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This study examined the effects of gender on RNA expression after ischemic stroke (IS). RNA obtained from blood of IS patients (n=51; 153 samples at 3, 5, and 24hours) and from matched controls (n=52) were processed on Affymetrix microarrays. Analyses of covariance for stroke versus control samples were performed separately for both genders and the regulated genes for females compared with males. In all, 242, 227, and 338 male-specific genes were regulated at 3, 5, and 24hours after IS, respectively, of which 59 were regulated at all time points. Overall, 774, 3,437, and 571 female-specific stroke genes were regulated at 3, 5, and 24hours, respectively, of which 152 were regulated at all time points. Male-specific stroke genes were associated with integrin, integrin-liked kinase, actin, tight junction, Wnt/β-catenin, RhoA, fibroblast growth factors (FGF), granzyme, and tumor necrosis factor receptor (TNFR)2 signaling. Female-specific stroke genes were associated with p53, high-mobility group box-1, hypoxia inducible factor (HIF)1α, interleukin (IL)1, IL6, IL12, IL18, acute-phase response, T-helper, macrophage, and estrogen signaling. Cell death signaling was overrepresented in both genders, although the molecules and pathways differed. Gender affects gene expression in the blood of IS patients, which likely implies gender differences in immune, inflammatory, and cell death responses to stroke.
Ischemic stroke (IS) is influenced by gender, with suggested differences in risk factors, outcomes, and etiology between men and women (Silva et al, 2010). For instance, men have increased stroke risk in middle age than do women, whereas women tend to have strokes at a later age and have more cardioembolic stroke. Possibly because of their longer life expectancy and higher incidence at older ages, women may be less likely to recover after IS and make up the majority of stroke deaths (Appelros et al, 2009; Reeves et al, 2008). However, women may derive greater benefit from thrombolysis compared with men, although this is controversial (Meseguer et al, 2009).
These gender differences in IS have been explained by both hormone-dependent and hormone-independent mechanisms (Liu et al, 2009; Vagnerova et al, 2008). For example, gender differences in peripheral and brain immune and inflammatory responses, cell apoptosis, and cell death may contribute (Libert et al, 2010; Siegel et al, 2010). However, the bases for many of the gender differences are still unclear.
Thus, to continue to explore the possible gender differences after IS, this study examined gene expression in whole blood of females compared with males at 3, 5, and 24hours after IS. We examined blood for a number of reasons. (1) We have previously shown changes of gene expression in all cells in blood of both females and males after IS (Du et al, 2006; Jickling et al, 2010a; Stamova et al, 2010). (2) The peripheral immune response affects outcome in stroke patients (Furuya et al, 2001) and experimental stroke (Herson and Hurn, 2010; Liu et al, 2009; Siegel et al, 2010). (3) Gene expression changes in the blood are a reflection of the immune response, effects of environmental factors, and genetic differences between females and males (Sharp et al, 2011). (4) Blood is easily accessible in humans, and can be studied as a function of the cause, risk factors, prognosis, and other parameters and can eventually be used to monitor therapies aimed at modulating the peripheral immune system.
This study examined RNA expression in blood using whole-genome microarrays to identify genes regulated in males after IS compared with male controls, and genes regulated in females after IS compared with female controls. This experimental design makes it possible to determine whether the expression of these genes is similar or different for both genders. As expected, many of the same genes were regulated in both males and females. However, large numbers of female- genes and male-specific genes were differentially expressed, thus suggesting that there are many sexually dimorphic molecular responses after IS in humans.
