Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repa... more Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg / kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.
The non-competitive NMDA receptor antagonists, including PCP (phencyclidine), ketamine, and MK-80... more The non-competitive NMDA receptor antagonists, including PCP (phencyclidine), ketamine, and MK-801 (dizocilpine) produce psychosis in humans and injure neurons in retrosplenial cortex in adult rodent brain. This study examined the effects of the metabotropic mGlu2/3 agonist LY379268 and antagonist LY341495 on cortical injury produced by systemic MK-801 (1 mg/kg i.p.) in adult female rats. Systemic injections of mGlu2/3 agonist LY379268, but not mGlu2/3 antagonist LY341495, decreased the injury in the retrosplenial cortex produced by systemic MK-801 as assessed by Hsp70 induction. Bilateral injections of LY379268, but not vehicle, into retrosplenial cortex or bilateral injections of LY379268 into anterior thalamus also decreased the injury in retrosplenial cortex produced by systemic MK-801. The data show that bilateral activation of mGlu2/3 glutamate receptors in cortex or anterior thalamus decreases the neuronal injury in retrosplenial cortex produced by systemic MK-801. Because antipsychotic medications decrease cortical injury produced by NMDA antagonists in rodents and decrease psychosis in humans, mGlu2/3 agonists that decrease cortical injury produced by NMDA antagonists in rodents might be evaluated for decreasing psychosis in people.
Neural progenitor cells (NPCs) Neuroblastoma cells (NBCs) mRNA microRNA (miRNA) Mature neurons (M... more Neural progenitor cells (NPCs) Neuroblastoma cells (NBCs) mRNA microRNA (miRNA) Mature neurons (MNs), neural progenitor cells (NPCs) and neuroblastoma cells (NBCs) are all neural-derived cells. However, MNs are unable to divide once differentiated; NPCs are able to divide a limited number of times and differentiate to normal brain cell types; whereas NBCs can divide an unlimited number of times but rarely differentiate. Here, we perform whole transcriptome (mRNA, miRNA) profiling of these cell types and compare expression levels of each cell type to the others. Integrated mRNA-miRNA functional analyses reveal that: 1) several very highly expressed genes (e.g., Robo1, Nrp1, Epha3, Unc5c, Dcc, Pak3, Limk4) and a few under-expressed miRNAs (e.g., miR-152, miR-146b, miR-339-5p) in MNs are associated with one important cellular process-axon guidance; 2) some very highly expressed mitogenic pathway genes (e.g., Map2k1, Igf1r, Rara, Runx1) and under-expressed miRNAs (e.g., miR-370, miR-9, miR-672) in NBCs are associated with cancer pathways. These results provide a library of negative mRNAmiRNA networks that are likely involved in the cellular processes of differentiation and division.
Using two microarray platforms, we identify HLA-DRB5 as the most highly expressed gene in MS comp... more Using two microarray platforms, we identify HLA-DRB5 as the most highly expressed gene in MS compared to healthy subjects. As expected, HLA-DRB5 expression was associated with the HLA-DRB1*1501 MS susceptibility allele. Besides HLA-DRB5, there were 1219 differentially expressed exons (pb 0.01, |fold change (FC)|>1.2) that differed between HLA-DRB1*1501 Positive multiple sclerosis subjects (MSP) compared to HLA-DRB1*1501 negative multiple sclerosis subjects (MSN). Analysis of the regulated genes revealed significantly different immune signaling pathways including IL-4 and IL-17 in these two MS genotypes. Different risk alleles appear to be associated with different patterns of gene expression that may reflect differences in pathophysiology of these two MS subtypes. These preliminary data will need to be confirmed in future studies.
The Histone Deacetylase 9 (HDAC9) polymorphism rs2107595 is associated with an increased risk for... more The Histone Deacetylase 9 (HDAC9) polymorphism rs2107595 is associated with an increased risk for large vessel atherosclerotic stroke (LVAS). In humans, there remains a need to better understand this HDAC9 polymorphism's contribution to large vessel stroke. In this pilot study, we evaluated whether the HDAC9 polymorphism rs2107595 is associated with differences in leukocyte gene expression in patients with LVAS. HDAC9 SNP rs2107595 was genotyped in 155 patients (43 LVAS and 112 vascular risk factor controls). RNA isolated from blood was processed on whole genome microarrays. Gene expression was compared between HDAC9 risk allele positive and risk allele negative LVAS patients and controls. Functional analysis identified canonical pathways and molecular functions associated with rs2107595 in LVAS. In HDAC9 SNP rs2107595 risk allele positive LVAS patients there were 155 genes differentially expressed compared to risk allele negative patients (fold change >|1.2|, p<0.05). The 155 genes separated the risk allele positive and negative LVAS patients on a Principal Components Analysis. Pathways associated with HDAC9 risk allele positive status involved IL6 signaling, cholesterol efflux, and platelet aggregation. These preliminary data suggest an association with the HDAC9 rs2107595 risk allele and peripheral immune, lipid, and clotting systems in LVAS. Further study is required to evaluate whether these differences are related to large vessel atherosclerosis and stroke risk.
Journal of Cerebral Blood Flow and Metabolism, Apr 13, 2018
Understanding how the blood transcriptome of human intracerebral hemorrhage (ICH) differs from is... more Understanding how the blood transcriptome of human intracerebral hemorrhage (ICH) differs from ischemic stroke (IS) and matched controls (CTRL) will improve understanding of immune and coagulation pathways in both disorders. This study examined RNA from 99 human whole-blood samples using GeneChip Õ HTA 2.0 arrays to assess differentially expressed transcripts of alternatively spliced genes between ICH, IS and CTRL. We used a mixed regression model with FDR-corrected p(Dx) < 0.2 and p < 0.005 and jFCj > 1.2 for individual comparisons. For time-dependent analyses, subjects were divided into four time-points: 0(CTRL), <24 h, 24-48 h, >48 h; 489 transcripts were differentially expressed between ICH and CTRL, and 63 between IS and CTRL. ICH had differentially expressed T-cell receptor and CD36 genes, and iNOS, TLR, macrophage, and T-helper pathways. IS had more non-coding RNA. ICH and IS both had angiogenesis, CTLA4 in T lymphocytes, CD28 in T helper cells, NFAT regulation of immune response, and glucocorticoid receptor signaling pathways. Self-organizing maps revealed 4357 transcripts changing expression over time in ICH, and 1136 in IS. Understanding ICH and IS transcriptomes will be useful for biomarker development, treatment and prevention strategies, and for evaluating how well animal models recapitulate human ICH and IS.
