Microvascular structure and network in the retina of patients with ischemic stroke.

BACKGROUND AND PURPOSE: Microvascular disease has been implicated in the pathogenesis of stroke. The retina provides a window to assess microcirculation noninvasively. We studied the association between quantitatively measured retinal microvascular characteristics and acute ischemic stroke. METHODS: We conducted a case-control study with acute ischemic stroke patients recruited from a tertiary hospital in Singapore and controls from the Singapore Epidemiology of Eye Disease program matched by 10-year age strata, sex, and race. Strokes were classified using modified Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. Retinal vascular parameters were measured from retinal fundus photographs using a computer program. Logistic regression models for stroke were constructed adjusting for age, sex, race, and additionally for smoking, hypertension, diabetes mellitus, and hypercholesterolemia. RESULTS: We included 557 ischemic stroke cases (261 lacunar, 185 large artery, and 54 cardioembolic stroke) and 557 controls. After adjusting for vascular risk factors, decreased arteriolar fractal dimension (odds ratio [OR] per standard deviation [SD] decrease, 2.28; 95% confidence interval [CI], 1.80-2.87) and venular fractal dimension (OR per SD decrease, 1.80; 95% CI, 1.46-2.23), increased arteriolar tortuosity (OR per SD increase, 1.56; 95% CI, 1.25-1.95), and venular tortuosity (OR per SD increase, 1.49; 95% CI, 1.27-1.76), narrower arteriolar caliber (OR per SD decrease, 2.79; 95% CI, 2.21-3.53), and wider venular caliber (OR per SD increase, 1.57; 95% CI, 1.27-1.95) were associated with stroke. Stratification by stroke subtypes and further adjustment for retinopathy signs revealed similar results. CONCLUSIONS: Patients with ischemic stroke have a sparser and more tortuous microvascular network in the retina. These findings provide insight into the structure and pattern of microcirculation changes in stroke.

Tumor necrosis factor-α induces aortic intima-media thickening via perivascular adipose tissue inflammation.

BACKGROUND/AIMS: Neointimal thickening results from inflammation in association with vascular smooth muscle cell (VSMC) proliferation. We studied the role of perivascular adipose tissue (PVAT) on VSMC proliferation and intima-media thickening (IMT) in a rodent model of chronic inflammation. METHODS: The abdominal aorta and surrounding PVAT of tumour necrosis factor (TNF)-α-injected mice were examined 28 days after administration. Plasma and PVAT cytokines were measured with Milliplex™ assays. Inflammatory cells were examined with immunofluorescence. Expression of transforming growth factor (TGF)-ß1, matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 was examined with immunohistochemistry, immunoblotting and zymography. IMT was determined. Cell proliferation and TGF-ß1 mRNA levels were examined after treating VSMC with PVAT homogenates ± MMP-2 inhibitors (batimastat, ARP 100 or TIMP-2) and SB-431542, a selective inhibitor of the TGF-ß-type 1 receptor. RESULTS: Significant increases in CD3, CD68, neutrophils, vascular cell adhesion molecule-1 and MMP-2 in PVAT, and TGF-ß1 and IMT of the aorta of TNF-α-injected mice were observed. PVAT of TNF-α-injected mice significantly up-regulated TGF-ß1 and increased cell proliferation in a dose-dependent manner and was attenuated by SB-431542, batimastat, ARP 100 and TIMP-2. CONCLUSIONS: Our study shows that chronic PVAT inflammation leads to MMP-mediated increase in TGF-ß1 and hence VSMC proliferation.

B vitamins and magnetic resonance imaging-detected ischemic brain lesions in patients with recent transient ischemic attack or stroke: the VITAmins TO Prevent Stroke (VITATOPS) MRI-substudy.