Subjects with acute IS were recruited through the combined approach to lysis utilizing eptifibatide and recombinant tissue-type plasminogen activator (rt-PA) (CLEAR) trial—a multi-center, randomized, double-blind safety study of recombinant-tissue plasminogen activator and eptifibatide (Pancioli et al, 2008) (NCT00250991 at http://clinicaltrials.gov/). This is part of the NINDS (National Institute of Neurological Disorders and Stroke)-funded SPOTRIAS (Specialized Programs of Translational Research in Acute Stroke) Network. The Internal Review Boards at each site approved this study. Written informed consent was obtained from all patients. Stroke patients had a diagnosis of acute IS, a NIHSS (National Institutes of Health Stroke Scale) >5 and were 18 to 80 years of age. Blood samples of each subject were drawn at 3, 5, and 24hours after their stroke. The first blood sample was drawn before 3hours after stroke onset and before treatment. Controls were subjects similar in age, gender, race, and vascular risk factors without symptomatic cardiovascular disease who were recruited from the University of Cincinnati, the University of California at Davis, the University of California at San Francisco, and the Wake Forest School of Medicine.
Blood sample collection, RNA isolation, and processing on Affymetrix U133 Plus 2.0. arrays (Affymetrix, Santa Clara, CA, USA) were performed as described previously (Stamova et al, 2010). Whole blood (15mL) was collected through venipuncture into six PAXgene Vacutainer tubes. These tubes contain a solution that immediately lyses all of the cells in whole blood and stabilizes the RNA. The RNA represents genes expressed in all white blood cells, immature red blood cells, and immature platelets. Blood samples were stored frozen at −80°C until processed.
Total RNA was isolated using the PAXgene Blood RNA kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and quantified with fiberoptic spectrophotometry using the Nanodrop ND-1000 (Nanodrop, Wilmington, DE, USA). Reverse transcription, amplification, and sample labeling were carried out using Nugen Ovation Whole-Blood reagents (Nugen, San Carlos, CA, USA). Affymetrix Genome U133 Plus 2.0 GeneChips were washed and processed on a Fluidics Station 450 and then scanned on a Genechip Scanner 3000.
Raw expression values were normalized using robust multichip averaging. To reduce false-positive results, probe sets with intensity lower than the background level in all samples were excluded (log2 <5.5), and probe sets identified as ‘absent' using the MAS 5.0 expression summary algorithm were also excluded. Of the original 54,000 probe sets, 35,361 were retained for further analysis.
A number of previous studies have examined sexually dimorphic differences of gene expression by directly comparing males with females (Heidecker et al, 2010). However, a number of these genes were located on sex chromosomes that could represent differences of transcript expression based on gender or differences based on the disease being studied. To eliminate this confound, we adopted a method used to study gender effects in Parkinson's disease (Cantuti-Castelvetri et al, 2007). Females with IS were compared with female controls, and males with IS were compared with male controls. Once the genes differentially regulated in females and males were identified, then these lists of genes were compared to identify stroke-induced, gender-related differential expression of genes. The female-specific IS genes were defined as genes that are differentially expressed in females with IS versus female controls after excluding any genes represented in males with IS versus control males gene list. The male-specific IS genes were defined as genes that are differentially expressed in males with IS versus male controls after excluding any genes represented in females with IS versus control females gene list.
Using the above approach, the analyses then addressed changes of gene expression at each time point (3, 5, and 24hours). An analysis of variance was performed to identify genes the expression of which indicated significant gender*diagnosis (stroke versus control) interaction in the blood of IS patients at 3hours (untreated), 5hours (treated), and 24hours (treated) after IS compared with nonstroke controls, adjusting for random effects of batch. Separate analyses were performed for females and males as described above, including the diagnosis main effect. Probe sets with a Benjamini–Hochberg false discovery rate (FDR) corrected P<0.05 and a fold change (FC) >1.5 were considered significant. A FDR approach was used to correct for multiple comparisons, with no more than 5% false positives (q<0.05) expected by chance. Differences in demographic data between groups were analyzed using Fisher's exact test and a two-tailed t-test where appropriate with P<0.05 considered significant.