Objective: To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes. Method... more Objective: To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes. Methods: A total of 212 patients were studied: 106 with acute ischemic stroke and 106 controls matched for risk factors. RNA from circulating leukocytes was isolated from blood collected in PAXgene tubes. Let7i microRNA expression was assessed using TaqMan quantitative reverse transcription PCR. To assess let7i regulation of gene expression in stroke, messenger RNA (mRNA) from leukocytes was measured by whole-genome Human Transcriptome Array Affymetrix microarray. Given microRNAs act to destabilize and degrade their target mRNA, mRNAs that inversely correlated with let7i were identified. To demonstrate let7i posttranscriptional regulation of target genes, a 39 untranslated region luciferase assay was performed. Target protein expression was assessed using ELISA. Results: Let7i was decreased in patients with acute ischemic stroke (fold change 21.70, p , 0.00001). A modest inverse correlation between let7i and NIH Stroke Scale score at admission (r 5 20.32, p 5 0.02), infarct volume (r 5 20.21, p 5 0.04), and plasma MMP9 (r 5 20.46, p 5 0.01) was identified. The decrease in let7i was associated with increased expression of several of its mRNA targets, including CD86, CXCL8, and HMGB1. In vitro studies confirm let7i posttranscriptional regulation of target genes CD86, CXCL8, and HMGB1. Functional analysis predicted let7i regulates pathways involved in leukocyte activation, recruitment, and proliferation including canonical pathways of CD86 signaling in T helper cells, HMGB1 signaling, and CXCL8 signaling. Conclusions: Let7i is decreased in circulating leukocytes of patients with acute ischemic stroke. Mechanisms by which let7i regulates inflammatory response post stroke include targeting CD86, CXCL8, and HMGB1. Neurology ® 2016;87:2198-2205 GLOSSARY cDNA 5 complementary DNA; HTA 5 Human Transcriptome Array; IL 5 interleukin; mRNA 5 messenger RNA; NIHSS 5 NIH Stroke Scale; 39UTR 5 39 untranslated region; TNF-a 5 tumor necrosis factor a.
Background: Since RNA expression differences have been reported in autism spectrum disorder (ASD)... more Background: Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume. Methods: RNA from blood was processed on whole genome exon arrays for 2-4-year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20). Results: A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. The only pathways significant after multiple comparison corrections (FDR <0.05) were the Nrf2-mediated reactive oxygen species (ROS) oxidative response (superoxide dismutase 2, catalase, peroxiredoxin 1, PIK3C3, DNAJC17, microsomal glutathione S-transferase 3) and superoxide radical degradation (SOD2, CAT). Conclusions: These data support differences in alternative splicing of mRNA in blood of ASD subjects compared to TD controls that differ related to head size. The findings are preliminary, need to be replicated in independent cohorts, and predicted alternative splicing differences need to be confirmed using direct analytical methods.
Background and Purpose-The cause of ischemic stroke remains unclear, or cryptogenic, in as many a... more 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 CHA 2 DS 2-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.
Introduction: Inflammation and infection are associated with cerebrovascular diseases including s... more Introduction: Inflammation and infection are associated with cerebrovascular diseases including stroke due to carotid atherosclerotic plaques. C-reactive protein (CRP), an acute-phase protein, is upregulated in the plasma of patients with carotid atherosclerotic plaques. However, little is known about whether bacterial molecules trigger inflammation or play a role in patients with carotid atherosclerotic plaques. Recently, it has been recognized that inflammation associated with atherosclerosis and morbidity and mortality in cardiovascular diseases may be due to lipopolysaccharide (LPS) that is found in the outer wall of all Gram-negative bacteria. These findings prompted this study to explore whether plasma levels of LPS and LPS-binding protein (LBP) are elevated and correlated with CRP levels in patients with asymptomatic carotid plaques (ACP). We also compared LBP levels in patients with ACP to large vessel (LV) strokes due to carotid plaques and to matched controls. Methods: Patients (n = 30) with ACP, LV stroke due to carotid atherosclerosis and age-, sex- matched healthy controls gave consent and had their blood drawn. Plasma was processed for LPS, LBP and CRP detection using separate ELISA for each. Results: Plasma LBP level in ACP (22.7 ± 2.92 μg/ml) was similar to LV stroke (21.6 ± 1.56 μg/ml, p = 0.74, ACP vs LV) but greater than controls (13.6 ± 1.43 μg/ml, p = 0.011, ACP vs controls). In ACP patients, plasma LPS level (159.5 ± 30.5 μg/ml) was greater than controls (42.6 ± 11.7 μg/ml, p = 0.001); plasma CRP levels (20.2 ± 6.2 μg/ml) was higher than controls (5.3 ± 2.1 μg/ml, p = 0.011). There was a positive correlation between LPS levels and LBP levels (r = 0.86, p &lt; 0.00001), LPS levels and CRP levels (r = 0.82, p = 0.00001), and LBP levels and CRP levels (r = 0.89, p &lt; 0.00001) in ACP cases. Conclusions: Plasma LPS, LBP and CRP associate with asymptomatic carotid plaques suggesting a pro-inflammatory state exists in patients with asymptomatic carotid plaques, a cause of large vessel stroke. LPS is postulated to directly upregulate both CRP and LBP. Elevated LBP in large vessel stroke patients suggests a Gram-negative bacteria associated post-stroke inflammatory state.
Intraventricular hemorrhage (IVH) is one of the most serious hemorrhagic brain diseases being ass... more Intraventricular hemorrhage (IVH) is one of the most serious hemorrhagic brain diseases being associated with blood brain barrier (BBB) disruption, brain edema, neuron loss, cognitive impairment and high mortality in humans. Increasing evidence suggests that neuronal cell death ensues when neurons attempt to re-enter the cell cycle. Our previous study has demonstrated that cell cycle inhibition via blocking Src family kinases (SFKs) prevents neuronal cell death. However, one of the concerns with cell cycle therapy might be that it would inhibit proliferation of neural progenitor cells (NPCs) and that inhibiting neurogenesis would produce cognitive effects related to the therapy itself. Using the rodent in vivo IVH model we show that i.p. injection of SFK inhibitor (PP2) prevents IVH-induced death of hippocampal neurons and cognitive deficits assessed on the Morris Water Maze. Moreover, PP2 (i.p.) alone did not affect proliferation of NPCs and did not affect cognition. Since there are several SFK gene family members in brain, we targeted specific SFK subtypes (e.g. Fyn, Lck and c-Src) using a newly developed in vivo nanoparticle-based siRNA transfection system. We show that nanoparticle-siRNA-Fyn plus nanoparticle-siRNA-Src attenuate IVH-induced neuron loss and cognitive deficits. Nanoparticle-siRNA-Fyn nor nanoparticle-siRNA-Src had no significant affects on population of NPCs or cognitive side effects, possibly because the nanoparticle-based siRNA transfection system only produces transient knockdown of the gene targets. This could provide a novel therapy for treating IVH patients as the nanoparticle-based siRNA approach provides heightened specificity for specific SFK gene(s) with less off target effects and this approach has been used in humans. Acknowledgements: This study was supported by AHA Beginning Grant-in-Aid 12BGIA12060381 (DZL) and NIH grant NS054652 (FRS). There were no conflicts of interest.
