The study protocol and all subsequent amendments are approved by the leading ethic committee of the Kyushu University Hospital (Kyushu University Institutional Review Board for Clinical Research, reference number #20-30, date of approval 9/26/2008) and the local ethics committees of the participating centers, National Hospital Organization Kyushu Medical Center (Kyushu Medical Center Institutional Review Board for Clinical Research, reference number # 07–38, date of approval 8/26/2007) and St Mary’s Hospital (St Mary’s Hospital Institutional Review Board for Clinical Research, date of approval 3/11/2008). The study is performed in accordance with the Declaration of Helsinki and its subsequent amendments, as well as the guidelines of Good Clinical Practice. The study is registered at the Fukuoka Stroke Registry (FSR), a multicenter observational study for acute brain infarction in Japan [12, 13]. The registration number is #22-164.
We recruited 171 patients with ischemic stroke who were hospitalized at Kyushu University Hospital, National Hospital Organization Kyushu Medical Center or St. Mary’s Hospital in Japan. On admission, the objectives, study design, risks and benefits were explained in detail to each patient or surrogate family members and written informed consent was obtained. Patients who consented to the study were prospectively enrolled and followed up to 3 months after the onset. Inclusion criteria were as follows: 1) ischemic stroke hospitalized within 24 hours after the onset, 2) definite diagnosis of stroke subtype. We excluded the patients who had severe complication, such as pneumonia or urinary tract infection, during the observational period.
An equal number of age- and gender-matched controls without a history of cardiovascular diseases, such as stroke, coronary heart diseases and atrial fibrillation, were enrolled as healthy subjects from the participants in the Hisayama Study, a worldwide well-known cohort study [14, 15].
Diagnosis of ischemic stroke and subtype
Stroke was defined as a sudden onset of focal neurological deficit persisting for more than 24 hours. The diagnosis of brain infarction was confirmed by brain imaging, including CT and MRI, in all patients. On the basis of the TOAST classification with minor modification,  we classified stroke patients into four categories (subtypes), i.e. atherothrombotic infarction (ATBI, n = 34), lacunar infarction (LAC, n = 45), cardioembolic infarction (CE, n = 49), and other type of brain infarction (OT, n = 43). We combined “Stroke of other determined etiology” and “stroke of undetermined etiology” into OT in this study.
Peripheral venous blood samples were collected from patients at five time points after the onset, days 0 (on admission), 3, 7, 14, and 90. Blood samples were mixed in an EDTA-containing tube, and were centrifuged at 1,400 × g for 10 min at 4 degrees immediately after they were drawn. The resultant plasma samples were frozen at 80 degrees below zero within 10 minutes. They were stored for about two weeks until the measurement of VEGF. For healthy control subjects, blood samples were obtained at one point at the enrollment. Plasma VEGF values were measured using the Human Multi-Analyte Profile (MAP) v1.6 provided by Rules-Based Medicine, Inc. (RBM, TX, USA) . A complete list of the analytes is available at http://www.myriadrbm.com/products-services/humanmap-services/.
Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic pressure ≥ 90 mmHg or as current treatment with antihypertensive drugs during the chronic stage of stroke or at enrollment for control subjects. Diabetes mellitus was determined by either a 75 g oral glucose tolerance test according to the diagnostic criteria of the World Health Organization in 1998,  casual blood glucose levels (≥ 11.1 mmol/L (200 mg/dL)), or a medical history of diabetes. Dyslipidemia was defined as either a cholesterol level ≥ 5.7 mmol/L (220 mg/dL), a low-density lipoprotein-cholesterol level ≥ 3.62 mmol/L (140 mg/dL), a high-density lipoprotein-cholesterol level < 1.03 mmol/L (40 mg/dL) or current treatment with a cholesterol-lowering drug. Atrial fibrillation was diagnosed based on electrocardiographic findings or medical history. Smoking was defined as having a previous or current smoking habit. Alcohol intake was defined as having a previous or current consumption including occasional drinking.
Evaluation of neurological severity and functional outcome
In analyses regarding neurological severity, patients were divided into tertiles (mild, moderate, and severe) in each stroke subtype, according to the National Institute of Health Stroke Scale (NIHSS). Functional outcome was evaluated by the modified Rankin Scale (mRS). Functional outcome was defined as poor (mRS score > 2) or good (mRS score ≤ 2). NIHSS was assessed at day 0 and 14, and mRS at day 14 and 90.
JMP software ver.8.0 (SAS Institute, Cary, NC) was used to perform all statistical analyses. In univariate analyses, categorical variables were compared by the χ
2 test, continuous variables by an unpaired Student t test or multiple comparisons as appropriate. In multivariate analyses, we performed logistic regression analyses with adjustment for confounding factors. P < 0.05 was considered significant.