BACKGROUND AND PURPOSE: Elevated concentrations of homocysteine are associated with cerebral small vessel disease (CSVD). B-vitamin supplementation with folate and vitamins B12 and B6 reduces homocysteine concentrations. In a substudy of the VITAmins TO Prevent Stroke (VITATOPS) trial, we assessed the hypothesis that the addition of once-daily supplements of B vitamins would reduce the progression of CSVD-related brain lesions. METHODS: A total of 359 patients with recent stroke or transient ischemic attack, who were randomly allocated to double-blind treatment with placebo or b vitamins, underwent brain MRI at randomization and after 2 years of B-vitamin supplementation. MR images were analyzed blinded to treatment allocation. Outcomes related to the prespecified hypothesis were progression of white matter hyperintensities and incident lacunes. We also explored the effect of B-vitamin supplementation on the incidence of other ischemic abnormalities. RESULTS: After 2 years of treatment with b vitamins or placebo, there was no significant difference in white matter hyperintensities volume change (0.08 vs 0.13 cm3; P=0.419) and incidence of lacunes (8.0% vs 5.9%, P=0.434; odds ratio=1.38). In a subanalysis of patients with MRI evidence of severe CSVD at baseline, b-vitamin supplementation was associated with a significant reduction in white matter hyperintensities volume change (0.3 vs 1.7 cm3; P=0.039). CONCLUSIONS: Daily B-vitamin supplementation for 2 years did not significantly reduce the progression of brain lesions resulting from presumed CSVD in all patients with recent stroke or transient ischemic attack but may do so in the subgroup of patients with recent stroke or transient ischemic attack and severe CSVD. CLINICAL TRIAL REGISTRATION: http://vitatops.highway1.com.au/. Unique identifier: NCT00097669 and ISRCTN74743444.

Long-Term Post-Stroke Functional Outcomes: A Comparison of Diabetics and Nondiabetics.

INTRODUCTION: Diabetes mellitus (DM) is known to influence outcomes in the short term following stroke. However, the impact of DM on long-term functional outcomes after stroke is unclear. We compared functional outcomes periodically over 7 years between diabetic and nondiabetic ischemic stroke patients, and investigated the impact of DM on the long-term trajectory of post-stroke functional outcomes. We also studied the influence of age on the diabetes-functional outcome association. METHODS: This is a longitudinal observational cohort study of 802 acute ischemic stroke patients admitted to the Singapore General Hospital from 2005 to 2007. Functional outcomes were assessed using the modified Rankin Scale (mRS) with poor functional outcome defined as mRS ≥3. Follow-up data were determined at 6 months and at median follow-up durations of 29 and 86 months. RESULTS: Among the 802 ischemic stroke patients studied (mean age 64 ± 12 years, male 63%), 42% had DM. In regression analyses adjusting for covariates, diabetic patients were more likely to have poor functional outcomes at 6 months (OR = 2.12, 95% CI: 1.23-3.67) and at median follow-up durations of 29 months (OR = 1.96, 95% CI: 1.37-2.81) and 86 months (OR = 2.27, 95% CI: 1.58-3.25). In addition, age modulated the effect of DM, with younger stroke patients (≤65 years) more likely to have long-term poor functional outcome at the 29-month (p = 0.0179) and 86-month (p = 0.0144) time points. CONCLUSIONS: DM was associated with poor functional outcomes following ischemic stroke in the long term, with the effect remaining consistent throughout the 7-year follow-up period. Age modified the effect of DM in the long term, with an observed increase in risk in the ≤65 age-group but not in the >65 age-group.

The prognostic effects of poststroke cognitive impairment no dementia and domain-specific cognitive impairments in nondisabled ischemic stroke patients.