MicroRNA (miRNA) are recently discovered small (~22 nucleotides), non-coding RNA that regulate tr... more MicroRNA (miRNA) are recently discovered small (~22 nucleotides), non-coding RNA that regulate translation of messenger RNA (mRNA) to protein. Though there are only hundreds of miRNAs, each of them can potentially regulate hundreds of target genes, via base-pairing with complementary sequences in mRNA. This provides one approach that targets a single miRNA to have effects on multiple genes. Our previous genomic studies have demonstrated that miR-122 decreased significantly in blood of experimental strokes produced by middle cerebral artery (MCA) occlusion in rats as well as in blood of patients with ischemic strokes. Therefore, we hypothesized that elevating blood miR-122 has the potential for treating stroke. Using the newly developed in vivo polyethylene glycol-liposome based miRNA transfection system and rat suture MCAO occlusion model, we show that injection of chemically modified mimic miR-122 (600ug/rat, i.v.) through tail vein immediately after MCAO occlusion significantly decreases the neurological impairment and significantly attenuates brain infarct volumes. Ongoing studies are identifying the target genes that are associated with the neuroprotective effects of miR-122 following stroke. Acknowledgements: This study was supported by NIH grant R01NS066845 (FRS). There were no conflicts of interest.
We aimed to determine if plasma levels of bacterial lipopolysaccharide (LPS) and lipoteichoic aci... more We aimed to determine if plasma levels of bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are associated with different causes of stroke and correlate with C-reactive protein (CRP), LPSbinding protein (LBP), and the NIH stroke scale (NIHSS). Ischemic stroke (cardioembolic (CE), large artery atherosclerosis (LAA), small vessel occlusion (SVO)), intracerebral hemorrhage (ICH), transient ischemic attack (TIA) and control subjects were compared (n = 205). Plasma LPS, LTA, CRP, and LBP levels were quantified by ELISA. LPS and CRP levels were elevated in ischemic strokes (CE, LAA, SVO) and ICH compared to controls. LBP levels were elevated in ischemic strokes (CE, LAA) and ICH. LTA levels were increased in SVO stroke compared to TIA but not controls. LPS levels correlated with CRP and LBP levels in stroke and TIA. LPS, LBP and CRP levels positively correlated with the NIHSS and WBC count but negatively correlated with total cholesterol. Plasma LPS and LBP associate with major causes of ischemic stroke and with ICH, whereas LPS/LBP do not associate with TIAs. LTA only associated with SVO stroke. LPS positively correlated with CRP, LBP, and WBC but negatively correlated with cholesterol. Higher LPS levels were associated with worse stroke outcomes. Stroke incidence increases with infection and inflammation prior to stroke 1. C-reactive protein (CRP) levels after stroke correlate with stroke severity 2 ; and, there is a whole genome immune response after stroke that differs for each stroke cause 3. This response includes TNF, IL1, IL6 and other cytokines downstream of TLR4 and TLR2 pathways. Thus, we explored whether LPS (Lipopolysaccharide) or LTA (Lipoteichoic acid) levels are elevated in different causes of stroke and correlated with CRP levels since TLR4 is the receptor for Gram-negative bacterial LPS and TLR2 is the receptor for Gram-positive bacterial LTA, respectively. LPS is increased in acute stroke and associated with poor short term outcome and long term mortality 4,5. However, LPS levels were measured with the limulus lysate enzymatic assay which detects total LPS activity without identifying LPS molecules 6. To solve this problem, we used a LPS specific ELISA for human plasma to quantify LPS, combined with an LPS binding protein (LBP) ELISA. LBP measurements, unlike LPS, are not subject to contamination. Thus, this study assessed plasma levels of LPS, LBP, LTA, and CRP in patients with different causes of ischemic stroke, intracerebral hemorrhage (ICH) and transient ischemic attacks (TIAs) compared to controls. We hypothesize that levels of LPS and LTA, the inflammatory molecules from Gram-negative bacteria and Gram-positive bacteria, respectively, might change in some types of stroke and the LPS and LTA levels might correlate with LBP or CRP levels since LBP and CRP are acute phase proteins whose plasma concentrations change in response to inflammation. Methods Subject recruitment. Subjects with ischemic stroke (cardioembolic (CE, n = 33), large artery atherosclerosis (LAA, n = 42), small-vessel/lacunar (SVO, n = 41), intracerebral hemorrhage (ICH, n = 36), transient ischemic attacks (TIAs, n = 31), and controls (n = 22) were recruited at the University of California, Davis. The study was approved by the UC Davis Institutional Review Board and adhered to all federal and state regulations related to the protection of human research subjects, including the Common Rule, the principles of the Belmont Report, and institutional policies and procedures. Written informed consent for participation was obtained from all participants or their proxy. Ischemic stroke, ICH, and TIA subjects were recruited within 72 h of symptom onset. Exclusion criteria for all subjects were cancer, recent infection (< 4 weeks) or chronic infection including HIV. Ischemic stroke, ICH, and TIA diagnoses were determined by two board-certified vascular neurologists. NIHSS, WBC count, lipid panel ((triglyceride (TG), total cholesterol (TC), high-density
Journal of Cerebral Blood Flow and Metabolism, Oct 1, 2016
Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our re... more Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.
Journal of Cerebral Blood Flow and Metabolism, Apr 9, 2019
Previous studies showed changes in mRNA levels in whole blood of rats and humans, and in miRNA in... more Previous studies showed changes in mRNA levels in whole blood of rats and humans, and in miRNA in whole blood of rats following intracerebral hemorrhage (ICH). Thus, this study assessed miRNA and their putative mRNA targets in whole blood of humans following ICH. Whole transcriptome profiling identified altered miRNA and mRNA levels in ICH patients compared to matched controls. Target mRNAs of the differentially expressed miRNAs were identified, and functional analysis of the miRNA-mRNA targets was performed. Twenty-nine miRNAs (22 down, 7 up) and 250 target mRNAs (136 up, 114 down), and 7 small nucleolar RNA changed expression after ICH compared to controls (FDR < 0.05, and fold change ! |1.2|). These included Let7i, miR-146a-5p, miR210-5p, miR-93-5p, miR-221, miR-874, miR-17-3p, miR-378a-5p, miR-532-5p, mir-4707, miR-4450, mir-1183, Let-7d-3p, miR-3937, miR-4288, miR-4741, miR-92a-1-3p, miR-4514, mir-4658, mir-3689d-1, miR-4760-3p, and mir-3183. Pathway analysis showed regulated miRNAs/ mRNAs were associated with toll-like receptor, natural killer cell, focal adhesion, TGF-b, phagosome, JAK-STAT, cytokine-cytokine receptor, chemokine, apoptosis, vascular smooth muscle, and RNA degradation signaling. Many of these pathways have been implicated in ICH. The differentially expressed miRNA and their putative mRNA targets and associated pathways may provide diagnostic biomarkers as well as point to therapeutic targets for ICH treatments in humans.
