Ischemic Stroke with Comorbid Cancer Has Specific miRNA-mRNA Networks in Blood That Vary by Ischemic Stroke Mechanism.

OBJECTIVE: Approximately half of ischemic strokes (IS) in cancer patients are cryptogenic, with many presumed cardioembolic. We evaluated whether there were specific miRNA and mRNA transcriptome architectures in peripheral blood of IS patients with and without comorbid cancer, and between cardioembolic versus noncardioembolic IS etiologies in comorbid cancer. METHODS: We studied patients with cancer and IS (CS; n = 42), stroke only (SO; n = 41), and cancer only (n = 28), and vascular risk factor-matched controls (n = 30). mRNA-Seq and miRNA-Seq data, analyzed with linear regression models, identified differentially expressed genes in CS versus SO and in cardioembolic versus noncardioembolic CS, and miRNA-mRNA regulatory pairs. Network-level analyses identified stroke etiology-specific responses in CS. RESULTS: A total of 2,085 mRNAs and 31 miRNAs were differentially expressed between CS and SO. In CS, 122 and 35 miRNA-mRNA regulatory pairs, and 5 and 3 coexpressed gene modules, were associated with cardioembolic and noncardioembolic CS, respectively. Complement, growth factor, and immune/inflammatory pathways showed differences between IS etiologies in CS. A 15-gene biomarker panel assembled from a derivation cohort (n = 50) correctly classified 81% of CS and 71% of SO participants in a validation cohort (n = 33). Another 15-gene panel correctly identified etiologies for 13 of 13 CS-cardioembolic and 11 of 11 CS-noncardioembolic participants upon cross-validation; 11 of 16 CS-cryptogenic participants were predicted cardioembolic. INTERPRETATION: We discovered unique mRNA and miRNA transcriptome architecture in CS and SO, and in CS with different IS etiologies. Cardioembolic and noncardioembolic etiologies in CS showed unique coexpression networks and potential master regulators. These may help distinguish CS from SO and identify IS etiology in cryptogenic CS patients. ANN NEUROL 2024.

Neuron-specific transcriptomic signatures indicate neuroinflammation and altered neuronal activity in ASD temporal cortex.

Autism spectrum disorder (ASD) is a highly heterogeneous disorder, yet transcriptomic profiling of bulk brain tissue has identified substantial convergence among dysregulated genes and pathways in ASD. However, this approach lacks cell-specific resolution. We performed comprehensive transcriptomic analyses on bulk tissue and laser-capture microdissected (LCM) neurons from 59 postmortem human brains (27 ASD and 32 controls) in the superior temporal gyrus (STG) of individuals ranging from 2 to 73 years of age. In bulk tissue, synaptic signaling, heat shock protein-related pathways, and RNA splicing were significantly altered in ASD. There was age-dependent dysregulation of genes involved in gamma aminobutyric acid (GABA) (GAD1 and GAD2) and glutamate (SLC38A1) signaling pathways. In LCM neurons, AP-1-mediated neuroinflammation and insulin/IGF-1 signaling pathways were upregulated in ASD, while mitochondrial function, ribosome, and spliceosome components were downregulated. GABA synthesizing enzymes GAD1 and GAD2 were both downregulated in ASD neurons. Mechanistic modeling suggested a direct link between inflammation and ASD in neurons, and prioritized inflammation-associated genes for future study. Alterations in small nucleolar RNAs (snoRNAs) associated with splicing events suggested interplay between snoRNA dysregulation and splicing disruption in neurons of individuals with ASD. Our findings supported the fundamental hypothesis of altered neuronal communication in ASD, demonstrated that inflammation was elevated at least in part in ASD neurons, and may reveal windows of opportunity for biotherapeutics to target the trajectory of gene expression and clinical manifestation of ASD throughout the human lifespan.

Monocyte, neutrophil, and whole blood transcriptome dynamics following ischemic stroke.