BACKGROUND AND PURPOSE: There is some evidence that poststroke dementia, cognitive impairment no dementia (CIND), and mild cognitive impairment predict for poor outcomes such as dementia, death, and institutionalization. However, few studies have examined the prognostic value of CIND, CIND severity, and domain impairments in a poststroke cohort. METHODS: A cohort of ischemic stroke patients with baseline cognitive assessments 3 months poststroke were followed up annually for outcomes of dependency, vascular events, and death for up to 5 years. Univariate and multivariate Cox proportional regression was performed to determine the ability CIND, CIND severity, and domain impairments to predict dependency, vascular outcomes, and death. RESULTS: Four-hundred nineteen patients without dementia (mean age 60±11 years, 32% female) were followed for a mean of 3.2 years. Older age, diabetes, more severe strokes, CIND-mild, and CIND-moderate were independently predictive of dependency. There were no independent predictors of recurrent vascular events. Older age, diabetes, and CIND-moderate were independently predictive of death. In analyses of individual cognitive domains, impairments in visuomotor speed were independently predictive of dependency. CONCLUSIONS: In poststroke patients, CIND predicts dependency and death, while CIND severity discriminates patients with poor survival. Impairments in visuomotor speed independently predict dependency. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique Identifier: NCT00161070.

Associations of retinal microvascular signs and intracranial large artery disease.

BACKGROUND AND PURPOSE: Intracranial large artery disease (ICLAD) is a major cause of ischemic stroke. Retinal microvascular changes are associated with stroke, including small vessel cerebral disease and extracranial carotid disease. We examined the relationship between ICLAD and retinal microvascular changes. METHODS: This is a prospective cohort of 802 acute ischemic stroke patients. Retinal changes were assessed from photographs by graders masked to clinical data. ICLAD was evaluated using prespecified criteria. RESULTS: ICLAD was not associated with ipsilateral retinal arteriolar/venular caliber, focal arteriolar narrowing, or arteriovenous nicking. Severe enhanced arteriolar light reflex was independently associated with any ICLAD (P=0.006) and severe ICLAD (P<0.001). CONCLUSIONS: Enhanced arteriolar light reflex, but not retinal vessel caliber, was related to ICLAD. These data suggest that retinal microvascular signs have specific associations with large cerebral vessel disease.

Intracranial Large Artery Disease is Independently Associated with Poor Functional Outcome in a Cohort of Ethnic South Asian Ischemic Stroke Patients.

BACKGROUND: Intracranial large artery disease (ICLAD) in ischemic stroke patients is associated with an increased risk for recurrent stroke; however, it is not known if ICLAD influences functional status following stroke. We studied the 6-month functional outcome in south Asian ischemic stroke patients and compared those with and without ICLAD. MATERIALS AND METHODS: This is a prospective cohort study of consecutive south Asian ischemic stroke patients. ICLAD was assessed with transcranial color-coded Doppler ultrasound or magnetic resonance angiography. Functional outcomes were obtained via telephone interviews with poor outcome defined as modified Rankin scale of 3-6. RESULTS: Of 216 ischemic stroke patients studied, 203 (93.9%) had follow-up data, of whom 50.7% (103) had ICLAD. Patients with ICLAD had a higher prevalence of hypertension (P < 0.001), hyperlipidemia (P = 0.047), ischemic heart disease (P = 0.030), and extracranial carotid disease (P = 0.005). A higher proportion of patients with ICLAD had poor functional outcome at 6 months (30.1%) versus those without ICLAD (13.0%) (P = 0.004). After adjusting for age, sex, hypertension, hyperlipidemia, diabetes, ischemic heart disease, atrial fibrillation, extracranial carotid stenosis, and recurrent vascular events, patients with ICLAD were 3.01 (95% confidence interval: 1.35-7.10) times more likely than those without ICLAD to have poor functional outcome. CONCLUSIONS: The presence of ICLAD rendered poorer functional prognosis after stroke. These findings support the specific evaluation of the benefits of known acute stroke treatments such as thrombolysis, as well as investigation of potential novel strategies such as acute stenting.

Differential associations of cortical and subcortical cerebral atrophy with retinal vascular signs in patients with acute stroke.

