Therefore, we studied the relationship of VEGF and KDR polymorphisms and moyamoya disease. VEGF is a major angiogenic factor and a prime regulator of endothelial cell proliferation [19]. The gene that encodes VEGF is located on chromosome 6 and is comprised of a 14-kb coding region with eight exons and seven introns [20]. VEGF is activated transcriptionally and posttranscriptionally by hypoxia in tumor necrosis and in various models of ischemia [21,22]. Ischemia stimulates VEGF expression in the brain suggesting that it may be important for the vascular response to cerebral ischemia [23,24,25]. Several single nucleotide polymorphisms (SNPs) have been described in the VEGF gene (National Center for Biotechnology Information, Gene association no: NT 007592). The VEGF gene includes at least 4 relatively common polymorphisms that may influence VEGF expression: 22578C.A (rs699947), 21154G.A (rs1570360), 2634G.C (rs2010963), and 936C.T (rs3025039) [26,27,28].
Three of these polymorphisms are located in the promoter region at 22578, 21154, and 2634 relative to the translation start site. The 22578A, 21154A, and 2634G alleles are all associated with decreased VEGF expression [26,27]. In addition to promoter region polymorphisms, the T allele of the common 936C.T polymorphism in the 39-untranslated region is also associated with significantly decreased serum VEGF levels [28]. Recently, several SNPs of the VEGF gene have been associated with cancer risk and prognosis [29], as well as coronary arterial disease [30]. Moreover, KDR 2604C, 1192A, and 1719A alleles of chromosome 4 were associated with decreased VEGF binding activity and coronary artery disease [31]. These results indicate the importance of VEGF-KDR signaling in human disease.
To our knowledge, no previous study has evaluated both KDR and VEGF polymorphisms with moyamoya disease and collateral vessel formation after surgery. The aim of present study was to evaluate the frequencies of the VEGF 22578C.A, 21154G.A, 2634G.C, and 936C.T and KDR 2604T.C (rs2071559), 1192G.A (rs2305948), and 1719T.A (rs1870377) polymorphisms in Korean patients with moyamoya disease in an effort to determine the relationship of these polymorphisms with moyamoya disease. Moyamoya disease has a bimodal pattern of incidence so we divided the patients into pediatric (,18 years) and adult ($18 years) groups. We further divided the moyamoya patients into ischemic or hemorrhagic groups based on clinical and MRI findings. We performed indirect bypass surgery in 64 patients and direct superficial temporal artery to middle cerebral artery bypass plus encephalo-duro-arterio-myo-synangiosis (STA-MCA plus EDAMS) in one patient. We graded newly developed collateral vessels according to the method of Matsushima et al. [32]. Briefly, Grade A represented synangiosis-induced filling of greater than two-thirds of MCA circulation, Grade B represented between onethird and two-thirds, and Grade C represented less than one-third. We further divided the 64 indirect bypass surgical patients by collateral vessel formation after 2 years into good (collateral grade A) and poor (collateral grade B, C) using MRA. Table 1 shows the demographic characteristics of the moyamoya patients and control subjects. All participants gave informed written consent prior to enrollment in the study. The institutional review boards of Severance Hospital (4-2008-0308) and CHA Bundang Medical Center (PBC09-103) approved this study.

VEGF Genotyping
We investigated four relevant single nucleotide polymorphism (SNP) candidates in the VEGF gene. We used the G-DEX blood extraction kit (iNtRON Biotechnology, Inc., Seongnam, South Korea) according to the manufacturer's instructions for DNA extraction. We obtained all SNP sequences from the HapMap database (www.hapmap.org) [33]. The VEGF -2578C.A, -1154G.A, -634G.C, and 936C.T polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.
We The VEGF 22578C.A and 2634G.C polymorphisms were identified by digesting the PCR product with the restriction endonuclease AvaII (New England Biolabs, Beverly, MA, USA). The VEGF 21154G.A polymorphism was identified by digesting the PCR product with the restriction endonuclease MnlI (New England Biolabs). The VEGF 936C.T polymorphism was identified by digesting the PCR product with the restriction endonuclease NlaIII (New England Biolabs). All restriction digests were performed at 37uC for 16 h.

