Vitamins Reverse Endothelial Dysfunction Through Regulation of eNOS and NAD(P)H Oxidase Activities

Antioxidant vitamins C and E have protective properties in genetic hypertension associated with enhanced oxidative stress. This study investigated whether vitamins C and/or E modulate vascular function by regulating enzymatic activities of endothelial nitric oxide synthase (eNOS) and NAD(P)H oxidase using thoracic aortas of 20- to 22-week-old male spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar-Kyoto rats (WKY). SHR aortas had impaired relaxant responses to acetylcholine but not to sodium nitroprusside, despite an 2-fold increase in eNOS activity and NO release. The levels of superoxide anion (O2 ), a potent NO scavenger, and NAD(P)H oxidase activity were also 2-fold higher in SHR aortas. Mechanical but not pharmacological inactivation of endothelium (by rubbing and 100 mol/L L-NAME, respectively) significantly abrogated O2 in both strains. Treatments of SHR aortas with NAD(P)H oxidase inhibitors, namely diphenyleneiodinium and apocynin, significantly diminished O2 production. The incubation of SHR aortas with different concentrations of vitamin C (10 to 100 mol/L) and specifically with high concentrations of vitamin E (100 mol/L) improved endothelial function, reduced superoxide production as well as NAD(P)H oxidase activity, and increased eNOS activity and NO generation in SHR aortas to the levels observed in vitamin C- and E-treated WKY aortas. Our results reveal endothelial NAD(P)H oxidase as the major source of vascular O2 in SHR and also show that vitamins C and E are critical in normalizing genetic endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities.

Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91-phox and p22-phox in Endothelial Cells

The production of reactive oxygen species (ROS) within endothelial cells may have several effects, including alterations in the activity of paracrine factors, gene expression, apoptosis, and cellular injury. Recent studies indicate that a phagocyte-type NAD(P)H oxidase is a major source of endothelial ROS. In contrast to the high-output phagocytic oxidase, the endothelial enzyme has much lower biochemical activity and a different substrate specificity (NADH.NADPH). In the present study, we (1) cloned and characterized the cDNA and predicted amino acid structures of the 2 major subunits of rat coronary microvascular endothelial cell NAD(P)H oxidase, gp91-phox and p22-phox; (2) undertook a detailed comparison with phagocytic NADPH oxidase sequences; and (3) studied the subcellular location of these subunits in endothelial cells. Although these studies revealed an overall high degree of homology (.90%) between the endothelial and phagocytic oxidase subunits, the endothelial gp91-phox sequence has potentially important differences in a putative NADPH-binding domain and in putative glycosylation sites. In addition, the subcellular location of the endothelial gp91-phox and p22-phox subunits is significantly different from that reported for the neutrophil oxidase, in that they are predominantly intracellular and collocated in the vicinity of the endoplasmic reticulum. This first detailed characterization of gp91-phox and p22-phox structure and location in endothelial cells provides new data that may account, in part, for the differences in function between the phagocytic and endothelial NAD(P)H oxidases.

Clinical outcome after first and recurrent hemorrhage in patients with untreated brain arteriovenous malformation

Background and Purpose: The morbidity from spontaneous hemorrhage of untreated brain arteriovenous malformations (AVM) is not well described. Methods: The 241 consecutive AVM patients (mean age 3716 years, 52% women) from the prospective Columbia AVM Databank initially presenting with hemorrhage were evaluated using the Rankin Scale (RS) and the National Institute of Health Stroke Scale (NIHSS). From the 241 AVM patients, 29 (12%) had subsequent intracranial hemorrhage during follow-up. For further comparisons, 84 non-AVM patients with intracerebral hemorrhage from the Northern Manhattan Study (NOMAS) served as a control group. Results: In 241 AVM patients presenting with hemorrhage the median RS was 2 and the median NIHSS was 1 (49% RS 0 to 1, 61% NIHSS 2). The median time between hemorrhage and clinical evaluation was 11 days (mean 219 days). Recurrent AVM hemorrhage during follow-up resulted in no significant increase in morbidity (median RS 2, P0.004; median NIHSS 3, P0.322; time between hemorrhage and study evaluation: median 55 days, mean 657 days). Among AVM-hemorrhage subtypes, parenchymatous AVM hemorrhage was associated with higher stroke morbidity (odds ratio, 2.9; 95% CI, 1.5 to 5.8 for NIHSS 2) than nonparenchymatous hemorrhages. Parenchymatous AVM hemorrhage had a significantly better outcome (median NIHSS 1) than non-AVM related hemorrhage (median NIHSS 12; P0.0001). Conclusions: Hemorrhage, either at initial presentation or during follow-up of untreated AVM patients appears to carry

