Xinhua Zhan

Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures

MicroRNAs (miRNAs) regulate gene expression and have a critical role in many biologic and pathologic processes. We hypothesized that miRNA expression profiles in injured brain (hippocampus) would show common as well as unique profiles when compared with those of blood. Adult, untouched, control rats were compared with rats with sham surgeries, ischemic strokes, brain hemorrhage (lysed blood, fresh blood, or thrombin), and kainate-induced seizures. Brain and whole-blood miRNA expression profiles were assessed 24 h later using TaqMan rodent miRNA arrays. MicroRNA response profiles were different for each condition. Many miRNAs changed more than 1.5-fold in brain and blood after each experimental manipulation, and several miRNAs were upregulated or downregulated in both brain and blood after a given injury. A few miRNAs (e.g., miR-298, miR-155, and miR-362-3p) were upregulated or downregulated more than twofold in both brain and blood after several different injuries. The results show the possible use of blood miRNAs as biomarkers for brain injury; that selected blood miRNAs may correlate with miRNA changes in the brain; and that many of the mRNAs, previously shown to be regulated in brain and blood after brain injury, are likely accounted for by changes in miRNA expression.

Hemorrhagic transformation after ischemic stroke in animals and humans.

Hemorrhagic transformation (HT) is a common complication of ischemic stroke that is exacerbated by thrombolytic therapy. Methods to better prevent, predict, and treat HT are needed. In this review, we summarize studies of HT in both animals and humans. We propose that early HT (<18 to 24 hours after stroke onset) relates to leukocyte-derived matrix metalloproteinase-9 (MMP-9) and brain-derived MMP-2 that damage the neurovascular unit and promote blood–brain barrier (BBB) disruption. This contrasts to delayed HT (>18 to 24 hours after stroke) that relates to ischemia activation of brain proteases (MMP-2, MMP-3, MMP-9, and endogenous tissue plasminogen activator), neuroinflammation, and factors that promote vascular remodeling (vascular endothelial growth factor and high-moblity-group-box-1). Processes that mediate BBB repair and reduce HT risk are discussed, including transforming growth factor beta signaling in monocytes, Src kinase signaling, MMP inhibitors, and inhibitors of reactive oxygen species. Finally, clinical features associated with HT in patients with stroke are reviewed, including approaches to predict HT by clinical factors, brain imaging, and blood biomarkers. Though remarkable advances in our understanding of HT have been made, additional efforts are needed to translate these discoveries to the clinic and reduce the impact of HT on patients with ischemic stroke.

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

BackgroundGene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization.MethodsWhole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms.ResultsReference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder).ConclusionThe reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Targeting neutrophils in ischemic stroke: translational insights from experimental studies

Neutrophils have key roles in ischemic brain injury, thrombosis, and atherosclerosis. As such, neutrophils are of great interest as targets to treat and prevent ischemic stroke. After stroke, neutrophils respond rapidly promoting blood–brain barrier disruption, cerebral edema, and brain injury. A surge of neutrophil-derived reactive oxygen species, proteases, and cytokines are released as neutrophils interact with cerebral endothelium. Neutrophils also are linked to the major processes that cause ischemic stroke, thrombosis, and atherosclerosis. Thrombosis is promoted through interactions with platelets, clotting factors, and release of prothrombotic molecules. In atherosclerosis, neutrophils promote plaque formation and rupture by generating oxidized-low density lipoprotein, enhancing monocyte infiltration, and degrading the fibrous cap. In experimental studies targeting neutrophils can improve stroke. However, early human studies have been met with challenges, and suggest that selective targeting of neutrophils may be required. Several properties of neutrophil are beneficial and thus may important to preserve in patients with stroke including antimicrobial, antiinflammatory, and neuroprotective functions.