Animal models of acute ischemic stroke have been criticized for failing to translate to human str... more Animal models of acute ischemic stroke have been criticized for failing to translate to human stroke. Nevertheless, animal models are necessary to improve our understanding of stroke pathophysiology and to guide the development of new stroke therapies. The rabbit embolic clot model is one animal model that has led to an effective therapy in human acute ischemic stroke, namely tissue plasminogen activator (tPA). We propose that potential compounds that demonstrate efficacy in non-rabbit animal models of acute ischemic stroke should also be tested in the rabbit embolic blood clot model and, where appropriate, compared to tPA prior to investigation in humans. Furthermore, the use of anesthesia needs to be considered as a major confounder in animal models of acute ischemic stroke, and death should be included as an outcome measure in animal stroke studies. These steps, along with the current STAIRs recommendations, may improve the successful translation of experimental therapies to clinical stroke treatments.
Annals of clinical and translational neurology, Aug 22, 2019
Objective: Though cigarette smoking (CS) is a well-known risk factor for ischemic stroke (IS), th... more Objective: Though cigarette smoking (CS) is a well-known risk factor for ischemic stroke (IS), there is no data on how CS affects the blood transcriptome in IS patients. Methods: We recruited IS-current smokers (IS-SM), ISnever smokers (IS-NSM), control-smokers (C-SM), and control-never smokers (C-NSM). mRNA expression was assessed on HTA-2.0 microarrays and unique as well as commonly expressed genes identified for IS-SM versus IS-NSM and C-SM versus C-NSM. Results: One hundred and fifty-eight genes were differentially expressed in IS-SM versus IS-NSM; 100 genes were differentially expressed in C-SM versus C-NSM; and 10 genes were common to both IS-SM and C-SM (P < 0.01; |fold change| ≥ 1.2). Functional pathway analysis showed the 158 IS-SM-regulated genes were associated with T-cell receptor, cytokine-cytokine receptor, chemokine, adipocytokine, tight junction, Jak-STAT, ubiquitin-mediated proteolysis, and adherens junction signaling. IS-SM showed more altered genes and functional networks than C-SM. Interpretation: We propose some of the 10 genes that are elevated in both IS-SM and C-SM (GRP15, LRRN3, CLDND1, ICOS, GCNT4, VPS13A, DAP3, SNORA54, HIST1H1D, and SCARNA6) might contribute to increased risk of stroke in current smokers, and some genes expressed by blood leukocytes and platelets after stroke in smokers might contribute to worse stroke outcomes that occur in smokers.
BACKGROUND AND PURPOSE: With advancing age, alterations occur to the immune system, including an ... more BACKGROUND AND PURPOSE: With advancing age, alterations occur to the immune system, including an increase in inflammation (inflammaging) and a reduced ability to respond to new immune challenges. The role of an aging immune system in patients with ischemic stroke remains unclear, although age is an important determinant of stroke risk and outcome. This study assessed the aging immune system in patients with acute ischemic stroke by differences in leukocyte gene expression in relationship to age. METHODS: Peripheral blood RNA from 2 cohorts with acute ischemic stroke was measured by whole-genome microarray, and genes associated with advancing age were identified (false discovery rate-corrected P<0.05, partial correlation coefficient <|0.3|). Genes were characterized by pathway analysis and compared with age-associated genes from nonstroke studies (n=3974). RESULTS: There were 166 genes associated with age in cohort 1 (derivation cohort, n=94). Sixty-nine of these age-associated genes were verified in cohort 2 (validation cohort, n=79). Identified genes included a decrease in CR2, CD27, CCR7, and NT5E. Genes were associated with altered B-cell receptor signaling, lymphocyte proliferation, and leukocyte homeostasis. Forty-three of the 69 age-associated genes in stroke were also associated with age in nonstroke studies. CONCLUSIONS: A relationship between leukocyte gene expression and age in patients with ischemic stroke was identified. The changes include alterations to the adaptive humoral immune system, which may influence age-related stroke risk and outcome.
Background This study identified early immune gene responses in peripheral blood associated with ... more Background This study identified early immune gene responses in peripheral blood associated with 90-day ischemic stroke (IS) outcomes. Methods Peripheral blood samples from the CLEAR trial IS patients at ≤ 3 h, 5 h, and 24 h after stroke were compared to vascular risk factor matched controls. Whole-transcriptome analyses identified genes and networks associated with 90-day IS outcome assessed using the modified Rankin Scale (mRS) and the NIH Stroke Scale (NIHSS). Results The expression of 467, 526, and 571 genes measured at ≤ 3, 5 and 24 h after IS, respectively, were associated with poor 90-day mRS outcome (mRS ≥ 3), while 49, 100 and 35 genes at ≤ 3, 5 and 24 h after IS were associated with good mRS 90-day outcome (mRS ≤ 2). Poor outcomes were associated with up-regulated genes or pathways such as IL-6, IL-7, IL-1, STAT3, S100A12, acute phase response, P38/MAPK, FGF, TGFA, MMP9, NF-kB, Toll-like receptor, iNOS, and PI3K/AKT. There were 94 probe sets shared for poor outcomes vs. co...
Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repa... more Poly (ADP-ribose) polymerase (PARP) is involved in various cellular functions, including DNA repair, the cell cycle and cell death. While PARP activation could play a critical role in repairing ischemic brain damage, PARP inactivation caused by caspase 3-cleavage may also be important for apoptotic execution. In this study we investigated the effects of transient global ischemia and kainic acid (KA) neurotoxicity, in gerbil and rat brains, respectively, on PARP gene expression and protein cleavage. PARP mRNA increased in the dentate gyrus of gerbil brains 4 h after 10 min of global ischemia, which returned to basal levels 8 h after ischemia. KA injection (10 mg / kg) also induced a marked elevation in PARP mRNA level selectively in the dentate gyrus of rat brains 1 h following the injection, which returned to basal levels 4 h after the injection. These observations provide the first evidence of altered PARP gene expression in brains subjected to ischemic and excitotoxic insults. Using both monoclonal and polyclonal antibodies to PARP cleavage products, little evidence of significant PARP cleavage was found in gerbil brains within the first 3 days after 10 min of global ischemia. In addition, there was little evidence of significant PARP cleavage in rat brains within 2 days after kainate (KA) injection. Though these findings show that caspase induced PARP cleavage is not substantially activated by global ischemia and excitotoxicity in whole brain, the PARP mRNA induction could suggest a role for PARP in repairing DNA following brain injury.
