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Ischemic brain and peripheral white blood cells release cytokines, chemokines and other molecules that activate the peripheral white blood cells after stroke. To assess gene expression in these peripheral white blood cells, whole blood was examined using oligonucleotide microarrays in 15 patients at 2.4 ± 0.5, 5 and 24 h after onset of ischemic stroke and compared with control blood samples. The 2.4 h blood samples were drawn before patients were treated either with tissue-type plasminogen activator (tPA) alone or with tPA plus Eptifibatide (the Combination approach to Lysis utilizing Eptifibatide And Recombinant tPA trial). Most genes induced in whole blood at 2 to 3 h were also induced at 5 and 24 h. Separate studies showed that the genes induced at 2 to 24 h after stroke were expressed mainly by polymorphonuclear leukocytes and to a lesser degree by monocytes. These genes included: matrix metalloproteinase 9; S100 calcium-binding proteins P, A12 and A9; coagulation factor V; arginase I; carbonic anhydrase IV; lymphocyte antigen 96 (cluster of differentiation (CD)96); monocarboxylic acid transporter (6); ets-2 (erythroblastosis virus E26 oncogene homolog 2); homeobox gene Hox 1.11; cytoskeleton-associated protein 4; N-formylpeptide receptor; ribonuclease-2; N-acetylneuraminate pyruvate lyase; BCL6; glycogen phosphorylase. The fold change of these genes varied from 1.6 to 6.8 and these 18 genes correctly classified 10/15 patients at 2.4 h, 13/15 patients at 5h and 15/15 patients at 24 h after stroke. These data provide insights into the inflammatory responses after stroke in humans, and should be helpful in diagnosis, understanding etiology and pathogenesis, and guiding acute treatment and development of new treatments for stroke.

Animals exposed to brief periods of moderate hypoxia (8% to 10% oxygen for 3 hours) are protected against cerebral and cardiac ischemia between 1 and 2 days later. This hypoxia preconditioning requires new RNA and protein synthesis. The mechanism of this hypoxia-induced tolerance correlates with the induction of the hypoxia-inducible factor (HIF), a transcription factor heterodimeric complex composed of inducible HIF-1α and constitutive HIF-1β proteins that bind to the hypoxia response elements in a number of HIF target genes. Our recent studies show that HIF-1α correlates with hypoxia induced tolerance in neonatal rat brain. HIF target genes, also induced following hypoxia-induced tolerance, include vascular endothelial growth factor, erythropoietin, glucose transporters, glycolytic enzymes, and many other genes. Some or all of these genes may contribute to hypoxia-induced protection against ischemia. HIF induction of the glycolytic enzymes accounts in part for the Pasteur effect in brain and other tissues. Hypoxia-induced tolerance is not likely to be equivalent to treatment with a single HIF target gene protein since other transcription factors including Egr-1 (NGFI-A) have been implicated in hypoxia regulation of gene expression. Understanding the mechanisms and genes involved in hypoxic tolerance may provide new therapeutic targets to treat ischemic injury and enhance recovery.

There are no biomarkers that differentiate cardioembolic from large-vessel atherosclerotic stroke, although the treatments differ for each and ~30% of strokes and transient ischemic attacks have undetermined etiologies using current clinical criteria. We aimed to define gene expression profiles in blood that differentiate cardioembolic from large-vessel atherosclerotic stroke. Peripheral blood samples were obtained from healthy controls and acute ischemic stroke patients (< 3, 5, and 24 h). RNA was purified, labeled, and applied to Affymetrix Human U133 Plus 2.0 Arrays. Expression profiles in the blood of cardioembolic stroke patients are distinctive from those of large-vessel atherosclerotic stroke patients. Seventy-seven genes differ at least 1.5-fold between them, and a minimum number of 23 genes differentiate the two types of stroke with at least 95.2% specificity and 95.2% sensitivity for each. Genes regulated in large-vessel atherosclerotic stroke are expressed in platelets and monocytes and modulate hemostasis. Genes regulated in cardioembolic stroke are expressed in neutrophils and modulate immune responses to infectious stimuli. This new method can be used to predict whether a stroke of unknown etiology was because of cardioembolism or large-vessel atherosclerosis that would lead to different therapy. These results have wide ranging implications for similar disorders.