Gene Expression Profiling of Blood for the Prediction of Ischemic Stroke

Background and Purpose— A blood-based biomarker of acute ischemic stroke would be of significant value in clinical practice. This study aimed to (1) replicate in a larger cohort our previous study using gene expression profiling to predict ischemic stroke; and (2) refine prediction of ischemic stroke by including control groups relevant to ischemic stroke. Methods— Patients with ischemic stroke (n=70, 199 samples) were compared with control subjects who were healthy (n=38), had vascular risk factors (n=52), and who had myocardial infarction (n=17). Whole blood was drawn ≤3 hours, 5 hours, and 24 hours after stroke onset and from control subjects. RNA was processed on whole genome microarrays. Genes differentially expressed in ischemic stroke were identified and analyzed for predictive ability to discriminate stroke from control subjects. Results— The 29 probe sets previously reported predicted a new set of ischemic strokes with 93.5% sensitivity and 89.5% specificity. Sixty- and 46-probe sets differentiated control groups from 3-hour and 24-hour ischemic stroke samples, respectively. A 97-probe set correctly classified 86% of ischemic strokes (3 hour+24 hour), 84% of healthy subjects, 96% of vascular risk factor subjects, and 75% with myocardial infarction. Conclusions— This study replicated our previously reported gene expression profile in a larger cohort and identified additional genes that discriminate ischemic stroke from relevant control groups. This multigene approach shows potential for a point-of-care test in acute ischemic stroke.

Signatures of cardioembolic and large-vessel ischemic stroke.

The cause of stroke remains unknown or cryptogenic in many patients. We sought to determine whether gene expression signatures in blood can distinguish between cardioembolic and large‐vessel causes of stroke, and whether these profiles can predict stroke etiology in the cryptogenic group.

microRNA Expression in Peripheral Blood Cells following Acute Ischemic Stroke and Their Predicted Gene Targets

Background microRNA (miRNA) are important regulators of gene expression. In patients with ischemic stroke we have previously shown that differences in immune cell gene expression are present. In this study we sought to determine the miRNA that are differentially expressed in peripheral blood cells of patients with acute ischemic stroke and thus may regulate immune cell gene expression. Methods miRNA from peripheral blood cells of forty-eight patients with ischemic stroke and vascular risk factor controls were compared. Differentially expressed miRNA in patients with ischemic stroke were determined by microarray with qRT-PCR confirmation. The gene targets and pathways associated with ischemic stroke that may be regulated by the identified miRNA were characterized. Results In patients with acute ischemic stroke, miR-122, miR-148a, let-7i, miR-19a, miR-320d, miR-4429 were decreased and miR-363, miR-487b were increased compared to vascular risk factor controls. These miRNA are predicted to regulate several genes in pathways previously identified by gene expression analyses, including toll-like receptor signaling, NF-κβ signaling, leukocyte extravasation signaling, and the prothrombin activation pathway. Conclusions Several miRNA are differentially expressed in blood cells of patients with acute ischemic stroke. These miRNA may regulate leukocyte gene expression in ischemic stroke including pathways involved in immune activation, leukocyte extravasation and thrombosis.

Prediction of Cardioembolic, Arterial, and Lacunar Causes of Cryptogenic Stroke by Gene Expression and Infarct Location

Background and Purpose— The cause of ischemic stroke remains unclear, or cryptogenic, in as many as 35% of patients with stroke. Not knowing the cause of stroke restricts optimal implementation of prevention therapy and limits stroke research. We demonstrate how gene expression profiles in blood can be used in conjunction with a measure of infarct location on neuroimaging to predict a probable cause in cryptogenic stroke. Methods— The cause of cryptogenic stroke was predicted using previously described profiles of differentially expressed genes characteristic of patients with cardioembolic, arterial, and lacunar stroke. RNA was isolated from peripheral blood of 131 cryptogenic strokes and compared with profiles derived from 149 strokes of known cause. Each sample was run on Affymetrix U133 Plus 2.0 microarrays. Cause of cryptogenic stroke was predicted using gene expression in blood and infarct location. Results— Cryptogenic strokes were predicted to be 58% cardioembolic, 18% arterial, 12% lacunar, and 12% unclear etiology. Cryptogenic stroke of predicted cardioembolic etiology had more prior myocardial infarction and higher CHA2DS2-VASc scores compared with stroke of predicted arterial etiology. Predicted lacunar strokes had higher systolic and diastolic blood pressures and lower National Institutes of Health Stroke Scale compared with predicted arterial and cardioembolic strokes. Cryptogenic strokes of unclear predicted etiology were less likely to have a prior transient ischemic attack or ischemic stroke. Conclusions— Gene expression in conjunction with a measure of infarct location can predict a probable cause in cryptogenic strokes. Predicted groups require further evaluation to determine whether relevant clinical, imaging, or therapeutic differences exist for each group.