BACKGROUND: After ischemic stroke (IS), peripheral leukocytes infiltrate the damaged region and modulate the response to injury. Peripheral blood cells display distinctive gene expression signatures post-IS and these transcriptional programs reflect changes in immune responses to IS. Dissecting the temporal dynamics of gene expression after IS improves our understanding of immune and clotting responses at the molecular and cellular level that are involved in acute brain injury and may assist with time-targeted, cell-specific therapy. METHODS: The transcriptomic profiles from peripheral monocytes, neutrophils, and whole blood from 38 ischemic stroke patients and 18 controls were analyzed with RNA-seq as a function of time and etiology after stroke. Differential expression analyses were performed at 0-24 h, 24-48 h, and >48 h following stroke. RESULTS: Unique patterns of temporal gene expression and pathways were distinguished for monocytes, neutrophils, and whole blood with enrichment of interleukin signaling pathways for different time points and stroke etiologies. Compared to control subjects, gene expression was generally upregulated in neutrophils and generally downregulated in monocytes over all times for cardioembolic, large vessel, and small vessel strokes. Self-organizing maps identified gene clusters with similar trajectories of gene expression over time for different stroke causes and sample types. Weighted Gene Co-expression Network Analyses identified modules of co-expressed genes that significantly varied with time after stroke and included hub genes of immunoglobulin genes in whole blood. CONCLUSIONS: Altogether, the identified genes and pathways are critical for understanding how the immune and clotting systems change over time after stroke. This study identifies potential time- and cell-specific biomarkers and treatment targets.

Early peripheral blood gene expression associated with good and poor 90-day ischemic stroke outcomes.

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. controls at all three time-points that correlated with 90-day mRS; 13 probe sets were shared for good outcomes vs. controls at all three time-points; and 46 probe sets were shared for poor vs. good outcomes at all three time-points that correlated with 90-day mRS. Weighted Gene Co-Expression Network Analysis (WGCNA) revealed modules significantly associated with 90-day outcome for mRS and NIHSS. Poor outcome modules were enriched with up-regulated neutrophil genes and with down-regulated T cell, B cell and monocyte-specific genes; and good outcome modules were associated with erythroblasts and megakaryocytes. Finally, genes identified by genome-wide association studies (GWAS) to contain significant stroke risk loci or loci associated with stroke outcome including ATP2B, GRK5, SH3PXD2A, CENPQ, HOXC4, HDAC9, BNC2, PTPN11, PIK3CG, CDK6, and PDE4DIP were significantly differentially expressed as a function of stroke outcome in the current study. CONCLUSIONS: This study suggests the immune response after stroke may impact functional outcomes and that some of the early post-stroke gene expression markers associated with outcome could be useful for predicting outcomes and could be targets for improving outcomes.

Progression of cerebral white matter hyperintensities is related to leucocyte gene expression.

Cerebral white matter hyperintensities are an important contributor to ageing brain pathology. Progression in white matter hyperintensity volume is associated with cognitive decline and gait impairment. Understanding the factors associated with white matter hyperintensity progression provides insight into pathogenesis and may identify novel treatment targets to improve cognitive health. We postulated that the immune system interaction with cerebral vessels and tissue may be associated with disease progression, and thus evaluated the relationship of blood leucocyte gene expression to progression of cerebral white matter hyperintensities. A brain MRI was obtained at baseline in 166 patients assessed for a cognitive complaint, and then repeated at regular intervals over a median of 5.9 years (interquartile range 3.5-8.2 years). White matter hyperintensity volumes were measured by semi-automated segmentation and percentage change in white matter hyperintensity per year calculated. A venous blood sample obtained at baseline was used to measure whole-genome expression by RNA sequencing. The relationship between change in white matter hyperintensity volumes over time and baseline leucocyte gene expression was analysed. The mean age was 77.8 (SD 7.5) years and 60.2% of participants were female. The median white matter hyperintensity volume was 13.4 ml (SD 17.4 ml). The mean change in white matter hyperintensity volume was 12% per year. Patients were divided in quartiles by percentage change in white matter hyperintensity volume, which was: -3.5% per year in quartile 1, 7.4% per year in quartile 2, 11.7% in quartile 3 and 33.6% per year in quartile 4. There were 148 genes associated with changing white matter hyperintensity volumes over time (P < 0.05 r > |0.2|). Genes and pathways identified have roles in endothelial dysfunction, extracellular matrix remodelling, altered remyelination, inflammation and response to ischaemia. ADAM8, CFD, EPHB4, FPR2, Wnt-B-catenin, focal adhesion kinase and SIGLEC1 were among the identified genes. The progression of white matter hyperintensity volumes over time is associated with genes involved in endothelial dysfunction, extracellular matrix remodelling, altered remyelination, inflammation and response to ischaemia. Further studies are needed to evaluate the role of peripheral inflammation in relation to rate of white matter hyperintensity progression and the contribution to cognitive decline.

Gene expression changes implicate specific peripheral immune responses to Deep and Lobar Intracerebral Hemorrhages in humans.