Ingenuity pathways analysis (IPA 8.0, Ingenuity Systems, Redwood, CA, USA) and the NIAID/NIH DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov) were used to identify statistically significant functional categories in the data set using a modified Fisher's exact test (P<0.05). The EASE score threshold (maximum probability)=0.1 was used to identify chromosome enrichment. The threshold for the EASE score used for the gene-enrichment analysis is based on a modified Fisher's exact P-value. Fisher's exact tests determined whether there were more genes per pathway or more genes per chromosome differentially expressed between the two genders after IS than would be expected by chance.
There were 51 IS subjects (n=51; 153 samples at 3, 5, and 24hours) and 52 matched control subjects in this study. Demographic and clinical characteristics of the subjects are shown in Table 1. There were no statistically significant differences in age, gender, race, and vascular risk factors between IS and control patients for each gender (P>0.05). There were no statistically significant differences in age, gender, race, cause of stroke, and NIHSS scores between male and female IS subjects, as well as between male and female controls (P>0.05).
Figure 1A shows 242 male-specific IS probe sets (representing 155 genes) differentially expressed at 3hours after IS before any treatment (FDR <0.05 and a FC>1.5). Of these, 151 probe sets were upregulated and 91 downregulated (Supplementary Table 1). There were 774 female-specific IS probe sets (representing 533 genes) that were differentially expressed at 3hours after IS before any treatment (FDR <0.05 and a FC>1.5) (Figure 1A). Of these, 415 probe sets were upregulated and 359 downregulated (Supplementary Table 2). There were 211 probe sets (representing 136 genes) differentially expressed by both females and males (FDR <0.05 and a FC>1.5) (Figure 1A, Supplementary Table 3) that are not discussed here.
Functional analyses of genes differentially regulated in males at 3hours showed significant numbers of genes in granzyme A, tight junction, integrin-linked kinase (ILK), TNFR2, integrin, and FGF signaling canonical pathways (Table 2A). The canonical pathways in females differed and included T-helper, nuclear factor (NF)-κB, interleukin (IL)12, IL6, p53, acute-phase response, high-mobility group box-1 (HMGB1), amyloid, and HIF1α signaling (Table 2B). Similarly, the gene ontology (GO) biologic processes differed in females than in males, with immune responses including cytokine and T-cell responses dominating female processes, and cell death, apoptosis, cell cycle, and tight junction and cell junction signaling characterizing male processes (Tables 2A and and2B2B).
There were 227 male-specific IS probe sets (representing 150 genes) differentially expressed at 5hours after IS (FDR <0.05 and a FC>1.5), with 134 probe sets being upregulated and 93 downregulated (Figure 1A; Supplementary Table 5). There were 3,437 female-specific IS probe sets (representing 2,078 genes) differentially regulated at 5hours (FDR <0.05 and a FC>1.5), with 1,278 probe sets upregulated and 2,169 downregulated (Figure 1A, Supplementary Table 6). There were 702 probe sets (representing 409 genes) differentially expressed by both females and males at 5hours (Figure 1A, Supplementary Table 7).
The functional analysis results of the male- and female-specific genes are listed in Supplementary Table 8. Notably, the female-specific IS genes involved in calcium-induced T-lymphocyte apoptosis processes were all downregulated (Supplementary Figure 1).
There were 338 male-specific IS probe sets (representing 230 genes) differentially expressed at 24hours after IS (FDR <0.05 and a FC>1.5), with 200 probe sets upregulated and 138 downregulated (Figure 1A, Supplementary Table 9). There were 571 female-specific IS probe sets (representing 405 genes) differentially regulated at 24hours (FDR <0.05 and a FC>1.5), with 378 probe sets upregulated and 193 downregulated (Figure 1A, Supplementary Table 10). There were 426 probe sets (representing 272 genes) that were differentially expressed by both females and males that are not discussed here (Figure 1A, Supplementary Table 11).
Notably, the top network associated with the female-specific genes regulated at 24hours was associated with inflammatory responses and apoptosis (Figure 2). Several key genes were upregulated in females after IS including interferon-, NAIP (neuronal apoptosis-inhibitory protein) delta (NLR family, apoptosis-inhibitory protein), CASP1 (caspase 1, apoptosis-related cysteine peptidase), and APAF1 (involved in formation of the apoptosome) (Figure 2).