The non-competitive NMDA receptor antagonists, including PCP (phencyclidine), ketamine, and MK-80... more The non-competitive NMDA receptor antagonists, including PCP (phencyclidine), ketamine, and MK-801 (dizocilpine) produce psychosis in humans and injure neurons in retrosplenial cortex in adult rodent brain. This study examined the effects of the metabotropic mGlu2/3 agonist LY379268 and antagonist LY341495 on cortical injury produced by systemic MK-801 (1 mg/kg i.p.) in adult female rats. Systemic injections of mGlu2/3 agonist LY379268, but not mGlu2/3 antagonist LY341495, decreased the injury in the retrosplenial cortex produced by systemic MK-801 as assessed by Hsp70 induction. Bilateral injections of LY379268, but not vehicle, into retrosplenial cortex or bilateral injections of LY379268 into anterior thalamus also decreased the injury in retrosplenial cortex produced by systemic MK-801. The data show that bilateral activation of mGlu2/3 glutamate receptors in cortex or anterior thalamus decreases the neuronal injury in retrosplenial cortex produced by systemic MK-801. Because antipsychotic medications decrease cortical injury produced by NMDA antagonists in rodents and decrease psychosis in humans, mGlu2/3 agonists that decrease cortical injury produced by NMDA antagonists in rodents might be evaluated for decreasing psychosis in people.
Neural progenitor cells (NPCs) Neuroblastoma cells (NBCs) mRNA microRNA (miRNA) Mature neurons (M... more Neural progenitor cells (NPCs) Neuroblastoma cells (NBCs) mRNA microRNA (miRNA) Mature neurons (MNs), neural progenitor cells (NPCs) and neuroblastoma cells (NBCs) are all neural-derived cells. However, MNs are unable to divide once differentiated; NPCs are able to divide a limited number of times and differentiate to normal brain cell types; whereas NBCs can divide an unlimited number of times but rarely differentiate. Here, we perform whole transcriptome (mRNA, miRNA) profiling of these cell types and compare expression levels of each cell type to the others. Integrated mRNA-miRNA functional analyses reveal that: 1) several very highly expressed genes (e.g., Robo1, Nrp1, Epha3, Unc5c, Dcc, Pak3, Limk4) and a few under-expressed miRNAs (e.g., miR-152, miR-146b, miR-339-5p) in MNs are associated with one important cellular process-axon guidance; 2) some very highly expressed mitogenic pathway genes (e.g., Map2k1, Igf1r, Rara, Runx1) and under-expressed miRNAs (e.g., miR-370, miR-9, miR-672) in NBCs are associated with cancer pathways. These results provide a library of negative mRNAmiRNA networks that are likely involved in the cellular processes of differentiation and division.
Using two microarray platforms, we identify HLA-DRB5 as the most highly expressed gene in MS comp... more Using two microarray platforms, we identify HLA-DRB5 as the most highly expressed gene in MS compared to healthy subjects. As expected, HLA-DRB5 expression was associated with the HLA-DRB1*1501 MS susceptibility allele. Besides HLA-DRB5, there were 1219 differentially expressed exons (pb 0.01, |fold change (FC)|>1.2) that differed between HLA-DRB1*1501 Positive multiple sclerosis subjects (MSP) compared to HLA-DRB1*1501 negative multiple sclerosis subjects (MSN). Analysis of the regulated genes revealed significantly different immune signaling pathways including IL-4 and IL-17 in these two MS genotypes. Different risk alleles appear to be associated with different patterns of gene expression that may reflect differences in pathophysiology of these two MS subtypes. These preliminary data will need to be confirmed in future studies.
The Histone Deacetylase 9 (HDAC9) polymorphism rs2107595 is associated with an increased risk for... more The Histone Deacetylase 9 (HDAC9) polymorphism rs2107595 is associated with an increased risk for large vessel atherosclerotic stroke (LVAS). In humans, there remains a need to better understand this HDAC9 polymorphism's contribution to large vessel stroke. In this pilot study, we evaluated whether the HDAC9 polymorphism rs2107595 is associated with differences in leukocyte gene expression in patients with LVAS. HDAC9 SNP rs2107595 was genotyped in 155 patients (43 LVAS and 112 vascular risk factor controls). RNA isolated from blood was processed on whole genome microarrays. Gene expression was compared between HDAC9 risk allele positive and risk allele negative LVAS patients and controls. Functional analysis identified canonical pathways and molecular functions associated with rs2107595 in LVAS. In HDAC9 SNP rs2107595 risk allele positive LVAS patients there were 155 genes differentially expressed compared to risk allele negative patients (fold change >|1.2|, p<0.05). The 155 genes separated the risk allele positive and negative LVAS patients on a Principal Components Analysis. Pathways associated with HDAC9 risk allele positive status involved IL6 signaling, cholesterol efflux, and platelet aggregation. These preliminary data suggest an association with the HDAC9 rs2107595 risk allele and peripheral immune, lipid, and clotting systems in LVAS. Further study is required to evaluate whether these differences are related to large vessel atherosclerosis and stroke risk.
Journal of Cerebral Blood Flow and Metabolism, Apr 13, 2018
Understanding how the blood transcriptome of human intracerebral hemorrhage (ICH) differs from is... more Understanding how the blood transcriptome of human intracerebral hemorrhage (ICH) differs from ischemic stroke (IS) and matched controls (CTRL) will improve understanding of immune and coagulation pathways in both disorders. This study examined RNA from 99 human whole-blood samples using GeneChip Õ HTA 2.0 arrays to assess differentially expressed transcripts of alternatively spliced genes between ICH, IS and CTRL. We used a mixed regression model with FDR-corrected p(Dx) < 0.2 and p < 0.005 and jFCj > 1.2 for individual comparisons. For time-dependent analyses, subjects were divided into four time-points: 0(CTRL), <24 h, 24-48 h, >48 h; 489 transcripts were differentially expressed between ICH and CTRL, and 63 between IS and CTRL. ICH had differentially expressed T-cell receptor and CD36 genes, and iNOS, TLR, macrophage, and T-helper pathways. IS had more non-coding RNA. ICH and IS both had angiogenesis, CTLA4 in T lymphocytes, CD28 in T helper cells, NFAT regulation of immune response, and glucocorticoid receptor signaling pathways. Self-organizing maps revealed 4357 transcripts changing expression over time in ICH, and 1136 in IS. Understanding ICH and IS transcriptomes will be useful for biomarker development, treatment and prevention strategies, and for evaluating how well animal models recapitulate human ICH and IS.