Granulocyte-colony-stimulating factor mobilizes bone marrow stem cells in patients with subacute ischemic stroke: the Stem Cell Trial of Recovery EnhanceMent After Stroke (STEMS) Pilot Randomized, Controlled Trial (ISRCTN 16784092)

Background and Purpose - Loss of motor function is common after stroke and leads to significant chronic disability. Stem cells are capable of self-renewal and of differentiating into multiple cell types, including neurones, glia, and vascular cells. We assessed the safety of granulocyte-colony-stimulating factor (G-CSF) after stroke and its effect on circulating CD34 stem cells. Methods - We performed a 2-center, dose-escalation, double-blind, randomized, placebo-controlled pilot trial (ISRCTN 16784092) of G-CSF (6 blocks of 1 to 10 g/kg SC, 1 or 5 daily doses) in 36 patients with recent ischemic stroke. Circulating CD34 stem cells were measured by flow cytometry; blood counts and measures of safety and functional outcome were also monitored. All measures were made blinded to treatment. Results - Thirty-six patients, whose mean SD age was 768 years and of whom 50% were male, were recruited. G-CSF (5 days of 10 g/kg) increased CD34 count in a dose-dependent manner, from 2.5 to 37.7 at day 5 (area under curve, P0.005). A dose-dependent rise in white cell count (P0.001) was also seen. There was no difference between treatment groups in the number of patients with serious adverse events: G-CSF, 7/24 (29%) versus placebo 3/12 (25%), or in their dependence (modified Rankin Scale, median 4, interquartile range, 3 to 5) at 90 days. Conclusions - ”G-CSF is effective at mobilizing bone marrow CD34 stem cells in patients with recent ischemic stroke. Administration is feasible and appears to be safe and well tolerated. The fate of mobilized cells and their effect on functional outcome remain to be determined. (Stroke. 2006;37:2979-2983.)

The Virtual International Stroke Trials Archive

Background and Purpose - Stroke has global importance and it causes an increasing amount of human suffering and economic burden, but its management is far from optimal. The unsuccessful outcome of several research programs highlights the need for reliable data on which to plan future clinical trials. The Virtual International Stroke Trials Archive aims to aid the planning of clinical trials by collating and providing access to a rich resource of patient data to perform exploratory analyses. Methods - Data were contributed by the principal investigators of numerous trials from the past 16 years. These data have been centrally collated and are available for anonymized analysis and hypothesis testing. Results - ”Currently, the Virtual International Stroke Trials Archive contains 21 trials. There are data on 15 000 patients with both ischemic and hemorrhagic stroke. Ages range between 18 and 103 years, with a mean age of 6912 years. Outcome measures include the Barthel Index, Scandinavian Stroke Scale, National Institutes of Health Stroke Scale, Orgogozo Scale, and modified Rankin Scale. Medical history and onset-to-treatment time are readily available, and computed tomography lesion data are available for selected trials. Conclusions - This resource has the potential to influence clinical trial design and implementation through data analyses that inform planning. (Stroke. 2007;38:1905-1910.)

Sex differences in quality of life in stroke survivors. Data from the Tinzaparin in Acute Ischaemic Stroke Trial (TAIST)