KDR Genotyping
We identified 3 well-known SNPs in the KDR gene, including one in the promoter region (2604) and two in the coding region (1192 and 1719). All SNP sequences were obtained from the HapMap database (http://www.hapmap.org) [33]. We used previously described primers and PCR-RFLP conditions for

Measurement of Whole Blood Nitric Oxide (NO)
Blood was collected from moyamoya patients into a tube containing anticoagulant 12 hours after a meal. NO production was evaluated by determining the circulating levels of nitrosylhemoglobin complexes. In the present study, the paramagnetic properties of nitrosyl-heme adducts were used to detect whole blood NO-hemoglobin derivatives (n = 33, 5.3066.93 arbitrary unit [AU]) by electron paramagnetic resonance (EPR) spectroscopy.

Statistical Analyses
To analyze the demographic characteristics of moyamoya disease, we used the Mann-Whitney and chi-square (x 2 ) tests for continuous and categorical data, respectively. The associations among pediatric and adult patients were estimated by computing the odds ratios (ORs) and 95% confidence intervals (CIs) using Fisher's exact test. The adjusted odds ratios (AORs) for VEGF and KDR polymorphisms were calculated using multiple logistic regression analyses using gender and age. The genotype distribution of each polymorphism was expected under Hardy-Weinberg equilibrium. Statistical analyses were performed using GraphPad  Figure 1 shows the linkage disequilibrium of VEGF and KDR polymorphisms from the present study. A comparison of genotype frequencies between moyamoya patients and control subjects of the VEGF 22578C.A, 21154G.A, 2634G.C, and 936C.T polymorphisms and the KDR 2604T.C, 1192G.A, and 1719T.A polymorphisms is shown in Table 2. There were no statistically significant differences between moyamoya patients and controls in any of the polymorphisms evaluated (Tables 2 and 3).

Results
In subgroup analyses (Tables 4 and 5), the CC genotype of the VEGF 2634 was less frequent in pediatric moyamoya disease (p = 0.040; CC vs. GG) and comparison with the GG+GC genotype was also significantly different in pediatric moyamoya patients. In the adult subgroup, the VEGF 2634CC genotype was more frequent in moyamoya disease (p = 0.024; CC vs. GG). The frequencies of the KDR polymorphisms in both the pediatric and adult subgroups were not significantly different. We conducted haplotype analyses of the VEGF and KDR polymorphisms ( Table 6). The C-G-C-C haplotype (VEGF 22578/21154/2634/936) in pediatric moyamoya patients was significantly different. In addition, the C-A-T haplotype (KDR 2604/1192/1719) increased the risk of pediatric moyamoya. In adult moyamoya, the A-A-G-C haplotype (VEGF 22578/21154/ 2634) was significantly different whereas other haplotypes were not. We have estimated and provided several haplotype frequencies in Table 6.
We performed indirect bypass surgery in 64 patients. The genotypes containing the VEGF 2634C allele had better collateral vessel formation after surgery whereas 2634GG was associated with poor collateral grade (Table 7). We also investigated the haplotype frequency differences related to collateral grades.
However, there were no statistical differences (Table S1). Our results potentially implicate the VEGF 2634G.C polymorphism in the development of collateral vessel formation in moyamoya disease. The other VEGF and KDR polymorphisms we studied did not exhibit statistically significant differences in collateral vessel formation.
To assess the clinical significance of the VEGF and KDR polymorphisms, we surveyed the association between the studied polymorphisms and various vascular risk factors (tHcy, FA, VB12, and NO). Blood tHcy, FA, VB12, and NO are essential for vascular homeostasis regulation [35]. There were no significant distinctions according to VEGF polymorphisms but the KDR 2604C (p = 0.017) and 1192A (p = 0.032) alleles were linked to decreased NO levels (Table S2). The association of the KDR 2604 and 1192 polymorphisms with NO levels suggests that KDR haplotypes containing 2604C or 1192A may adversely affect vascular homeostasis.