Objective: To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes. Method... more Objective: To evaluate microRNA let7i in ischemic stroke and its regulation of leukocytes. Methods: A total of 212 patients were studied: 106 with acute ischemic stroke and 106 controls matched for risk factors. RNA from circulating leukocytes was isolated from blood collected in PAXgene tubes. Let7i microRNA expression was assessed using TaqMan quantitative reverse transcription PCR. To assess let7i regulation of gene expression in stroke, messenger RNA (mRNA) from leukocytes was measured by whole-genome Human Transcriptome Array Affymetrix microarray. Given microRNAs act to destabilize and degrade their target mRNA, mRNAs that inversely correlated with let7i were identified. To demonstrate let7i posttranscriptional regulation of target genes, a 39 untranslated region luciferase assay was performed. Target protein expression was assessed using ELISA. Results: Let7i was decreased in patients with acute ischemic stroke (fold change 21.70, p , 0.00001). A modest inverse correlation between let7i and NIH Stroke Scale score at admission (r 5 20.32, p 5 0.02), infarct volume (r 5 20.21, p 5 0.04), and plasma MMP9 (r 5 20.46, p 5 0.01) was identified. The decrease in let7i was associated with increased expression of several of its mRNA targets, including CD86, CXCL8, and HMGB1. In vitro studies confirm let7i posttranscriptional regulation of target genes CD86, CXCL8, and HMGB1. Functional analysis predicted let7i regulates pathways involved in leukocyte activation, recruitment, and proliferation including canonical pathways of CD86 signaling in T helper cells, HMGB1 signaling, and CXCL8 signaling. Conclusions: Let7i is decreased in circulating leukocytes of patients with acute ischemic stroke. Mechanisms by which let7i regulates inflammatory response post stroke include targeting CD86, CXCL8, and HMGB1. Neurology ® 2016;87:2198-2205 GLOSSARY cDNA 5 complementary DNA; HTA 5 Human Transcriptome Array; IL 5 interleukin; mRNA 5 messenger RNA; NIHSS 5 NIH Stroke Scale; 39UTR 5 39 untranslated region; TNF-a 5 tumor necrosis factor a.
Background: Since RNA expression differences have been reported in autism spectrum disorder (ASD)... more Background: Since RNA expression differences have been reported in autism spectrum disorder (ASD) for blood and brain, and differential alternative splicing (DAS) has been reported in ASD brains, we determined if there was DAS in blood mRNA of ASD subjects compared to typically developing (TD) controls, as well as in ASD subgroups related to cerebral volume. Methods: RNA from blood was processed on whole genome exon arrays for 2-4-year-old ASD and TD boys. An ANCOVA with age and batch as covariates was used to predict DAS for ALL ASD (n=30), ASD with normal total cerebral volumes (NTCV), and ASD with large total cerebral volumes (LTCV) compared to TD controls (n=20). Results: A total of 53 genes were predicted to have DAS for ALL ASD versus TD, 169 genes for ASD_NTCV versus TD, 1 gene for ASD_LTCV versus TD, and 27 genes for ASD_LTCV versus ASD_NTCV. These differences were significant at P <0.05 after false discovery rate corrections for multiple comparisons (FDR <5% false positives). A number of the genes predicted to have DAS in ASD are known to regulate DAS (SFPQ, SRPK1, SRSF11, SRSF2IP, FUS, LSM14A). In addition, a number of genes with predicted DAS are involved in pathways implicated in previous ASD studies, such as ROS monocyte/macrophage, Natural Killer Cell, mTOR, and NGF signaling. The only pathways significant after multiple comparison corrections (FDR <0.05) were the Nrf2-mediated reactive oxygen species (ROS) oxidative response (superoxide dismutase 2, catalase, peroxiredoxin 1, PIK3C3, DNAJC17, microsomal glutathione S-transferase 3) and superoxide radical degradation (SOD2, CAT). Conclusions: These data support differences in alternative splicing of mRNA in blood of ASD subjects compared to TD controls that differ related to head size. The findings are preliminary, need to be replicated in independent cohorts, and predicted alternative splicing differences need to be confirmed using direct analytical methods.
Background and Purpose-The cause of ischemic stroke remains unclear, or cryptogenic, in as many a... more 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 CHA 2 DS 2-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.
Introduction: Inflammation and infection are associated with cerebrovascular diseases including s... more Introduction: Inflammation and infection are associated with cerebrovascular diseases including stroke due to carotid atherosclerotic plaques. C-reactive protein (CRP), an acute-phase protein, is upregulated in the plasma of patients with carotid atherosclerotic plaques. However, little is known about whether bacterial molecules trigger inflammation or play a role in patients with carotid atherosclerotic plaques. Recently, it has been recognized that inflammation associated with atherosclerosis and morbidity and mortality in cardiovascular diseases may be due to lipopolysaccharide (LPS) that is found in the outer wall of all Gram-negative bacteria. These findings prompted this study to explore whether plasma levels of LPS and LPS-binding protein (LBP) are elevated and correlated with CRP levels in patients with asymptomatic carotid plaques (ACP). We also compared LBP levels in patients with ACP to large vessel (LV) strokes due to carotid plaques and to matched controls. Methods: Patients (n = 30) with ACP, LV stroke due to carotid atherosclerosis and age-, sex- matched healthy controls gave consent and had their blood drawn. Plasma was processed for LPS, LBP and CRP detection using separate ELISA for each. Results: Plasma LBP level in ACP (22.7 ± 2.92 μg/ml) was similar to LV stroke (21.6 ± 1.56 μg/ml, p = 0.74, ACP vs LV) but greater than controls (13.6 ± 1.43 μg/ml, p = 0.011, ACP vs controls). In ACP patients, plasma LPS level (159.5 ± 30.5 μg/ml) was greater than controls (42.6 ± 11.7 μg/ml, p = 0.001); plasma CRP levels (20.2 ± 6.2 μg/ml) was higher than controls (5.3 ± 2.1 μg/ml, p = 0.011). There was a positive correlation between LPS levels and LBP levels (r = 0.86, p &lt; 0.00001), LPS levels and CRP levels (r = 0.82, p = 0.00001), and LBP levels and CRP levels (r = 0.89, p &lt; 0.00001) in ACP cases. Conclusions: Plasma LPS, LBP and CRP associate with asymptomatic carotid plaques suggesting a pro-inflammatory state exists in patients with asymptomatic carotid plaques, a cause of large vessel stroke. LPS is postulated to directly upregulate both CRP and LBP. Elevated LBP in large vessel stroke patients suggests a Gram-negative bacteria associated post-stroke inflammatory state.