Additional functional analysis results of the female- and male-specific genes are shown in Supplementary Table 12. Actin cytoskeleton assembly, actin filament, cell motion, and tight junction processes were associated with male-specific IS genes. Immune and inflammatory processes differed between the genders. B-cell receptors, CD27 (a TNF receptor) signaling in lymphocytes, and lymphotoxin β-receptor signaling were associated with male-specific IS genes. T-helper cell differentiation, cytotoxic T lymphocyte-mediated apoptosis, and altered T- and B-cell signaling in rheumatoid arthritis were associated with female-specific genes. Cell apoptosis and cell death processes were overrepresented in both male- and female- specific IS genes, but were associated with different pathways (Supplementary Table 12).
Of the 242, 227, and 338 male-specific IS probe sets differentially regulated at 3, 5, 24hours after IS, respectively, 59 probe sets (representing 44 genes) were common to all 3 time points (FDR <0.05 and a FC>1.5) (Figures 1B and and3A;3A; Supplementary Table 13). In all, 18 of the 59 were upregulated and 41 were downregulated. Of the 774, 3,437, and 571 female-specific IS probe sets differentially regulated at 3, 5, and 24hours, respectively, there were 152 (representing 119 genes) common to all 3 time points (FDR <0.05 and a FC>1.5) (Figures 1C and and3B;3B; Supplementary Table 14). In all, 95 of the 152 probe sets were upregulated and 57 were downregulated at all 3 time points.
The functional analyses for these female- and male-specific genes that were differentially expressed at all three time points after IS are shown in Tables 3A and and3B.3B. Many of the canonical pathways and biologic processes are similar to those observed at the 3-hour time point (Table 2). Pathways for males for all three time points included Wnt/β-catenin signaling, actin cytoskeleton signaling, and RhoA signaling (Table 3A). Pathways for females included sphingolipid metabolism and Toll-like receptor signaling in addition to those seen at 3hours (Tables 2B and and3B).3B). It must be noted that apoptosis and cell death are common to male and female biologic processes for genes differentially expressed at all three times after IS (Table 3A and and3B3B).
Of the 211, 702, and 426 probe sets differentially expressed in both males and females at 3, 5, and 24hours after IS (Figure 1A), 107 probe sets (representing 71 genes) were common to both genders at all 3 time points (FDR <0.05 and a FC>1.5) (Supplementary Figure 2, Supplementary Table 15). In all, 97 of these 107 probe sets were upregulated, whereas only 10 were downregulated. These genes represent those whose expressions change after IS and are independent of gender and the time after IS. It must be noted that FCs in females are generally greater than those in males. Over half of these genes overlapped our previously reported IS genes (Stamova et al, 2010). Thus, the current common IS genes, especially the 107 probe sets common to both genders at all three time points (Supplementary Table 15), could be potentially used for IS predictors within 24hours after stroke onset.
Supplementary Table 4 lists the chromosomes on which there was an overrepresentation of genes from each of the above gene lists. None of the gene lists were found to have overrepresentation on the X or Y chromosomes. This may be accounted for in part by the very strict statistical cutoffs used for analyses in this study.
This is the first study to show gender-associated changes of gene expression in whole blood of humans after IS at the whole-genome level. There were a number of changes of gene expression in both genders after IS, some of which are gender specific and some of which are common to both genders. Although this study cannot answer whether these changes represent a cause or an effect of the stroke, they support gender-specific changes of gene expression that may reflect differences in the immune system, inflammatory responses, and cell death for the different genders after IS.