Discussion
In this study, we found VEGF or KDR polymorphisms influence moyamoya disease in subgroup analyses as well as the formation of revascularization after bypass surgery. VEGF is involved in vasculogenesis in different intracranial lesions [11], is an endothelial cell mitogen that induces transient vascular leakage, and a potent angiogenic factor [36]. VEGF promotes angiogenesis in cerebral ischemia [11,37] and causes pathologic vessel formation [12]. Moyamoya disease is characterized by the angiographic findings of arterial stenosis and occlusion of the circle of Willis [38]. It can lead to transient ischemic attacks or a cerebral infarction pattern in juveniles [39] and a hemorrhagic stroke pattern in adults [40,41]. Takekawa et al. [18] reported increased VEGF expression in autopsy specimens from adults with moyamoya disease and Sakamoto et al. reported that the total meningeal cellularity and VEGF expression in the moyamoya dura was significantly higher in moyamoya patients compared to controls [11]. Although increased VEGF concentrations have been demonstrated in moyamoya disease [11,18], the specific role of VEGF in moyamoya genetics remains unclear. We therefore reasoned that mutations and genetic polymorphisms of the VEGF gene may cause cerebral ischemia in moyamoya disease.
Vascular endothelial growth factor A (VEGF-A) is a disulfidebonded dimeric glycoprotein that is a member of a protein family that includes VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PGF). The gene that encodes VEGF-A is located on human chromosome 6 and comprises a 14-kb coding region with eight exons. VEGF cellular signaling activity depends on specific membrane receptors. The receptors include fms like tyrosine kinase-1 (Flt-1 or VEGFR-1), KDR (also known as VEGFR-2) [42,43], and Flt-4 (also known as VEGFR-3) [44,45]. VEGF acts on endothelial cells particularly through Flt-1 and KDR [46,47]. Epidermal growth factor (EGF) arising from hypoxia stimulates Flt-1 expression and inhibits KDR expression [48].
VEGF binding to KDR activates multiple signaling cascades that affect angiogenesis as well as endothelial cell survival, proliferation, and migration. Recently, several SNPs in the VEGF gene have been linked to cancer risk and prognosis [29] and coronary arterial disease [30], indicating the importance of the VEGF-KDR signaling pathway in human disease. Oh et al. reported the SNP 1719T allele conferred ischemic stroke risk in a dose dependent manner [49].
Revascularization after bypass surgery and the formation of new pial vessels may play a role in moyamoya disease. Several studies have suggested that endogenous VEGF production mediates compensatory revascularization during various physiological and pathological processes [37,50,51,52].
In addition, as suggested by the data in our study, VEGF 2634 G.C may be a possible prognostic biomarker after bypass surgery. Taken together, we can speculate that VEGF polymorphisms influence moyamoya disease as well as the formation of synangiosis-induced collateral vessel after bypass surgery. Although revascularization with pial synangiosis helps ameliorate ischemic symptoms, some patients progress to cerebral infarction or hemorrhage, even after surgery. This likely reflects the degree of synangiosis and is dependent on the genetic characteristics of the patient. Therefore, VEGF or KDR polymorphisms can be used as prognostic factors after revascularization surgery.
There are some limitations in correlating VEGF or KDR polymorphisms with our clinical findings. Our data should be interpreted with caution because of the relatively small sample size. Patients require long-term follow up to assess clinical outcomes and variation in the clinical characteristics of moyamoya disease makes it difficult to identify specific genes that are associated with the disease. In addition, moyamoya disease is characterized by genetic heterogeneity and complex interactions between genes and other factors. Although there are limitations with sample size and long-term follow-up clinical findings, it is important to determine the relationship of VEGF or KDR polymorphisms with moyamoya disease and collateral vessel formation after surgery.
In summary, no differences in VEGF 22578, 21154, 2634, and 936 or KDR 2604, 1192, and 1719 polymorphisms were observed between total moyamoya disease patients and control subjects. However, in subgroup analyses, we found that the CC genotype in VEGF 2634 occurred less frequently in pediatric patients (p = 0.040) and occurred more often in adult moyamoya patients (p = 0.024). The genotypes including the VEGF 2634C allele had better collateral vessel formation after surgery. In addition, the C-G-C-C (VEGF 22578/21154/2634/936) haplotype and the C-A-T (KDR -604/1192/1719) haplotype in pediatric patients, as well as the A-A-G-C (VEGF 22578/ 21154/2634/936) in adult moyamoya patients, had significant differences. Therefore, these results suggest that VEGF or KDR polymorphisms influence moyamoya disease as well as the formation of synangiosis-induced collateral vessel after bypass surgery.

Supporting Information
Table S1 Haplotype analyses of VEGF and KDR polymorphisms according to collateral score. *P-values after 10,000 permutation test. (DOC)