Intraventricular hemorrhage (IVH) is one of the most serious hemorrhagic brain diseases being ass... more Intraventricular hemorrhage (IVH) is one of the most serious hemorrhagic brain diseases being associated with blood brain barrier (BBB) disruption, brain edema, neuron loss, cognitive impairment and high mortality in humans. Increasing evidence suggests that neuronal cell death ensues when neurons attempt to re-enter the cell cycle. Our previous study has demonstrated that cell cycle inhibition via blocking Src family kinases (SFKs) prevents neuronal cell death. However, one of the concerns with cell cycle therapy might be that it would inhibit proliferation of neural progenitor cells (NPCs) and that inhibiting neurogenesis would produce cognitive effects related to the therapy itself. Using the rodent in vivo IVH model we show that i.p. injection of SFK inhibitor (PP2) prevents IVH-induced death of hippocampal neurons and cognitive deficits assessed on the Morris Water Maze. Moreover, PP2 (i.p.) alone did not affect proliferation of NPCs and did not affect cognition. Since there are several SFK gene family members in brain, we targeted specific SFK subtypes (e.g. Fyn, Lck and c-Src) using a newly developed in vivo nanoparticle-based siRNA transfection system. We show that nanoparticle-siRNA-Fyn plus nanoparticle-siRNA-Src attenuate IVH-induced neuron loss and cognitive deficits. Nanoparticle-siRNA-Fyn nor nanoparticle-siRNA-Src had no significant affects on population of NPCs or cognitive side effects, possibly because the nanoparticle-based siRNA transfection system only produces transient knockdown of the gene targets. This could provide a novel therapy for treating IVH patients as the nanoparticle-based siRNA approach provides heightened specificity for specific SFK gene(s) with less off target effects and this approach has been used in humans. Acknowledgements: This study was supported by AHA Beginning Grant-in-Aid 12BGIA12060381 (DZL) and NIH grant NS054652 (FRS). There were no conflicts of interest.
MicroRNA (miRNA) are recently discovered small (~22 nucleotides), non-coding RNA that regulate tr... more MicroRNA (miRNA) are recently discovered small (~22 nucleotides), non-coding RNA that regulate translation of messenger RNA (mRNA) to protein. Though there are only hundreds of miRNAs, each of them can potentially regulate hundreds of target genes, via base-pairing with complementary sequences in mRNA. This provides one approach that targets a single miRNA to have effects on multiple genes. Our previous genomic studies have demonstrated that miR-122 decreased significantly in blood of experimental strokes produced by middle cerebral artery (MCA) occlusion in rats as well as in blood of patients with ischemic strokes. Therefore, we hypothesized that elevating blood miR-122 has the potential for treating stroke. Using the newly developed in vivo polyethylene glycol-liposome based miRNA transfection system and rat suture MCAO occlusion model, we show that injection of chemically modified mimic miR-122 (600ug/rat, i.v.) through tail vein immediately after MCAO occlusion significantly decreases the neurological impairment and significantly attenuates brain infarct volumes. Ongoing studies are identifying the target genes that are associated with the neuroprotective effects of miR-122 following stroke. Acknowledgements: This study was supported by NIH grant R01NS066845 (FRS). There were no conflicts of interest.
We aimed to determine if plasma levels of bacterial lipopolysaccharide (LPS) and lipoteichoic aci... more We aimed to determine if plasma levels of bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are associated with different causes of stroke and correlate with C-reactive protein (CRP), LPSbinding protein (LBP), and the NIH stroke scale (NIHSS). Ischemic stroke (cardioembolic (CE), large artery atherosclerosis (LAA), small vessel occlusion (SVO)), intracerebral hemorrhage (ICH), transient ischemic attack (TIA) and control subjects were compared (n = 205). Plasma LPS, LTA, CRP, and LBP levels were quantified by ELISA. LPS and CRP levels were elevated in ischemic strokes (CE, LAA, SVO) and ICH compared to controls. LBP levels were elevated in ischemic strokes (CE, LAA) and ICH. LTA levels were increased in SVO stroke compared to TIA but not controls. LPS levels correlated with CRP and LBP levels in stroke and TIA. LPS, LBP and CRP levels positively correlated with the NIHSS and WBC count but negatively correlated with total cholesterol. Plasma LPS and LBP associate with major causes of ischemic stroke and with ICH, whereas LPS/LBP do not associate with TIAs. LTA only associated with SVO stroke. LPS positively correlated with CRP, LBP, and WBC but negatively correlated with cholesterol. Higher LPS levels were associated with worse stroke outcomes. Stroke incidence increases with infection and inflammation prior to stroke 1. C-reactive protein (CRP) levels after stroke correlate with stroke severity 2 ; and, there is a whole genome immune response after stroke that differs for each stroke cause 3. This response includes TNF, IL1, IL6 and other cytokines downstream of TLR4 and TLR2 pathways. Thus, we explored whether LPS (Lipopolysaccharide) or LTA (Lipoteichoic acid) levels are elevated in different causes of stroke and correlated with CRP levels since TLR4 is the receptor for Gram-negative bacterial LPS and TLR2 is the receptor for Gram-positive bacterial LTA, respectively. LPS is increased in acute stroke and associated with poor short term outcome and long term mortality 4,5. However, LPS levels were measured with the limulus lysate enzymatic assay which detects total LPS activity without identifying LPS molecules 6. To solve this problem, we used a LPS specific ELISA for human plasma to quantify LPS, combined with an LPS binding protein (LBP) ELISA. LBP measurements, unlike LPS, are not subject to contamination. Thus, this study assessed plasma levels of LPS, LBP, LTA, and CRP in patients with different causes of ischemic stroke, intracerebral hemorrhage (ICH) and transient ischemic attacks (TIAs) compared to controls. We hypothesize that levels of LPS and LTA, the inflammatory molecules from Gram-negative bacteria and Gram-positive bacteria, respectively, might change in some types of stroke and the LPS and LTA levels might correlate with LBP or CRP levels since LBP and CRP are acute phase proteins whose plasma concentrations change in response to inflammation. Methods Subject recruitment. Subjects with ischemic stroke (cardioembolic (CE, n = 33), large artery atherosclerosis (LAA, n = 42), small-vessel/lacunar (SVO, n = 41), intracerebral hemorrhage (ICH, n = 36), transient ischemic attacks (TIAs, n = 31), and controls (n = 22) were recruited at the University of California, Davis. The study was approved by the UC Davis Institutional Review Board and adhered to all federal and state regulations related to the protection of human research subjects, including the Common Rule, the principles of the Belmont Report, and institutional policies and procedures. Written informed consent for participation was obtained from all participants or their proxy. Ischemic stroke, ICH, and TIA subjects were recruited within 72 h of symptom onset. Exclusion criteria for all subjects were cancer, recent infection (< 4 weeks) or chronic infection including HIV. Ischemic stroke, ICH, and TIA diagnoses were determined by two board-certified vascular neurologists. NIHSS, WBC count, lipid panel ((triglyceride (TG), total cholesterol (TC), high-density
Journal of Cerebral Blood Flow and Metabolism, Oct 1, 2016
Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our re... more Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.