Blood samples for this study were obtained before treatment and after treatment for stroke. Thus, the discussion below will focus primarily on genes and pathways induced before treatment (3hours) to avoid the possible effects of treatment on the interpretation of results, with the most reliable genes being expressed at all three times points. Moreover, genes and pathways induced within the 3-hour window after acute IS likely represent immune responses that could serve as targets for acute treatment and for understanding acute responses to brain injury (Tang et al, 2006). Genes and pathways induced at 5 and 24hours could be attributed to treatment, or some interaction between gender and treatment, both of which would be interesting for future studies. Some genes related to cell death at 24hours in females are discussed because of their relevance and similarity to recent animal studies (Siegel et al, 2010).
Overall, there were more female-specific genes regulated compared with male-specific genes at each time point after IS. The explanation for this is unclear, but is certainly one indicator of the gender-dependent differences of the immune response to IS. One or several epigenetic mechanisms might lead to the observed differences including DNA methylation or histone deacetylation (both of which suppress gene expression), DNA sumoylation, and microRNAs that generally downregulate expression of their many targets (Dharap et al, 2009; Liu et al, 2010). Future studies will be required to determine whether there are gender-specific modifications of DNA methylation, histone deacetylation, and/or microRNA responses after stroke.
There were similar numbers of genes regulated at all time points in males after IS, whereas there were many more genes regulated at 5hours compared with 3 and 24hours in females. Notably, most of the female-specific IS genes at 5hours were downregulated compared with controls, whereas most of the female-specific IS genes at 3 and 24hours were upregulated. This could indicate a unique female-specific response to stroke by 5hours after IS. Alternatively, this could mean females and males respond quite differently to the treatment with recombinant-tissue plasminogen activator±eptifibatide that was administered by 3hours after stroke. Changes of gene expression have been described in blood owing to recombinant-tissue plasminogen activator in our rodent stroke model, although these studies did not determine whether there were gender-specific differences (Jickling et al, 2010b).
A surprising result was the overrepresentation of hormone-related pathways and regulation of selected genes potentially related to estrogen in the blood of women after IS (Table 2B, Supplementary Tables 2 and 6). One such gene was an estrogen receptor, G protein-coupled estrogen receptor 1 (GPER) (also called GPR30), which was upregulated in females at 3 and 5hours after IS and not in males. G protein-coupled estrogen receptor, originally an orphan G protein-coupled receptor, is expressed on the cell surface of leukocytes, endothelial cells, and smooth muscle cells (Feldman and Gros, 2011). G protein-coupled estrogen receptor signals through phosphoinositide kinase-3 and protein kinase A to modulate ERK (extracellular signal-regulated kinase), which mediates ERK-dependent apoptosis of smooth muscles cells, which is balanced with antiapoptotic classical estrogen receptors (namely ERα, ERβ). However, there is also rapid signaling that mediates vasodilation and decreases blood pressure through effects on vascular smooth muscle possibly mediated by myosin light-chain phosphorylation (Feldman and Gros, 2011). Aldosterone also activates GPER. Although the role of GPER in leukocytes after stroke is uncertain, it presumably signals estrogen- and aldosterone-mediated influences that induce rapid intracellular G protein-mediated events specific to females.
Another estrogen/hormone-related gene upregulated in females was STAT5B (also know as STAT5). Glucocorticoids act on GCR-α (glucocorticoid receptor-α) to activate signal transducer and activator of transcription (STAT)5 and 17β--estradiol phosphorylates the STAT5 transcription factor, which then binds to the promoters of its downstream target genes and activates them (Xu et al, 2009). Here, STAT5 is also activated by platelet-derived growth factor (PDGF) receptors, Erk, JAK1/2/3, c-src, Lyn, Tyk2 (IL12/IL23 related), c-MPL (thrombopoietin receptor), growth hormone, Epo receptor through CrkL, p300, CBP, IL2 receptor through Syk, and by angiopoietin 1 through the TIE2 receptor. The STAT5 downstream genes include many genes involved in apoptosis, cell proliferation, cell cycle, and cell survival (Heltemes-Harris et al, 2011).