BACKGROUND AND PURPOSE: The relationship of cortical and subcortical cerebral atrophy to cerebral microvascular disease is unclear. We aimed to assess the associations of retinal vascular signs with cortical and subcortical atrophy in patients with acute stroke. METHODS: In the Multi-Centre Retinal Stroke Study, 1360 patients with acute stroke admitted to 2 Australian and 1 Singaporean tertiary hospital during 2005 to 2007 underwent neuroimaging and retinal photography. Cortical and subcortical cerebral atrophy were graded based on standard CT scans. A masked assessment of retinal photographs identified focal retinal vascular signs, including retinopathy and retinal arteriolar wall signs (ie, focal arteriolar narrowing, arteriovenous nicking, arteriolar wall light reflex) and measured quantitative signs (retinal arteriolar and venular caliber). RESULTS: After adjusting for age, gender, study site, hypertension, hypercholesterolemia, diabetes, and smoking status, none of the retinal vascular signs assessed were associated with cortical atrophy, whereas retinopathy (OR, 1.9; CI, 1.2 to 3.0) and enhanced arteriolar light reflex (OR, 2.0; CI, 1.2 to 3.2) were significantly associated with subcortical atrophy. CONCLUSIONS: Our finding that certain retinal vascular signs are associated with subcortical but not cortical atrophy, suggests a differential pathophysiology between these 2 cerebral atrophy subtypes and a potential role for small vessel disease underlying subcortical cerebral atrophy.

Ethnicity does not affect the homocysteine-lowering effect of B-vitamin therapy in Singaporean stroke patients.

BACKGROUND AND PURPOSE: Increased total homocysteine (tHcy) is a risk factor for stroke. This study examines whether the efficacy of B-vitamins in reducing tHcy is modified by ethnicity in a Singaporean ischemic stroke population. METHODS: 505 patients (419 Chinese, 41 Malays and 45 Indians) with ischemic stroke were randomized to receive placebo or B-vitamins. Fasting blood samples collected at baseline and 1 year were assayed for tHcy. MTHFR polymorphisms were genotyped. RESULTS: Ethnicity did not independently determine tHcy at baseline. The magnitude of tHcy reduction by B-vitamin treatment was consistent across ethnic groups (Chinese -3.8+/-4.5, Malay -4.9+/-4.2, and Indian -3.3+/-3.6 micromol/L) despite ethnic differences in MTHFR genotype and baseline folic acid (FA) and vitamin B(12) (vitB(12)) concentrations. CONCLUSIONS: Ethnicity does not appear to affect the tHcy-lowering effect of B-vitamins, despite differences in dietary intake and prevalence of MTHFR polymorphisms. This suggests that the effect of B-vitamins in lowering tHcy is generalizable across Asian populations. However, due to relatively small numbers of non-Chinese studied, confirmation in other populations is required.

Retinal vascular caliber and extracranial carotid disease in patients with acute ischemic stroke: the Multi-Centre Retinal Stroke (MCRS) study.

BACKGROUND AND PURPOSE: Previous studies show that both retinal vascular caliber and carotid disease predict incident stroke in the general population, but the exact relationship between these 2 microvascular and macrovascular structural risk factors is unclear. We studied the relationship between retinal vascular caliber and carotid disease in patients presenting with acute ischemic stroke. METHODS: We conducted a cross-sectional study of patients with acute ischemic stroke recruited from 3 centers (Melbourne, Sydney, Singapore). The caliber of retinal arterioles and venules was measured from digital retinal photographs. Severe extracranial carotid disease was defined as stenosis >or=75% or occlusion determined by carotid Doppler using North American Symptomatic Carotid Endarterectomy Trial-based criteria. RESULTS: Among the 1029 patients with acute stroke studied, 7% of the population had severe extracranial carotid disease. Retinal venular caliber was associated with ipsilateral severe carotid disease (P<0.001 in multivariate models). Patients with wider retinal venular caliber were more likely to have severe ipsilateral carotid disease (multivariable-adjusted OR, 3.81; 95% CI, 1.80 to 8.07, comparing the largest and smallest venular caliber quartiles). The retinal venular caliber-carotid disease association remained significant in patients with large artery stroke. CONCLUSIONS: In patients with acute stroke, retinal venular widening was strongly associated with ipsilateral severe extracranial carotid disease. Our findings suggest concomitant retinal and cerebral microvascular disease may be present in patients with carotid stenosis or occlusion disease. The pathogenesis of stroke due to carotid disease may thus be partially mediated by microvascular disease.