Exposure to moderate hypoxia alone does not cause neuronal death as long as blood pressure and cerebral blood flow are maintained in mammals. In neonatal and adult mammals including rats and mice, carotid occlusion in combination with hypoxia produces neuronal death and brain infarction. However, preexposure to 8% oxygen for 3 h protects the brain and likely other organs of neonatal and adult rats against combined hypoxia-ischemia 24 h later. In this paper, the possible mechanisms of this so-called hypoxia-induced tolerance to ischemia is discussed. One mechanism likely involves hypoxia-inducible factor-1α (HIF-1α). HIF-1α is a transcription factor that – during hypoxia – binds with a second protein (HIF-1β) in the nucleus to promoter elements in hypoxia-responsive target genes. This causes upregulation of HIF target genes including VEGF, erythropoietin, iNOS, glucose transporter-1, glycolytic enzymes, and many other genes to protect the brain against ischemia 24 h later. In addition, non-HIF pathways including MTF-1, Egr-1 and others act directly or indirectly on other target genes to also promote hypoxia-induced preconditioning. Hypoxia preconditioning can be mimicked by iron chelators like desferrioxamine and transition metals like cobalt chloride that inhibit prolyl hydroxylases, increase HIF-1α levels in the brain, and produce protection of the brain against combined hypoxia-ischemia 24 h later. This hypoxia preconditioning has potential clinical usefulness in protecting high-risk newborns or to provide protection prior to surgery.

After intracerebral hemorrhage (ICH), many changes of gene transcription occur that may be important because they will contribute to understanding mechanisms of injury and recovery. Therefore, gene expression was assessed using Affymetrix microarrays in the striatum and the overlying cortex at 24 h after intracranial infusions of blood into the striatum of adult rats. Intracerebral hemorrhage regulated 369 of 8,740 transcripts as compared with saline-injected controls, with 104 regulated genes shared by the striatum and cortex. There were 108 upregulated and 126 downregulated genes in striatum, and 170 upregulated and 69 downregulated genes in the cortex. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed upregulation of IL-1-beta, Lipcortin 1 (annexin) and metallothionein 1,2, and downregulation of potassium voltage-gated channel, shaker-related subfamily, beta member 2 (Kcnab2). Of the functional groups of genes modulated by ICH, many metabolism and signal-transduction-related genes decreased in striatum but increased in adjacent cortex. In contrast, most enzyme, cytokine, chemokine, and immune response genes were upregulated in both striatum and in the cortex after ICH, likely in response to foreign proteins from the blood. A number of these genes may contribute to brain edema and cellular apoptosis caused by ICH. In addition, downregulation of growth factor pathways and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway could also contribute to perihematoma cell death/apoptosis. Intracerebral hemorrhage-related downregulation of GABA-related genes and potassium channels might contribute to perihematoma cellular excitability and increased risk of post-ICH seizures. These genomic responses to ICH potentially provide new therapeutic targets for treatment.

Understanding transcriptional changes in brain after ischemia may provide therapeutic targets for treating stroke and promoting recovery. To study these changes on a genomic scale, oligonucleotide arrays were used to assess RNA samples from periinfarction cortex of adult Sprague-Dawley rats 24 h after permanent middle cerebral artery occlusions. Of the 328 regulated transcripts in ischemia compared with sham-operated animals, 264 were upregulated, 64 were downregulated, and 163 (49.7%) had not been reported in stroke. Of the functional groups modulated by ischemia: G-protein–related genes were the least reported; and cytokines, chemokines, stress proteins, and cell adhesion and immune molecules were the most highly expressed. Quantitative reverse transcription polymerase chain reaction of 20 selected genes at 2, 4, and 24 h after ischemia showed early upregulated genes (2 h) including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, JAK2, CELF, Tenascin, and DAF. Late upregulated genes (24 h) included Cathepsin C, Cip-26, Cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein. Glycerol 3-phosphate dehydrogenase, which is involved in mitochondrial reoxidation of glycolysis derived NADH, was regulated more than 60-fold. Plasticity-related transcripts were regulated, including Narp, agrin, and Cpg21. A newly reported lung pathway was also regulated in ischemic brain: C/EBP induction of Egr-1 (NGFI-A) with downstream induction of PAI-1, VEGF, ICAM, IL1, and MIP1. Genes regulated acutely after stroke may modulate cell survival and death; also, late regulated genes may be related to tissue repair and functional recovery.