The mechanisms for the upregulation of the above hormone-related genes in females is unclear because almost all of the women with IS except two in this study were postmenopausal and thus their estrogen levels should be low (Masood et al, 2010). Perhaps the most likely explanation is that the above genes were activated by upstream signals other than estrogen, particularly STAT5, which is activated by a large number of molecules. However, because administration of estrogens in postmenopausal women in the HERS (Heart and Estrogen/progestin Replacement Study) and WHI (Women's Health Initiative) studies suggest an increase in cardiovascular disease (Masood et al, 2010), this implies that estrogen signaling mechanisms are still in place in spite of the decrease in estrogen levels after menopause. Indeed, decreases of an agonist virtually always lead to upregulation of the receptors for the agonist and sensitization of the associated downstream pathways. Thus, if stroke was to alter estrogen levels, even small amounts in women, this might affect some of these pathways. Further study is required to determine whether stroke itself changes circulating estrogen, estrogen metabolites, or other molecules in postmenopausal women that activate estrogen-related molecules and pathways discussed above.
Although cell death and apoptosis occur after IS in both genders, the mechanisms of injury could be different. Previous studies have shown that ischemic cell death pathways are different in the male and female brains, females often showing caspase-mediated cell death of individual neurons, whereas males are more sensitive to caspase-independent cell death (Siegel et al, 2010).
Indeed, in this study of blood, several of the major caspase-mediated pathways were evident in the peripheral leukocytes of females. Caspase 1, APAF1, and NAIP delta were overexpressed in females at one day. Caspase 1 cleaves cytokines interleukin 1β and IL18 into active mature peptides, and is involved in the formation of the inflammasome and activation of inflammation. Caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby functioning as an initiator for activation of caspase 3. APAF1 is an integral part of the apoptosome, which activates/cleaves caspase 3, which is one of the main executioner caspases in the intrinsic apoptotic pathways. The NAIP, which is also induced in females, is an inhibitor of procaspase 9 and thus modulates formation of the apoptosome.
There are also female-associated necrosis cell death-related pathways. For example, HMGB1 signaling was prominent in the pathways activated in females (Tables 2B and and3B).3B). High-mobility group box-1 signaling enhances angiogenesis and restores cardiac function and can stimulate formation of regeneration of cardiomyocytes. It can also stimulate ischemia-induced angiogenesis and enhance collateral blood flow in ischemic hind limbs of diabetic mice through a vascular endothelial growth factor-dependent manner (Biscetti et al, 2011).
The p53 signaling pathway is also represented in genes induced in females after IS (Tables 2B and and3B).3B). P53 functions are complex including activating DNA repair proteins (survival), inducing growth arrest to allow for DNA repair, and initiating apoptosis if DNA damage is excessive. The p53-regulated gene AKT3 mediates vascular endothelial growth factor stimulation of mitochondrial biogenesis and can prevent certain types of apoptosis (Shao and Aplin, 2010). It is noteworthy that GADD45 (a p53 downstream gene) and GSK3B (interacts with p53), were upregulated in females. GADD45, which detects DNA damage, links nuclear factor-κB to mitogen-activated protein kinase cascades (Yang et al, 2009). GSK3B links WNT (also in males) and FGF signaling (also in males) to regulate β-catenin and Snail signaling.
Nuclear factor-κB signaling was also prominent in females after IS (Tables 2B and and3B),3B), which usually promotes cell survival but can also promote death. In blood, both nuclear factor-κB and p38-mitogen-activated protein kinase regulate the neutrophil apoptotic program (Dyugovskaya and Polyakov, 2010). Inhibitors of apoptosis proteins not only inhibit caspases but also act as ubiquitin-E3 ligases (the RING domain of the inhibitors of apoptosis) regulating nuclear factor-κB signaling and cell survival (Lopez and Meier, 2010).
Granzyme A signaling is associated with male cell death pathways—including the perforin 1 and HMGB2 genes. The perforin protein, which is found within the granules of all cytotoxic T cells and NK cells, is a pore-forming protein that permits entry of granzymes into targeted cells (Lieberman, 2010). As perforin is induced, and HMGB2 is a granzyme A target gene, this suggests activation of granzyme A pathways in males.