Enhanced sensitivity of celecoxib in human glioblastoma cells: Induction of DNA damage leading to p53-dependent G1 cell cycle arrest and autophagy.

BACKGROUND: Selective cyclooxygenase (COX)-2 inhibitors elicit anti-proliferative responses in various tumours, however the underlying anti-tumour mechanisms are unclear. Mutational inactivation of the tumour suppressor p53 gene is frequent in malignant gliomas. The role of p53 mutation in the anti-tumour responses of the selective COX-2 inhibitor celecoxib in human glioblastoma cells is unknown. In this study, we used human glioblastoma cells with various p53 status; U87MG (with high and low p53 functional levels), LN229 (functional p53) and U373MG (mutant p53) cells. Inhibition of p53 was achieved in U87MG cells transfected with E6 oncoprotein (U87MG-E6) and treated with pifithrin-alpha, a reversible inhibitor of p53 (U87MG-PFT). We investigated whether the anti-glioblastoma responses of celecoxib were p53-dependent, and whether celecoxib induced DNA damage leading to p53-dependent G1 cell cycle arrest, followed by autophagy or apoptosis. RESULTS: Our findings demonstrated that celecoxib concentration-dependently reduced glioblastoma cell viability, following 24 and 72 hours of treatment. Inhibition of functional p53 in glioblastoma cells significantly reduced the anti-proliferative effect of celecoxib. In U87MG cells, celecoxib (8 and 30 muM) significantly induced DNA damage and inhibited DNA synthesis, corresponding with p53 activation. Celecoxib induced G1-phase cell cycle arrest, accompanied with p21 activation in U87MG cells. Cell cycle progression of U87MG-E6 and U87MG-PFT cells was not affected by celecoxib. In parallel, celecoxib induced G1 cell cycle arrest in LN229 cells, but not in U373MG cells. Autophagy was induced by celecoxib in U87MG and LN229 cells, as shown by the significantly greater population of acridine orange-stained cells and increased levels of LC3-II protein (in comparison with non-treated controls). Celecoxib did not induce significant autophagy in U87MG-PFT, U87MG-E6 and U373MG cells, which lack functional p53. Regardless of p53 status, celecoxib caused no significant difference in apoptosis level of U87MG, U87MG-PFT, U87MG-E6 and U373MG cells. CONCLUSION: Our findings reveal that p53 increases human glioblastoma sensitivity to celecoxib. Celecoxib inhibits glioblastoma cell viability by induction of DNA damage, leading to p53-dependent G1 cell cycle arrest and p53-dependent autophagy, but not apoptosis.

Retinal microvasculature in acute lacunar stroke: a cross-sectional study.