Introduction: Female sex is predictive of poor functional outcome in stroke, even after correction for prognostic factors. Poor quality of life (QoL) is observed in stroke survivors, with lower scores seen in the most disabled patients. We used data from the TAIST trial to assess the relationship between sex and QoL after ischaemic stroke. Methods: TAIST was a randomised controlled trial assessing the safety and efficacy of tinzaparin versus aspirin in 1,484 patients with acute ischaemic stroke. QoL was measured at 180 days post randomisation using the short-form 36 health survey which assesses QoL across eight domains. The relationship between sex and each domain was assessed using ordinal regression, both unadjusted and adjusted for key prognostics factors. Results: Of the 1,484 patients randomised into TAIST, 216 had died at 180 days post randomisation. 1,268 survivors were included in this analysis, 694 males (55%), 574 females (45%). Females tended to score lower than males across all QoL domains (apart from general health); statistically significant lower scores were seen for physical functioning (odds ratio (OR) 0.58, 95% confidence interval (CI) 0.47-0.72), vitality (OR 0.79, 95% CI 0.64-0.98) and mental health (OR 0.75, 95% CI 0.61-0.93). The results for physical functioning and mental health remained significant after adjustment for prognostic variables (OR 0.73, 95% CI 0.58-0.92; OR 0.76, 95% CI 0.60-0.95 respectively). Conclusions: QoL, in particular physical function and mental health domains, is lower in female patients after stroke. This difference persists even after correction for known prognostic factors such as age and stroke severity.

Empirical evidence of bias in the design of experimental stroke studies: a metaepidemiologic approach

Background and Purpose: At least part of the failure in the transition from experimental to clinical studies in stroke has been attributed to the imprecision introduced by problems in the design of experimental stroke studies. Using a metaepidemiologic approach, we addressed the effect of randomization, blinding, and use of comorbid animals on the estimate of how effectively therapeutic interventions reduce infarct size. Methods: Electronic and manual searches were performed to identify meta-analyses that described interventions in experimental stroke. For each meta-analysis thus identified, a reanalysis was conducted to estimate the impact of various quality items on the estimate of efficacy, and these estimates were combined in a meta meta-analysis to obtain a summary measure of the impact of the various design characteristics. Results: Thirteen meta-analyses that described outcomes in 15 635 animals were included. Studies that included unblinded induction of ischemia reported effect sizes 13.1% (95% CI, 26.4% to 0.2%) greater than studies that included blinding, and studies that included healthy animals instead of animals with comorbidities overstated the effect size by 11.5% (95% CI, 21.2% to 1.8%). No significant effect was found for randomization, blinded outcome assessment, or high aggregate CAMARADES quality score. Conclusions: We provide empirical evidence of bias in the design of studies, with studies that included unblinded induction of ischemia or healthy animals overestimating the effectiveness of the intervention. This bias could account for the failure in the transition from bench to bedside of stroke therapies.

Can we improve the statistical analysis of stroke trials? Statistical re-analysis of functional outcomes in stroke trials

Background: Most large acute stroke trials have been neutral. Functional outcome is usually analysed using a yes or no answer, e.g. death or dependency vs. independence. We assessed which statistical approaches are most efficient in analysing outcomes from stroke trials. Methods: Individual patient data from acute, rehabilitation and stroke unit trials studying the effects of interventions which alter functional outcome were assessed. Outcomes included modified Rankin Scale, Barthel Index, and ‘3 questions’. Data were analysed using a variety of approaches which compare two treatment groups. The results for each statistical test for each trial were then compared. Results: Data from 55 datasets were obtained (47 trials, 54,173 patients). The test results differed substantially so that approaches which use the ordered nature of functional outcome data (ordinal logistic regression, t-test, robust ranks test, bootstrapping the difference in mean rank) were more efficient statistically than those which collapse the data into 2 groups (chi square) (ANOVA p<0.001). The findings were consistent across different types and sizes of trial and for the different measures of functional outcome. Conclusions: When analysing functional outcome from stroke trials, statistical tests which use the original ordered data are more efficient and more likely to yield reliable results. Suitable approaches included ordinal logistic regression, t-test, and robust ranks test.

Use of ordinal outcomes in vascular prevention trials: comparison with binary outcomes in published trials