Granzyme A signaling induces caspase-independent cell death that is morphologically indistinguishable from apoptosis (Lieberman, 2010). Granzyme A, localized to cytotoxic T cells, cleaves a component of the electron transport chain complex I (NDUF33), resulting in overproduction of superoxide. Granzyme A and superoxide drive the endoplasmic reticulum-associated complex SET into the nucleus where it activates the NM23-H1 endonuclease to produce DNA nicks. At the same time, granzyme A cleaves and inactivates HMGB2 and Ape 1 to interfere with BER (base excision repair pathway for DNA). Granzyme A also interferes with recognition of damaged DNA by cleaving Ku70 and PARP-1, which is a key gene in caspase-independent cell death pathways (Lieberman, 2010).
Other genes associated with cell death and apoptosis in males are shown in Tables 2A and and3A.3A. The mechanism and effects of upregulation of α-synuclein (SNCA) in leukocytes in males after stroke is not known, but SNCA is implicated in cell death in Parkinson's disease. Death-associated protein-related apoptotic kinase-2 (DRAK2) (STK17B), a member of the death-associated protein-like family of serine/threonine kinases, is highly expressed in lymphoid organs and is a negative regulator of T-cell activation and as a p53 target gene induces apoptosis. SERPINB2 (plasminogen activator inhibitor type-2) alters gene expression, influences the rate of cell proliferation and differentiation, and can inhibit apoptosis. Adenomatous polyposis coli, also known as deleted in polyposis 2.5, is part of the Wnt signaling pathway, forms a complex with glycogen synthase kinase 3-β (B) and axin which controls β-catenin (Wnt signaling pathway), prevents genes that stimulate cell division from being turned on too often, and prevents cell overproliferation. The pathway can promote apoptosis or promote cell survival and proliferation depending on the cellular context.
The relationship of these cell death pathways in leukocytes to the ischemic brain of males and females cannot be answered from this study. It seems likely that a number of genes expressed in immune cells in blood would be similar to the genes expressed in immune cells in the brain (microglia and perhaps astrocytes). Thus, these male–female differences in the peripheral immune response may mirror, at least to some degree, the responses in the brain. This would imply that apoptotic, necrotic, and other mechanisms of cell death occur in both females and males, but that the precise molecules and pathways differ to some extent in blood and possibly in the brain.
A number of pathways activated in males after stroke were associated with the cytoskeleton, cell adhesion, and potential interactions of leukocytes with the blood–brain barrier (BBB). The functional pathways included integrin signaling, ILK signaling, tight junction signaling, actin cytoskeleton signaling, and RhoA signaling (Tables 2A and and3A).3A). Notably, TLN1 (talin 1, a cytoskeletal protein-encoded gene) and MYH10 (myosin, heavy chain 10, nonmuscle), which have significant roles in the assembly of actin filaments and in cell adhesion (TLN1) and actin binding (MYH10), were upregulated in males after IS. This is consistent with reported gender differences in the regulation of the actin cytoskeleton network, which results in gender differences of cell motility (Giretti and Simoncini, 2008). Integrins are cell surface molecules that link the cell to the extracellular matrix. Integrin-linked kinase signaling has a key role in signaling from the cell to structurally modify the basal lamina and extracellular matrix (Huang et al, 2006). The ILKs downstream pathways modulate the actions of matrix metalloproteinase (MMPs), target leukocytes to specific tissues and entry across the BBB, are activated by oxidative stress, regulate endothelial cells survival, and control vasculogenesis by recruitment of endothelial cells to the ischemic tissue Thus, these pathways are associated with leukocyte adhesion, infiltration, and breakdown of the BBB after stroke (Huang et al, 2006). These male-specific increases in integrins, ILKs, and cell adhesion molecules in human stroke may relate to reports of markedly increased leukocyte adhesion and subendothelial migration in atherosclerotic lesions of male compared with female rabbits (Nathan et al, 1999). The functional consequences of these gender-specific differences of integrin, ILK, and cytoskeletal and BBB signaling are not known in so far as human stroke pathophysiology.