BACKGROUND: Lacunar stroke accounts for a quarter of cases of acute ischaemic stroke; however, its underlying pathophysiology remains unclear. Our aim was to establish whether there is an association between changes in the retinal microvasculature and lacunar stroke that might provide clues to the pathology of cerebral small vessel disease. METHODS: In this cross-sectional study, we recruited patients who presented with acute stroke at three centres in two countries (Sydney and Melbourne, Australia, and Singapore). Each patient had standardised clinical assessments, retinal photography, and CT or MRI of the brain. Changes in the retinal microvasculature were assessed from retinal photographs by graders who were masked to the patients' clinical details. Lacunar stroke was diagnosed according to a modified version of the TOAST criteria (Treatment of Acute Stroke Trial) or the OCSP criteria (Oxfordshire Community Stroke Project) and by MRI findings. FINDINGS: We recruited 1321 patients aged 19 to 94 years with acute ischaemic stroke; 410 (31%) had lacunar stroke. Patients with acute lacunar stroke were no more likely to have hypertension (p=0.12), diabetes (p=0.51), or hypercholesterolaemia (p=0.91) than were patients with other types of ischaemic stroke. However, patients with lacunar stroke were more likely to have retinal microvessel signs, particularly when stroke subtype was confirmed using diffusion-weighted MRI, than were patients with other stroke subtypes. After adjustment for age, sex, study site, smoking history, hypertension, and diabetes, the patients with lacunar stroke were more likely than those with other stroke subtypes to have microvessel signs, and when stroke subtype was confirmed by diffusion-weighted MRI the odds ratios were: 3.55 (95% CI 1.77-7.12) for focal arteriolar narrowing; 1.96 (1.19-3.24) for arteriovenous nipping; 2.32 (1.42-3.79) for enhanced light reflex of the arteriolar wall; 1.33 (0.74-2.41) for generalised retinal arteriolar narrowing; 1.45 (0.84-2.51) for small retinal arteriole:venule ratio; and 1.35 (0.80-2.26) for retinal venular widening. INTERPRETATION: Our findings suggest that acute lacunar stroke is a manifestation of non-atherothrombotic occlusive small vessel disease, which might have implications for the prevention and treatment of this stroke subtype. FUNDING: National Health and Medical Research Council of Australia; National Medical Research Council of Singapore; Scottish Funding Council; New South Wales Health.

Serum erythrocyte sedimentation rate is higher among ethnic South Asian compared to ethnic Chinese ischemic stroke patients. Is this attributable to metabolic syndrome or central obesity?

Inflammation, a vascular risk factor, is more pronounced among ethnic South Asians compared to ethnic Chinese in the general population. We compared serum erythrocyte sedimentation rate (ESR) levels between ethnic South Asian and Chinese acute ischemic stroke patients, and further investigated if metabolic syndrome or central obesity could account for any difference detected. We prospectively recruited consecutive ischemic stroke patients within seven days of onset. Measurement of serum ESR was performed within two days of admission. Median serum ESR was higher among the 55 ethnic South Asian (16 mm/h IQR 3-35) compared to the 165 ethnic Chinese patients (9 mm/h IQR 4-19), p=0.004). Serum ESR was correlated with age. Higher serum ESR was associated with female gender, non-smokers, patients with central obesity and low high-density lipoprotein (HDL) cholesterol. Using regression analysis, South Asian ethnicity remained significantly associated with serum ESR, independent of age, gender, smoking status, metabolic syndrome, central obesity and low HDL. Ethnic South Asian ischemic stroke patients have a higher inflammatory state compared to ethnic Chinese patients. As the higher inflammatory state is independent of demographic and risk factors, we propose an underlying genetic or cultural basis for the ethnic difference.

Metabolic syndrome is associated with intracranial large artery disease among ethnic Chinese patients with stroke.

BACKGROUND: The association of metabolic syndrome (MetS) and intracranial large artery disease (ICLAD) has been described, but only in cohorts of ethnic Korean patients with stroke. We investigated the relationship of MetS and ICLAD among ethnic Chinese patients. METHODS: This is a prospective study of consecutive ethnic Chinese patients with acute ischemic stroke living in Singapore. ICLAD was diagnosed on transcranial color-coded Doppler or magnetic resonance angiography. RESULTS: Among the 135 patients studied, the frequency of MetS was higher among patients with ICLAD (39% v 16%, P = .003). This association was independent of age, hypertension, and diabetes (P = .003), and individual MetS criteria (P = .004). The prevalence of ICLAD positively correlated with the number of MetS criteria fulfilled (P = .021). CONCLUSIONS: Our findings of an association between MetS and ICLAD among ethnic Chinese patients with stroke concur with findings among ethnic Korean patients. This validation provides a basis for further investigation into the pathophysiologic link between MetS and ICLAD.