The peripheral immune system response to Intracerebral Hemorrhage (ICH) may differ with ICH in different brain locations. Thus, we investigated peripheral blood mRNA expression of Deep ICH, Lobar ICH, and vascular risk factor-matched control subjects (n = 59). Deep ICH subjects usually had hypertension. Some Lobar ICH subjects had cerebral amyloid angiopathy (CAA). Genes and gene networks in Deep ICH and Lobar ICH were compared to controls. We found 774 differentially expressed genes (DEGs) and 2 co-expressed gene modules associated with Deep ICH, and 441 DEGs and 5 modules associated with Lobar ICH. Pathway enrichment showed some common immune/inflammatory responses between locations including Autophagy, T Cell Receptor, Inflammasome, and Neuroinflammation Signaling. Th2, Interferon, GP6, and BEX2 Signaling were unique to Deep ICH. Necroptosis Signaling, Protein Ubiquitination, Amyloid Processing, and various RNA Processing terms were unique to Lobar ICH. Finding amyloid processing pathways in blood of Lobar ICH patients suggests peripheral immune cells may participate in processes leading to perivascular/vascular amyloid in CAA vessels and/or are involved in its removal. This study identifies distinct peripheral blood transcriptome architectures in Deep and Lobar ICH, emphasizes the need for considering location in ICH studies/clinical trials, and presents potential location-specific treatment targets.

Bacterial lipopolysaccharide is associated with stroke.

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), LPS-binding 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.

Aging Immune System in Acute Ischemic Stroke: A Transcriptomic Analysis.

[Figure: see text].

Distinct peripheral blood monocyte and neutrophil transcriptional programs following intracerebral hemorrhage and different etiologies of ischemic stroke.

Understanding cell-specific transcriptome responses following intracerebral hemorrhage (ICH) and ischemic stroke (IS) will improve knowledge of the immune response to brain injury. Transcriptomic profiles of 141 samples from 48 subjects with ICH, different IS etiologies, and vascular risk factor controls were characterized using RNA-seq in isolated neutrophils, monocytes and whole blood. In both IS and ICH, monocyte genes were down-regulated, whereas neutrophil gene expression changes were generally up-regulated. The monocyte down-regulated response to ICH included innate, adaptive immune, dendritic, NK cell and atherosclerosis signaling. Neutrophil responses to ICH included tRNA charging, mitochondrial dysfunction, and ER stress pathways. Common monocyte and neutrophil responses to ICH included interferon signaling, neuroinflammation, death receptor signaling, and NFAT pathways. Suppressed monocyte responses to IS included interferon and dendritic cell maturation signaling, phagosome formation, and IL-15 signaling. Activated neutrophil responses to IS included oxidative phosphorylation, mTOR, BMP, growth factor signaling, and calpain proteases-mediated blood-brain barrier (BBB) dysfunction. Common monocyte and neutrophil responses to IS included JAK1, JAK3, STAT3, and thrombopoietin signaling. Cell-type and cause-specific approaches will assist the search for future IS and ICH biomarkers and treatments.

Molecular Correlates of Hemorrhage and Edema Volumes Following Human Intracerebral Hemorrhage Implicate Inflammation, Autophagy, mRNA Splicing, and T Cell Receptor Signaling.

Intracerebral hemorrhage (ICH) and perihematomal edema (PHE) volumes are major determinants of ICH outcomes as is the immune system which plays a significant role in damage and repair. Thus, we performed whole-transcriptome analyses of 18 ICH patients to delineate peripheral blood genes and networks associated with ICH volume, absolute perihematomal edema (aPHE) volume, and relative PHE (aPHE/ICH; rPHE). We found 440, 266, and 391 genes correlated with ICH and aPHE volumes and rPHE, respectively (p < 0.005, partial-correlation > |0.6|). These mainly represented inflammatory pathways including NF-κB, TREM1, and Neuroinflammation Signaling-most activated with larger volumes. Weighted Gene Co-Expression Network Analysis identified seven modules significantly correlated with these measures (p < 0.05). Most modules were enriched in neutrophil, monocyte, erythroblast, and/or T cell-specific genes. Autophagy, apoptosis, HIF-1α, inflammatory and neuroinflammatory response (including Toll-like receptors), cell adhesion (including MMP9), platelet activation, T cell receptor signaling, and mRNA splicing were represented in these modules (FDR p < 0.05). Module hub genes, potential master regulators, were enriched in neutrophil-specific genes in three modules. Hub genes included NCF2, NCF4, STX3, and CSF3R, and involved immune response, autophagy, and neutrophil chemotaxis. One module that correlated negatively with ICH volume correlated positively with rPHE. Its genes and hubs were enriched in T cell-specific genes including hubs LCK and ITK, Src family tyrosine kinases whose modulation improved outcomes and reduced BBB dysfunction following experimental ICH. This study uncovers molecular underpinnings associated with ICH and PHE volumes and pathophysiology in human ICH, where knowledge is scarce. The identified pathways and hub genes may represent novel therapeutic targets.