Background and Purpose—Vascular prevention trials mostly count “yes/no” (binary) outcome events, eg, stroke/no stroke. Analysis of ordered categorical vascular events (eg, fatal stroke/nonfatal stroke/no stroke) is clinically relevant and could be more powerful statistically. Although this is not a novel idea in the statistical community, ordinal outcomes have not been applied to stroke prevention trials in the past. Methods—Summary data on stroke, myocardial infarction, combined vascular events, and bleeding were obtained by treatment group from published vascular prevention trials. Data were analyzed using 10 statistical approaches which allow comparison of 2 ordinal or binary treatment groups. The results for each statistical test for each trial were then compared using Friedman 2-way analysis of variance with multiple comparison procedures. Results—Across 85 trials (335 305 subjects) the test results differed substantially so that approaches which used the ordinal nature of stroke events (fatal/nonfatal/no stroke) were more efficient than those which combined the data to form 2 groups (P0.0001). The most efficient tests were bootstrapping the difference in mean rank, Mann–Whitney U test, and ordinal logistic regression; 4- and 5-level data were more efficient still. Similar findings were obtained for myocardial infarction, combined vascular outcomes, and bleeding. The findings were consistent across different types, designs and sizes of trial, and for the different types of intervention. Conclusions—When analyzing vascular events from prevention trials, statistical tests which use ordered categorical data are more efficient and are more likely to yield reliable results than binary tests. This approach gives additional information on treatment effects by severity of event and will allow trials to be smaller. (Stroke. 2008;39:000-000.)

The relationship between baseline blood pressure and computed tomography findings in acute stroke: data from the Tinzaparin in Acute Ischaemic Stroke Trial (TAIST)

Background and Purpose—High blood pressure (BP) is present in 80% of patients with acute ischemic stroke and is independently associated with poor outcome. There are few data examining the relationship between admission BP and acute CT findings. Methods—TAIST was a randomized controlled trial assessing 10 days of treatment with tinzaparin versus aspirin in 1489 patients with acute ischemic stroke (48 hr) with admission BP of 220/120 mm Hg. CT brain scans were performed before randomization and after 10 days. The relationships between baseline BP and adjudicated CT findings were assessed. Odds ratios per 10 mm Hg change in BP were calculated. Results—Higher systolic BP (SBP) was associated with abnormal CT scans because of independent associations with chronic changes of leukoariosis (OR, 1.12; 95% CI, 1.05–1.17) and old infarction (OR, 1.12; 95% CI, 1.06 –1.17) at baseline, and signs of visible infarction at day 10 (OR, 1.06; 95% CI, 1.00 –1.13). A lower SBP was associated with signs of acute infarction (OR, 0.94; 95% CI, 0.89–0.99). Hemorrhagic transformation, dense middle cerebral artery sign, mass effect, and cerebral edema at day 10 were not independently associated with baseline BP. Conclusion—Although high baseline BP is independently associated with a poor outcome after stroke, this was not shown to be through an association with increased hemorrhagic transformation, cerebral edema, or mass effect; trial design may be suboptimal to detect this. Higher SBP is associated with visible infarction on day 10 scans. The influence of changing BP in acute stroke on CT findings is still to be ascertained.

Can we improve the statistical analysis of stroke trials? Statistical reanalysis of functional outcomes in stroke trials

Background and Purpose—Most large acute stroke trials have been neutral. Functional outcome is usually analyzed using a yes or no answer, eg, death or dependency versus independence. We assessed which statistical approaches are most efficient in analyzing outcomes from stroke trials. Methods—Individual patient data from acute, rehabilitation and stroke unit trials studying the effects of interventions which alter functional outcome were assessed. Outcomes included modified Rankin Scale, Barthel Index, and “3 questions”. Data were analyzed using a variety of approaches which compare 2 treatment groups. The results for each statistical test for each trial were then compared. Results—Data from 55 datasets were obtained (47 trials, 54 173 patients). The test results differed substantially so that approaches which use the ordered nature of functional outcome data (ordinal logistic regression, t test, robust ranks test, bootstrapping the difference in mean rank) were more efficient statistically than those which collapse the data into 2 groups (2; ANOVA, P0.001). The findings were consistent across different types and sizes of trial and for the different measures of functional outcome. Conclusions—When analyzing functional outcome from stroke trials, statistical tests which use the original ordered data are more efficient and more likely to yield reliable results. Suitable approaches included ordinal logistic regression, test, and robust ranks test.

Good laboratory practice: preventing introduction of bias at the bench

Background and Purpose—As a research community, we have failed to demonstrate that drugs which show substantial efficacy in animal models of cerebral ischemia can also improve outcome in human stroke. Summary of Review—Accumulating evidence suggests this may be due, at least in part, to problems in the design, conduct and reporting of animal experiments which create a systematic bias resulting in the overstatement of neuroprotective efficacy. Conclusions—Here, we set out a series of measures to reduce bias in the design, conduct and reporting of animal experiments modeling human stroke.