Journal of Cerebral Blood Flow and Metabolism, Apr 9, 2019
Previous studies showed changes in mRNA levels in whole blood of rats and humans, and in miRNA in... more Previous studies showed changes in mRNA levels in whole blood of rats and humans, and in miRNA in whole blood of rats following intracerebral hemorrhage (ICH). Thus, this study assessed miRNA and their putative mRNA targets in whole blood of humans following ICH. Whole transcriptome profiling identified altered miRNA and mRNA levels in ICH patients compared to matched controls. Target mRNAs of the differentially expressed miRNAs were identified, and functional analysis of the miRNA-mRNA targets was performed. Twenty-nine miRNAs (22 down, 7 up) and 250 target mRNAs (136 up, 114 down), and 7 small nucleolar RNA changed expression after ICH compared to controls (FDR < 0.05, and fold change ! |1.2|). These included Let7i, miR-146a-5p, miR210-5p, miR-93-5p, miR-221, miR-874, miR-17-3p, miR-378a-5p, miR-532-5p, mir-4707, miR-4450, mir-1183, Let-7d-3p, miR-3937, miR-4288, miR-4741, miR-92a-1-3p, miR-4514, mir-4658, mir-3689d-1, miR-4760-3p, and mir-3183. Pathway analysis showed regulated miRNAs/ mRNAs were associated with toll-like receptor, natural killer cell, focal adhesion, TGF-b, phagosome, JAK-STAT, cytokine-cytokine receptor, chemokine, apoptosis, vascular smooth muscle, and RNA degradation signaling. Many of these pathways have been implicated in ICH. The differentially expressed miRNA and their putative mRNA targets and associated pathways may provide diagnostic biomarkers as well as point to therapeutic targets for ICH treatments in humans.
Animal models of acute ischemic stroke have been criticized for failing to translate to human str... more Animal models of acute ischemic stroke have been criticized for failing to translate to human stroke. Nevertheless, animal models are necessary to improve our understanding of stroke pathophysiology and to guide the development of new stroke therapies. The rabbit embolic clot model is one animal model that has led to an effective therapy in human acute ischemic stroke, namely tissue plasminogen activator (tPA). We propose that potential compounds that demonstrate efficacy in non-rabbit animal models of acute ischemic stroke should also be tested in the rabbit embolic blood clot model and, where appropriate, compared to tPA prior to investigation in humans. Furthermore, the use of anesthesia needs to be considered as a major confounder in animal models of acute ischemic stroke, and death should be included as an outcome measure in animal stroke studies. These steps, along with the current STAIRs recommendations, may improve the successful translation of experimental therapies to clinical stroke treatments.
Annals of clinical and translational neurology, Aug 22, 2019
Objective: Though cigarette smoking (CS) is a well-known risk factor for ischemic stroke (IS), th... more Objective: Though cigarette smoking (CS) is a well-known risk factor for ischemic stroke (IS), there is no data on how CS affects the blood transcriptome in IS patients. Methods: We recruited IS-current smokers (IS-SM), ISnever smokers (IS-NSM), control-smokers (C-SM), and control-never smokers (C-NSM). mRNA expression was assessed on HTA-2.0 microarrays and unique as well as commonly expressed genes identified for IS-SM versus IS-NSM and C-SM versus C-NSM. Results: One hundred and fifty-eight genes were differentially expressed in IS-SM versus IS-NSM; 100 genes were differentially expressed in C-SM versus C-NSM; and 10 genes were common to both IS-SM and C-SM (P < 0.01; |fold change| ≥ 1.2). Functional pathway analysis showed the 158 IS-SM-regulated genes were associated with T-cell receptor, cytokine-cytokine receptor, chemokine, adipocytokine, tight junction, Jak-STAT, ubiquitin-mediated proteolysis, and adherens junction signaling. IS-SM showed more altered genes and functional networks than C-SM. Interpretation: We propose some of the 10 genes that are elevated in both IS-SM and C-SM (GRP15, LRRN3, CLDND1, ICOS, GCNT4, VPS13A, DAP3, SNORA54, HIST1H1D, and SCARNA6) might contribute to increased risk of stroke in current smokers, and some genes expressed by blood leukocytes and platelets after stroke in smokers might contribute to worse stroke outcomes that occur in smokers.
BACKGROUND AND PURPOSE: With advancing age, alterations occur to the immune system, including an ... more BACKGROUND AND PURPOSE: With advancing age, alterations occur to the immune system, including an increase in inflammation (inflammaging) and a reduced ability to respond to new immune challenges. The role of an aging immune system in patients with ischemic stroke remains unclear, although age is an important determinant of stroke risk and outcome. This study assessed the aging immune system in patients with acute ischemic stroke by differences in leukocyte gene expression in relationship to age. METHODS: Peripheral blood RNA from 2 cohorts with acute ischemic stroke was measured by whole-genome microarray, and genes associated with advancing age were identified (false discovery rate-corrected P<0.05, partial correlation coefficient <|0.3|). Genes were characterized by pathway analysis and compared with age-associated genes from nonstroke studies (n=3974). RESULTS: There were 166 genes associated with age in cohort 1 (derivation cohort, n=94). Sixty-nine of these age-associated genes were verified in cohort 2 (validation cohort, n=79). Identified genes included a decrease in CR2, CD27, CCR7, and NT5E. Genes were associated with altered B-cell receptor signaling, lymphocyte proliferation, and leukocyte homeostasis. Forty-three of the 69 age-associated genes in stroke were also associated with age in nonstroke studies. CONCLUSIONS: A relationship between leukocyte gene expression and age in patients with ischemic stroke was identified. The changes include alterations to the adaptive humoral immune system, which may influence age-related stroke risk and outcome.
Background This study identified early immune gene responses in peripheral blood associated with ... more Background This study identified early immune gene responses in peripheral blood associated with 90-day ischemic stroke (IS) outcomes. Methods Peripheral blood samples from the CLEAR trial IS patients at ≤ 3 h, 5 h, and 24 h after stroke were compared to vascular risk factor matched controls. Whole-transcriptome analyses identified genes and networks associated with 90-day IS outcome assessed using the modified Rankin Scale (mRS) and the NIH Stroke Scale (NIHSS). Results The expression of 467, 526, and 571 genes measured at ≤ 3, 5 and 24 h after IS, respectively, were associated with poor 90-day mRS outcome (mRS ≥ 3), while 49, 100 and 35 genes at ≤ 3, 5 and 24 h after IS were associated with good mRS 90-day outcome (mRS ≤ 2). Poor outcomes were associated with up-regulated genes or pathways such as IL-6, IL-7, IL-1, STAT3, S100A12, acute phase response, P38/MAPK, FGF, TGFA, MMP9, NF-kB, Toll-like receptor, iNOS, and PI3K/AKT. There were 94 probe sets shared for poor outcomes vs. co...
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Papers by Frank Sharp