Alternative Splicing of Putative Stroke/Vascular Risk Factor Genes Expressed in Blood Following Ischemic Stroke Is Sexually Dimorphic and Cause-Specific.

Genome-wide association studies have identified putative ischemic stroke risk genes, yet, their expression after stroke is unexplored in spite of growing interest in elucidating their specific role and identifying candidate genes for stroke treatment. Thus, we took an exploratory approach to investigate sexual dimorphism, alternative splicing, and etiology in putative risk gene expression in blood following cardioembolic, atherosclerotic large vessel disease and small vessel disease/lacunar causes of ischemic stroke in each sex compared to controls. Whole transcriptome arrays assessed 71 putative stroke/vascular risk factor genes for blood RNA expression at gene-, exon-, and alternative splicing-levels. Male (n = 122) and female (n = 123) stroke and control volunteers from three university medical centers were matched for race, age, vascular risk factors, and blood draw time since stroke onset. Exclusion criteria included: previous stroke, drug abuse, subarachnoid or intracerebral hemorrhage, hemorrhagic transformation, infection, dialysis, cancer, hematological abnormalities, thrombolytics, anticoagulants or immunosuppressants. Significant differential gene expression (fold change > |1.2|, p < 0.05, partial correlation > |0.4|) and alternative splicing (false discovery rate p < 0.3) were assessed. At gene level, few were differentially expressed: ALDH2, ALOX5AP, F13A1, and IMPA2 (males, all stroke); ITGB3 (females, cardioembolic); ADD1 (males, atherosclerotic); F13A1, IMPA2 (males, lacunar); and WNK1 (females, lacunar). GP1BA and ITGA2B were alternatively spliced in both sexes (all patients vs. controls). Six genes in males, five in females, were alternatively spliced in all stroke compared to controls. Alternative splicing and exon-level analyses associated many genes with specific etiology in either sex. Of 71 genes, 70 had differential exon-level expression in stroke patients compared to control subjects. Among stroke patients, 24 genes represented by differentially expressed exons were male-specific, six were common between sexes, and two were female-specific. In lacunar stroke, expression of 19 differentially expressed exons representing six genes (ADD1, NINJ2, PCSK9, PEMT, SMARCA4, WNK1) decreased in males and increased in females. Results demonstrate alternative splicing and sexually dimorphic expression of most putative risk genes in stroke patients' blood. Since expression was also often cause-specific, sex, and etiology are factors to consider in stroke treatment trials and genetic association studies as society trends toward more personalized medicine.

Genetic variation contributes to gene expression response in ischemic stroke: an eQTL study.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) contribute to complex disorders such as ischemic stroke (IS). Since SNPs could affect IS by altering gene expression, we studied the association of common SNPs with changes in mRNA expression (i.e. expression quantitative trait loci; eQTL) in blood after IS. METHODS: RNA and DNA were isolated from 137 patients with acute IS and 138 vascular risk factor controls (VRFC). Gene expression was measured using Affymetrix HTA 2.0 microarrays and SNP variants were assessed with Axiom Biobank Genotyping microarrays. A linear model with a genotype (SNP) × diagnosis (IS and VRFC) interaction term was fit for each SNP-gene pair. RESULTS: The eQTL interaction analysis revealed significant genotype × diagnosis interaction for four SNP-gene pairs as cis-eQTL and 70 SNP-gene pairs as trans-eQTL. Cis-eQTL involved in the inflammatory response to IS included rs56348411 which correlated with neurogranin expression (NRGN), rs78046578 which correlated with CXCL10 expression, rs975903 which correlated with SMAD4 expression, and rs62299879 which correlated with CD38 expression. These four genes are important in regulating inflammatory response and BBB stabilization. SNP rs148791848 was a strong trans-eQTL for anosmin-1 (ANOS1) which is involved in neural cell adhesion and axonal migration and may be important after stroke. INTERPRETATION: This study highlights the contribution of genetic variation to regulating gene expression following IS. Specific inflammatory response to stroke is at least partially influenced by genetic variation. This has implications for progressing toward personalized treatment strategies. Additional research is required to investigate these genes as therapeutic targets.

