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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  |0.6|). 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

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.

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.

BackgroundIntracerebral 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.MethodsWe 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.ResultsOf 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  |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.ConclusionsThis 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.

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)  1.2 for individual comparisons. For time-dependent analyses, subjects were divided into four time-points: 0(CTRL), 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.

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

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.