Females, in contrast to males, showed a preponderance of immune and inflammatory pathways. The canonical pathways included IL6, IL12, Toll-like receptor, T-helper cell differentiation, role of macrophages in rheumatoid arthritis, and HMGB1 signaling (Tables 2B and and3B).3B). The GO biologic processes included defense response, inflammatory and immune responses, innate and adaptive and acute inflammatory responses, leukocyte differentiation, and regulation of T-cell activation (Tables 2B and and3B3B).
The IL6 signaling pathway stimulates the immune response through STAT3, JAK1, ERK1/2, and other molecules. The IL12 cytokine signals through Tyk2, STAT3, and STAT4 to induce interferon-γ which promotes Th1 cell differentiation. Interleukin-12 signaling through STAT4 also promotes natural killer cell toxicity (through perforin), T-cell proliferation (through IL2Ra), and cell adhesion (P-selectin). Interleukin-18 also signals through STAT4 to activate similar pathways. Plasma IL18 levels are increased in IS patients (Yuen et al, 2007), although no gender differences are reported.
The innate immune response pathways were overrepresented in females including many cytokine and complement genes (Tables 2B and and3B).3B). Of those, antagonists of IL1 are in clinical trial for stroke. Complement receptor 1, CD35 is a membrane receptor for C3b and C4b expressed on leukocytes and has an important role in the removal of immune complexes and cells coated with C3b and C4b, part of the B cell-mediated antibody killing response pathway. PGLYRP1 is one of a family of secreted proteins expressed in polymorphonuclear leukocytes (PGLYRP1), that recognize bacterial peptidoglycan and likely damaged host antigens including the ischemic brain, and mediate proinflammatory/cell killing responses by neutrophils (Dziarski and Gupta, 2010).
One of the other inflammatory/immune molecules associated with females after IS is ALOX5 (lipoxygenase 5). This enzyme catalyzes two steps in the biosynthesis of leukotrienes, lipid mediators of inflammation derived from arachidonic acid which function in normal host defense, and are implicated in causing atherosclerosis (Radmark and Samuelsson, 2010). Notably, genetic variants of ALOX5AP (ALOX5-activating protein) are involved in the pathogenesis of stroke by increasing leukotriene production and inflammation. Another is CXCL1, a chemokine released by neutrophils, which acts on vascular endothelium causing BBB dysfunction and increased permeability (DiStasi and Ley, 2009).
This is a preliminary discovery-type study. Our matching for demographic variables including age, race, and vascular risk factors showed no statistical differences of the parameters assessed between the groups. Nevertheless, there may be baseline differences between control males and control females, or other modest demographic differences between groups that could have affected the results particularly because of the relatively small sample size. Thus, the results will need to be confirmed in a future independent study with larger sample size.
As patients were treated after the 3-hour blood draw, some of the sexually dimorphic IS genes observed at 5 and 24hours may be related to treatment. Indeed, treatment may have masked some changes of gene expression at these time points. Thus, the discussion generally focused on those genes and pathways induced before treatment. It is important to emphasize that the expression of any given gene should be viewed with caution; and because many genes support the role of a pathway, the gender-specific pathways are likely to be more reliable and more likely to be replicated. The only accepted way to account for the multiple comparisons and false positives in a whole-genome study like this one is to replicate a given gene or pathway in a separate future study with an independent cohort of subjects.
Dr Glen Jickling is a fellow of the Canadian Institutes of Health Research (CIHR). A detailed listing of all references for all statements made in the discussion can be provided upon request as the total number of references was limited by the journal.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)
This study was supported by NS056302 (FRS), PO21040N635110 (JPB), and KO2 NS058760 (CB) from NIH/NINDS and the American Heart Association Bugher Foundation (FRS).