MicroRNA and their target mRNAs change expression in whole blood of patients after intracerebral hemorrhage.

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-ß, 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.

mRNA Expression Profiles from Whole Blood Associated with Vasospasm in Patients with Subarachnoid Hemorrhage.

BACKGROUND: Though there are many biomarker studies of plasma and serum in patients with aneurysmal subarachnoid hemorrhage (SAH), few have examined blood cells that might contribute to vasospasm. In this study, we evaluated inflammatory and prothrombotic pathways by examining mRNA expression in whole blood of SAH patients with and without vasospasm. METHODS: Adult SAH patients with vasospasm (n = 29) and without vasospasm (n = 21) were matched for sex, race/ethnicity, and aneurysm treatment method. Diagnosis of vasospasm was made by angiography. mRNA expression was measured by Affymetrix Human Exon 1.0 ST Arrays. SAH patients with vasospasm were compared to those without vasospasm by ANCOVA to identify differential gene, exon, and alternatively spliced transcript expression. Analyses were adjusted for age, batch, and time of blood draw after SAH. RESULTS: At the gene level, there were 259 differentially expressed genes between SAH patients with vasospasm compared to patients without (false discovery rate < 0.05, |fold change| ≥ 1.2). At the exon level, 1210 exons representing 1093 genes were differentially regulated between the two groups (P < 0.005, ≥ 1.2 |fold change|). Principal components analysis segregated SAH patients with and without vasospasm. Signaling pathways for the 1093 vasospasm-related genes included adrenergic, P2Y, ET-1, NO, sildenafil, renin-angiotensin, thrombin, CCR3, CXCR4, MIF, fMLP, PKA, PKC, CRH, PPARα/RXRα, and calcium. Genes predicted to be alternatively spliced included IL23A, RSU1, PAQR6, and TRIP6. CONCLUSIONS: This is the first study to demonstrate that mRNA expression in whole blood distinguishes SAH patients with vasospasm from those without vasospasm and supports a role of coagulation and immune systems in vasospasm.

Cancer-Related Ischemic Stroke Has a Distinct Blood mRNA Expression Profile.

Background and Purpose- Comorbid cancer is common in patients with acute ischemic stroke (AIS). As blood mRNA profiles can distinguish AIS mechanisms, we hypothesized that cancer-related AIS would have a distinctive gene expression profile. Methods- We evaluated 4 groups of 10 subjects prospectively enrolled at 3 centers from 2009 to 2018. This included the group of interest with active solid tumor cancer and AIS and 3 control groups with active cancer only, AIS only, or vascular risk factors only. Subjects in the AIS-only and cancer-only groups were matched to subjects in the cancer-stroke group by age, sex, and cancer type (if applicable). Subjects in the vascular risk factor group were matched to subjects in the cancer-stroke and stroke-only groups by age, sex, and vascular risk factors. Blood was drawn 72 to 120 hours after stroke. Total RNA was processed using 3' mRNA sequencing. ANOVA and Fisher least significant difference contrast methods were used to estimate differential gene expression between groups. Results- In the cancer-stroke group, 50% of strokes were cryptogenic. All groups had differentially expressed genes that could distinguish among them. Comparing the cancer-stroke group to the stroke-only group and after accounting for cancer-only genes, 438 genes were differentially expressed, including upregulation of multiple genes/pathways implicated in autophagy signaling, immunity/inflammation, and gene regulation, including IL (interleukin)-1, interferon, relaxin, mammalian target of rapamycin signaling, SQSTMI1 (sequestosome-1), and CREB1 (cAMP response element binding protein-1). Conclusions- This study provides evidence for a distinctive molecular signature in blood mRNA expression profiles of patients with cancer-related AIS. Future studies should evaluate whether blood mRNA can predict detection of occult cancer in patients with AIS. Clinical Trial Registration- URL: https://clinicaltrials.gov. Unique identifier: NCT02604667.

Smoking affects gene expression in blood of patients with ischemic stroke.

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), IS-never 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.

