Background and Purpose Though peripheral blood mRNA and microRNA change following ischemic stroke, any role for long noncoding RNA (lncRNA), which comprise most of the genome and have been implicated in various diseases, is unknown. Thus, we hypothesized that lncRNA expression also changes following stroke. Methods lncRNA expression was assessed in 266 whole-blood RNA samples drawn once per individual from ischemic stroke patients and matched vascular risk factor controls. Differential lncRNA expression was assessed by Analysis of Covariance (ANCOVA, p-value < 0.005; fold change > |1.2|), principal components analysis and hierarchical clustering on a derivation set (n=176) and confirmed on a validation set (n=90). Post-stroke temporal lncRNA expression changes were assessed using ANCOVA with confounding factor correction (p<0.005; partial correlation with time since event >|0.4|). Because sexual dimorphism exists in stroke, analyses were performed for each sex separately. Results 299 lncRNAs were differentially expressed between stroke and control males, whereas 97 lncRNAs were differentially expressed between stroke and control females. Significant changes of lncRNA expression with time after stroke were detected for 49 lncRNAs in males and 31 lncRNAs in females. Some differentially expressed lncRNAs mapped close to genomic locations of previously identified putative stroke-risk genes, including Lipoprotein, Lp(A)-Like 2, ABO blood group, Prostaglandin 12 Synthase, and α-Adducins. Conclusions This study provides evidence of altered and sexually dimorphic lncRNA expression in peripheral blood of stroke patients compared to controls and suggests lncRNAs have potential for stroke biomarker development. Some regulated lncRNA could regulate some previously identified putative stroke-risk genes.

Because our recent studies have demonstrated that miR-122 decreased in whole blood of patients and in whole blood of rats following ischemic stroke, we tested whether elevating blood miR-122 would improve stroke outcomes in rats. Young adult rats were subjected to a temporary middle cerebral artery occlusion (MCAO) or sham operation. A polyethylene glycol-liposome-based transfection system was used to administer a miR-122 mimic after MCAO. Neurological deficits, brain infarction, brain vessel integrity, adhesion molecule expression and expression of miR-122 target and indirect-target genes were examined in blood at 24 h after MCAO with or without miR-122 treatment. miR-122 decreased in blood after MCAO, whereas miR-122 mimic elevated miR-122 in blood 24 h after MCAO. Intravenous but not intracerebroventricular injection of miR-122 mimic decreased neurological deficits and brain infarction, attenuated ICAM-1 expression, and maintained vessel integrity after MCAO. The miR-122 mimic also down-regulated direct target genes (e.g. Vcam1, Nos2, Pla2g2a) and indirect target genes (e.g. Alox5, Itga2b, Timp3, Il1b, Il2, Mmp8) in blood after MCAO which are predicted to affect cell adhesion, diapedesis, leukocyte extravasation, eicosanoid and atherosclerosis signaling. The data show that elevating miR-122 improves stroke outcomes and we postulate this occurs via downregulating miR-122 target genes in blood leukocytes.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12974-019-1433-4) contains supplementary material, which is available to authorized users.

Whole transcriptome studies have used 3′-biased expression microarrays to study genes regulated in the blood of stroke patients. However, alternatively spliced messenger RNA isoforms have not been investigated for ischemic stroke or intracerebral hemorrhage (ICH) in animals or humans. Alternative splicing is the mechanism whereby different combinations of exons of a single gene produce distinct mRNA and protein isoforms. Here, we used RNA sequencing (RNA-seq) to determine if alternative splicing differs for ICH and cardioembolic, large vessel and lacunar causes of ischemic stroke compared to controls. RNA libraries from 20 whole blood samples were sequenced to 200 M 2 × 100 bp reads using Illumina sequencing-by-synthesis technology. Differential alternative splicing was assessed using one-way analysis of variance (ANOVA), and differential exon usage was calculated. Four hundred twelve genes displayed differential alternative splicing among the groups (false discovery rate, FDR; p < 0.05). They were involved in cellular immune response, cell death, and cell survival pathways. Distinct expression signatures based on usage of 308 exons (292 genes) differentiated the groups (p < 0.0005; fold change >|1.2|). This pilot study demonstrates that alternatively spliced genes from whole blood differ in ICH compared to ischemic stroke and differ between different ischemic stroke etiologies. These results require validation in a separate cohort.Electronic supplementary materialThe online version of this article (doi:10.1007/s12975-015-0407-9) contains supplementary material, which is available to authorized users.

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.

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-β, 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.

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.