Src Kinase Inhibition Improves Acute Outcomes After Experimental Intracerebral Hemorrhage

Background and Purpose— The mechanisms by which intracerebral hemorrhages produce changes of blood flow and metabolism, cell death, and behavioral abnormalities are complex. In this study, we begin to test the hypothesis that intracerebral hemorrhage activates Src kinases that phosphorylate other molecules to produce cell injury and behavioral deficits after intracerebral hemorrhage (ICH). Methods— ICH was produced in adult Sprague Dawley rats by direct injection of autologous blood (50 &mgr;L) into striatum. Src kinase activity, glucose hypermetabolic areas around the ICH, TUNEL-stained cells, and apomorphine-induced rotational behaviors were assessed in animals with ICH pretreated with the Src kinase inhibitor, PP1, or with vehicle. Results— PP1 (3 mg/kg) blocked increases of Src kinase activity (5-fold) at 3 hours after ICH. PP1 also blocked the areas of glucose hypermetabolism and decreased the numbers of TUNEL-stained cells surrounding the ICH at 24 hours. Finally, apomorphine-induced (1 mg/kg) rotation at 24 hours after ICH was markedly attenuated by previous treatment with PP1 (3 mg/kg intraperitoneal). Conclusions— PP1 decreases Src kinase activation, glucose metabolic activation, cell death, and behavioral abnormalities after ICH in striatum of adult rats. It is hypothesized that intracerebral hemorrhage, possibly via thrombin activation of protease-activated receptors, activates Src that phosphorylates NMDA receptors, matrix metalloproteinases, and other proteins that mediate injury after ICH.

Gram-negative bacterial molecules associate with Alzheimer disease pathology

Objective: We determined whether Gram-negative bacterial molecules are associated with Alzheimer disease (AD) neuropathology given that previous studies demonstrate Gram-negative Escherichia coli bacteria can form extracellular amyloid and Gram-negative bacteria have been reported as the predominant bacteria found in normal human brains. Methods: Brain samples from gray and white matter were studied from patients with AD (n = 24) and age-matched controls (n = 18). Lipopolysaccharide (LPS) and E coli K99 pili protein were evaluated by Western blots and immunocytochemistry. Human brain samples were assessed for E coli DNA followed by DNA sequencing. Results: LPS and E coli K99 were detected immunocytochemically in brain parenchyma and vessels in all AD and control brains. K99 levels measured using Western blots were greater in AD compared to control brains (p < 0.01) and K99 was localized to neuron-like cells in AD but not control brains. LPS levels were also greater in AD compared to control brain. LPS colocalized with Aβ1-40/42 in amyloid plaques and with Aβ1-40/42 around vessels in AD brains. DNA sequencing confirmed E coli DNA in human control and AD brains. Conclusions: E coli K99 and LPS levels were greater in AD compared to control brains. LPS colocalized with Aβ1-40/42 in amyloid plaques and around vessels in AD brain. The data show that Gram-negative bacterial molecules are associated with AD neuropathology. They are consistent with our LPS-ischemia-hypoxia rat model that produces myelin aggregates that colocalize with Aβ and resemble amyloid-like plaques.