Inflammatory, regulatory, and autophagy co-expression modules and hub genes underlie the peripheral immune response to human intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) has a high morbidity and mortality. The peripheral immune system and cross-talk between peripheral blood and brain have been implicated in the ICH immune response. Thus, we delineated the gene networks associated with human ICH in the peripheral blood transcriptome. We also compared the differentially expressed genes in blood following ICH to a prior human study of perihematomal brain tissue. METHODS: We performed peripheral blood whole-transcriptome analysis of ICH and matched vascular risk factor control subjects (n = 66). Gene co-expression network analysis identified groups of co-expressed genes (modules) associated with ICH and their most interconnected genes (hubs). Mixed-effects regression identified differentially expressed genes in ICH compared to controls. RESULTS: Of seven ICH-associated modules, six were enriched with cell-specific genes: one neutrophil module, one neutrophil plus monocyte module, one T cell module, one Natural Killer cell module, and two erythroblast modules. The neutrophil/monocyte modules were enriched in inflammatory/immune pathways; the T cell module in T cell receptor signaling genes; and the Natural Killer cell module in genes regulating alternative splicing, epigenetic, and post-translational modifications. One erythroblast module was enriched in autophagy pathways implicated in experimental ICH, and NRF2 signaling implicated in hematoma clearance. Many hub genes or module members, such as IARS, mTOR, S1PR1, LCK, FYN, SKAP1, ITK, AMBRA1, NLRC4, IL6R, IL17RA, GAB2, MXD1, PIK3CD, NUMB, MAPK14, DDX24, EVL, TDP1, ATG3, WDFY3, GSK3B, STAT3, STX3, CSF3R, PIP4K2A, ANXA3, DGAT2, LRP10, FLOT2, ANK1, CR1, SLC4A1, and DYSF, have been implicated in neuroinflammation, cell death, transcriptional regulation, and some as experimental ICH therapeutic targets. Gene-level analysis revealed 1225 genes (FDR p < 0.05, fold-change > |1.2|) have altered expression in ICH in peripheral blood. There was significant overlap of the 1225 genes with dysregulated genes in human perihematomal brain tissue (p = 7 × 10-3). Overlapping genes were enriched for neutrophil-specific genes (p = 6.4 × 10-08) involved in interleukin, neuroinflammation, apoptosis, and PPAR signaling. CONCLUSIONS: This study delineates key processes underlying ICH pathophysiology, complements experimental ICH findings, and the hub genes significantly expand the list of novel ICH therapeutic targets. The overlap between blood and brain gene responses underscores the importance of examining blood-brain interactions in human ICH.

HDAC9 Polymorphism Alters Blood Gene Expression in Patients with Large Vessel Atherosclerotic Stroke.

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 risk allele-negative LVAS patients on a principal component analysis. Pathways associated with HDAC9 risk allele-positive status involved IL-6 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.

The intracerebral hemorrhage blood transcriptome in humans differs from the ischemic stroke and vascular risk factor control blood transcriptomes.

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® 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 |FC| > 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.

Lipopolysaccharide Associates with Amyloid Plaques, Neurons and Oligodendrocytes in Alzheimer's Disease Brain: A Review.

This review proposes that lipopolysaccharide (LPS, found in the wall of all Gram-negative bacteria) could play a role in causing sporadic Alzheimer's disease (AD). This is based in part upon recent studies showing that: Gram-negative E. coli bacteria can form extracellular amyloid; bacterial-encoded 16S rRNA is present in all human brains with over 70% being Gram-negative bacteria; ultrastructural analyses have shown microbes in erythrocytes of AD patients; blood LPS levels in AD patients are 3-fold the levels in control; LPS combined with focal cerebral ischemia and hypoxia produced amyloid-like plaques and myelin injury in adult rat cortex. Moreover, Gram-negative bacterial LPS was found in aging control and AD brains, though LPS levels were much higher in AD brains. In addition, LPS co-localized with amyloid plaques, peri-vascular amyloid, neurons, and oligodendrocytes in AD brains. Based upon the postulate LPS caused oligodendrocyte injury, degraded Myelin Basic Protein (dMBP) levels were found to be much higher in AD compared to control brains. Immunofluorescence showed that the dMBP co-localized with ß amyloid (Aß) and LPS in amyloid plaques in AD brain, and dMBP and other myelin molecules were found in the walls of vesicles in periventricular White Matter (WM). These data led to the hypothesis that LPS acts on leukocyte and microglial TLR4-CD14/TLR2 receptors to produce NFkB mediated increases of cytokines which increase Aß levels, damage oligodendrocytes and produce myelin injury found in AD brain. Since Aß1-42 is also an agonist for TLR4 receptors, this could produce a vicious cycle that accounts for the relentless progression of AD. Thus, LPS, the TLR4 receptor complex, and Gram-negative bacteria might be treatment or prevention targets for sporadic AD.