Is routine retinal examination useful in patients with acute ischemic stroke?

BACKGROUND AND PURPOSE: Patients with ischemic stroke have a high prevalence of hypertension and diabetes, which are major risk factors for potentially blinding retinal diseases. We studied the prevalence of retinal diseases, and the need for an ophthalmology referral, among persons with acute ischemic stroke. METHODS: We conducted a prospective study of 300 consecutive patients with acute ischemic stroke. Retinal photographs were taken and assessed in a masked fashion. Patients were advised and referred if they required an ophthalmology evaluation. RESULTS: Of the 286 patients with gradable photographs, retinal abnormalities were detected in 59%. Ophthalmology evaluation was advised for 3% of patients on an urgent basis and 28% on a nonurgent basis and resulted in either acute treatment or active follow-up for all who were subsequently reviewed. CONCLUSIONS: Patients with acute ischemic stroke have a high prevalence of retinal abnormalities. This study suggests that a routine retinal examination may provide an opportunity to detect potentially vision-threatening retinal diseases.

Characterization of a side population of astrocytoma cells in response to temozolomide.

OBJECT: Cancer progenitor-like cells isolated by Hoechst 33342 dye efflux (termed the "side population" [SP]) have been studied in a variety of cancers, including malignant brain tumors. In this study, the authors investigate the nature of the SP phenotype in 2 glioma cell lines, U87MG and T98G, and their response to temozolomide. The roles of several adenosine triphosphate-binding cassette (ABC) multidrug transporters expressed by SP cells, in particular ABCG2, are also examined. METHODS: Using fluorescence-activated cell sorting, the cells were separated into SP and non-SP fractions and analyzed for progenitor cell-like properties with immunofluorescence staining, quantitative real-time polymerase chain reaction, and their ability to reform glioma mass in an immune-compromised mouse. The response of the SP cells to temozolomide was investigated at the cellular and molecular levels. Small interfering RNA knockdown was used to examine the specific role of the ABCG2 transporter, and the cells' tumorigenic potential was measured using the soft agar clonogenic assay. RESULTS: Side population cells are characterized by the presence of progenitor cell-like properties: increased expression of nestin, musashi-1, and ABCG2 were observed. In addition, only SP cells were able to reconstitute cellular heterogeneity; these cells were also more invasive than the non-SP cells, and possessed tumorigenic capacity. Temozolomide treatment increased the number of SP cells, and this corresponded to more progenitor-like cells, concurrent with elevated expression of several ABC transporters. CONCLUSIONS: Knockdown of ABCG2 transporters did not abrogate the SP cell response to temozolomide. Upregulation of several other ABC drug transporter genes is proposed to account for this chemoresistance.

Concomitant coronary artery disease among Asian ischaemic stroke patients.

INTRODUCTION: Coronary artery disease (CAD) is the leading cause of death following ischaemic stroke. We aimed to study the prevalence and associations of concomitant CAD among ischaemic stroke patients in Singapore. MATERIALS AND METHODS: We prospectively studied 2686 consecutive Asian ischaemic stroke patients. RESULTS: CAD was prevalent among 24% of the study patients. Older age, hypertension, diabetes, hyperlipidaemia, atrial fibrillation, large stroke and South Asian ethnicity were independently associated with CAD. CONCLUSIONS: The variables found to be associated with CAD are known atherosclerotic risk factors (older age, hypertension, diabetes, hyperlipidaemia) or associations of cardioembolic stroke (atrial fibrillation, large stroke). The over-representation of South Asians with concomitant CAD is consistent with the high burden of CAD in this ethnic group.

South Asian patients with ischemic stroke: intracranial large arteries are the predominant site of disease.