Heme oxygenase-1 (HO-1) is an inducible and cytoprotective enzyme that provides a defense against oxidant damage. The present study screened 137 HO-1/interacting proteins using a profound co-immunoprecipitation (Co-IP) coupled with proteomics, and profiled the global HO-1 interactome network, including oxidative phosphorylation, endoplasmic reticulum and transport vesicle functions. Among these molecules, we observed that a novel interactor, emopamil-binding protein (EBP), is closely related to the cholesterol metabolism process. This study demonstrated that cholesterol promotes excessive oxidative stress and alters the energy metabolism in cardiomyocytes, further triggering numerous cardiovascular diseases. We observed that cholesterol caused the overexpression of EBP and HO-1 by the activation of AKT and Nrf2/mTOR pathways. In addition, HO-1 and EBP performed a myocardial protective function. The overexpression of HO-1 alleviated the cholesterol-induced excessive oxidative stress status by inhibition of the carbohydrate metabolism. Notably, we also confirmed that the loss of partial HO-1 activity aggravated the oxidative damage and cardiac systolic function induced by a high-fat diet in HO-1 heterozygous (HO-1+/−) mice. These findings indicate that the HO-1/EBP interaction plays a protective role in alleviating the dysfunction of oxidative stress and cardiac systolic function induced by cholesterol stimulation.

This study determined whether stroke and other types of insults produced a gene expression profile in blood that correlated with the presence of neuronal injury. Adult rats were subjected to ischemic stroke, intracerebral hemorrhage, status epilepticus, and insulin-induced hypoglycemia and compared with untouched, sham surgery, and hypoxia animals that had no brain injury. One day later, microarray analyses showed that 117 genes were upregulated and 80 genes were downregulated in mononuclear blood cells of the \"injury\" (n = 12) compared with the \"no injury\" (n = 9) animals. A second experiment examined the whole blood genomic response of adult rats after global ischemia and kainate seizures. Animals with no brain injury were compared with those with brain injury documented by TUNEL and PANT staining. One day later, microarray analyses showed that 37 genes were upregulated and 67 genes were downregulated in whole blood of the injury (n = 4) animals compared with the no-injury (n = 4) animals. Quantitative reverse transcription-polymerase chain reaction confirmed that the vesicular monoamine transporter-2 increased 2.3- and 1.6-fold in animals with severe and mild brain injury, respectively, compared with no-injury animals. Vascular tyrosine phosphatase-1 increased 2.0-fold after severe injury compared with no injury. The data support the hypothesis that there is a peripheral blood genomic response to neuronal injury, and that this blood response is associated with a specific blood mRNA gene expression profile that can be used as a marker of the neuronal damage.

This study determined whether stroke and other types of insults produced a gene expression profile in blood that correlated with the presence of neuronal injury. Adult rats were subjected to ischemic stroke, intracerebral hemorrhage, status epilepticus, and insulin-induced hypoglycemia and compared with untouched, sham surgery, and hypoxia animals that had no brain injury. One day later, microarray analyses showed that 117 genes were upregulated and 80 genes were downregulated in mononuclear blood cells of the “injury” (n = 12) compared with the “no injury” (n = 9) animals. A second experiment examined the whole blood genomic response of adult rats after global ischemia and kainate seizures. Animals with no brain injury were compared with those with brain injury documented by TUNEL and PANT staining. One day later, microarray analyses showed that 37 genes were upregulated and 67 genes were downregulated in whole blood of the injury (n = 4) animals compared with the no-injury (n = 4) animals. Quantitative reverse transcription–polymerase chain reaction confirmed that the vesicular monoamine transporter-2 increased 2.3- and 1.6-fold in animals with severe and mild brain injury, respectively, compared with no-injury animals. Vascular tyrosine phosphatase-1 increased 2.0-fold after severe injury compared with no injury. The data support the hypothesis that there is a peripheral blood genomic response to neuronal injury, and that this blood response is associated with a specific blood mRNA gene expression profile that can be used as a marker of the neuronal damage.

Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.