BACKGROUND AND PURPOSE: South Asians are the most prevalent ethnic group in the world. Intracranial disease is the most common vascular lesion worldwide. METHODS: We prospectively studied 200 consecutive ethnic South Asian patients with acute ischemic stroke in Singapore. RESULTS: Intracranial large-artery disease was prevalent among 54% of all stroke subtypes and was independently associated with hypertension and higher serum erythrocyte sedimentation rate. CONCLUSIONS: Among ethnic South Asian patients with ischemic stroke, intracranial large arteries are the predominant site of disease.

Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor celecoxib: inhibition of tumor angiogenesis with extensive tumor necrosis.

PURPOSE: Toward improved glioblastoma multiforme treatment, we determined whether celecoxib, a selective cyclooxygenase (COX)-2 inhibitor, could enhance glioblastoma radiosensitivity by inducing tumor necrosis and inhibiting tumor angiogenesis. METHODS AND MATERIALS: U-87MG cells treated with celecoxib, irradiation, or both were assayed for clonogenic survival and angiogenic factor protein analysis (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor [VEGF]). In vivo, survival of mice intracranially implanted with U-87MG cells and treated with celecoxib and/or irradiation was monitored. Isolated tumors were assessed for tumor necrosis and tumor microvascular density by von Williebrand's factor (vWF) immunohistochemical staining. RESULTS: Celecoxib (4 and 30 microM; 24, 48, and 72 h) enhanced U-87MG cell radiosensitivity by significantly reducing clonogenic survival of irradiated cells. Angiopoietin-1 and VEGF proteins were decreased, whereas angiopoietin-2 expression increased after 72 h of celecoxib alone and when combined with irradiation. In vivo, median survival of control mice intracranially implanted with U-87MG cells was 18 days. Celecoxib (100 mg/kg/day, 2 weeks) significantly extended median survival of irradiated mice (24 Gy total) from 34 to 41 days, with extensive tumor necrosis [24.5 +/- 8.6% of tumor region, compared with irradiation alone (2.7 +/- 1.8%)]. Tumor microvascular density was significantly reduced in combined celecoxib and irradiated tumors (52.5 +/- 2.9 microvessels per mm2 tumor region), compared with irradiated tumors alone (65.4 +/- 4.0 microvessels per mm2). CONCLUSION: Celecoxib significantly enhanced glioblastoma radiosensitivity, reduced clonogenic survival, and prolonged survival of glioblastoma-implanted mice by inhibition of tumor angiogenesis with extensive tumor necrosis.

A novel methodology to probe endothelial differential gene expression profile reveals novel genes.

Endothelial dysfunction is a major feature of vascular diseases. A practical, minimally invasive method to effectively "probe" gene transcription for an individual patient's endothelium has potential to "customize" assessment for an individual at risk of vascular disease as well as pathophysiologic insight in an in vivo human, clinical context. Published literature lacks a methodology to identify endothelial differential gene expression in individuals with vascular disease. We describe a methodology to do so. The aim of this study was to specifically utilize (a) cutaneous microvascular biopsy, (b) laser capture microdissection, (c) cDNA amplification, (d) suppression subtractive hybridization, (e) high-throughput sequencing techniques, (f) real-time polymerase chain reaction (PCR), and (g) in combination of these methods, to profile differential gene expression in the context of cardiovascular and cerebrovascular disease. Endothelial cells were obtained by laser capture microdissection from a patient and a healthy sibling's microvascular biopsy tissues. Endothelial RNA was extracted, reverse transcribed, and amplified to ds cDNA. Suppression subtractive hybridization was used to establish an endothelial differential gene expression library. Real-time PCR confirmed SERP1, caspase 8, IGFBP7, S100A4, F85, and F147 up-regulation between 1.4- and 3.47-fold. The authors have successfully established a methodology to profile endothelial differential gene expression and identified six differentially expressed genes. This minimally invasive novel method has potential wide application in the customized assessment of many patients suffering vascular diseases.