MMP2 and MMP9 Activity Is Crucial for Adult Visual Cortex Plasticity in Healthy and Stroke-Affected Mice.

A fundamental regulator of neuronal network development and plasticity is the extracellular matrix (ECM) of the brain. The ECM provides a scaffold stabilizing synaptic circuits, while the proteolytic cleavage of its components and cell surface proteins are thought to have permissive roles in the regulation of plasticity. The enzymatic proteolysis is thought to be crucial for homeostasis between stability and reorganizational plasticity and facilitated largely by a family of proteinases named matrix metalloproteinases (MMPs). Here, we investigated whether MMP2 and MMP9 play a role in mediating adult primary visual cortex (V1) plasticity as well as stroke-induced impairments of visual cortex plasticity in mice. In healthy adult mice, selective inhibition of MMP2/9 for 7 d suppressed ocular dominance plasticity. In contrast, brief inhibition of MMP2/9 after a cortical stroke rescued compromised plasticity. Our data indicate that the proteolytic activity of MMP2 and MMP9 is critical and required to be within a narrow range to allow adult visual plasticity.SIGNIFICANCE STATEMENT Learning and recovery from injuries depend on the plasticity of neuronal connections. The brain's extracellular matrix (ECM) provides a scaffold for stabilizing synaptic circuits, while its enzymatic proteolysis is hypothesized to regulate homeostasis between stability and reorganizational plasticity. ECM digestion is facilitated by a family of matrix metalloproteinases (MMPs). Here, we show that treatments that inhibit MMP2/9 can either inhibit or rescue cortical plasticity depending on cortical state: in the visual cortex of healthy adult mice, inhibition of MMP2/9 suppressed cortical plasticity. In contrast, brief inhibition of MMP2/9 after a stroke rescued compromised plasticity. Our data provide strong evidence that an optimal level of MMP2/9 proteolytic activity is crucial for adult visual plasticity.

Inactivation of mouse transmembrane prolyl 4-hydroxylase increases blood brain barrier permeability and ischemia-induced cerebral neuroinflammation.

Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm-/- mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm-/- cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm-/- mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.

Does Src Kinase Mediated Vasoconstriction Impair Penumbral Reperfusion?

Despite successful recanalization, a significant number of patients with ischemic stroke experience impaired local brain tissue reperfusion with adverse clinical outcome. The cause and mechanism of this multifactorial complication are yet to be understood. At the current moment, major attention is given to dysfunction in blood-brain barrier and capillary blood flow but contribution of exaggerated constriction of cerebral arterioles has also been suggested. In the brain, arterioles significantly contribute to vascular resistance and thus control of perfusion. Accordingly, pathological changes in arteriolar wall function can, therefore, limit sufficient reperfusion in ischemic stroke, but this has not yet received sufficient attention. Although an increased vascular tone after reperfusion has been demonstrated in several studies, the mechanism behind it remains to be characterized. Importantly, the majority of conventional mechanisms controlling vascular contraction failed to explain elevated cerebrovascular tone after reperfusion. We propose here that the Na,K-ATPase-dependent Src kinase activation are the key mechanisms responsible for elevation of cerebrovascular tone after reperfusion. The Na,K-ATPase, which is essential to control intracellular ion homeostasis, also executes numerous signaling functions. Under hypoxic conditions, the Na,K-ATPase is endocytosed from the membrane of vascular smooth muscle cells. This initiates the Src kinase signaling pathway that sensitizes the contractile machinery to intracellular Ca2+ resulting in hypercontractility of vascular smooth muscle cells and, thus, elevated cerebrovascular tone that can contribute to impaired reperfusion after stroke. This mechanism integrates with cerebral edema that was suggested to underlie impaired reperfusion and is further supported by several studies, which are discussed in this article. However, final demonstration of the molecular mechanism behind Src kinase-associated arteriolar hypercontractility in stroke remains to be done.

Cyclin-Dependent Kinases (CDK) and Their Role in Diseases Development-Review.

Cyclin-dependent kinases (CDKs) are involved in many crucial processes, such as cell cycle and transcription, as well as communication, metabolism, and apoptosis. The kinases are organized in a pathway to ensure that, during cell division, each cell accurately replicates its DNA, and ensure its segregation equally between the two daughter cells. Deregulation of any of the stages of the cell cycle or transcription leads to apoptosis but, if uncorrected, can result in a series of diseases, such as cancer, neurodegenerative diseases (Alzheimer's or Parkinson's disease), and stroke. This review presents the current state of knowledge about the characteristics of cyclin-dependent kinases as potential pharmacological targets.

Efficacy of Clopidogrel-Aspirin Therapy for Stroke Does Not Exist in CYP2C19 Loss-of-Function Allele Noncarriers With Overweight/Obesity.

Background and Purpose- The role of dual-antiplatelet therapy with clopidogrel plus aspirin has been demonstrated to substantially decrease the risk of recurrent stroke among patients with minor stroke and transient ischemic attack. We aimed to determine whether the efficacy of clopidogrel-aspirin therapy among patients with minor stroke / transient ischemic attack was influenced by the stratification of CYP2C19 genotype and body mass index (BMI). Methods- CYP2C19 loss-of-function allele (LoFA) carriers were defined as patients with either LoFA of *2 or *3. Low/normal weight and overweight/obesity was defined as BMI <25 and ≥25 kg/m2, respectively. Primary outcome was defined as stroke recurrence at 3 months. Results- In a total of 2933 patients, there were 1726 (58.8%) LoFA carriers and 1275 (43.5%) patients with overweight/obesity (BMI ≥25 kg/m2). Stratified analyses by LoFA carrying status and BMI, hazard ratios (hazard ratios 95% CIs) of the clopidogrel-aspirin therapy for stroke recurrence were 0.90 (0.60-1.36), 0.87 (0.56-1.35), 0.65 (0.39-1.09), and 0.40 (0.22-0.71) among subgroups of LoFA carriers with overweight/obesity, LoFA carriers with low/normal weight, LoFA noncarriers with overweight/obesity, and LoFA noncarriers with low/normal weight, respectively, with P=0.049 for interaction. Conclusions- Efficacy of clopidogrel-aspirin therapy in reducing the risk of stroke recurrence is not present in CYP2C19 LoFA noncarriers with overweight/obesity. Our study suggests that BMI significantly influences the correlation between CYP2C19 genotype and efficacy of clopidogrel-aspirin therapy. Clinical Trial Registration- URL: https://www.clinicaltrials.gov. Unique identifier: NCT00979589.

Transforming Growth Factor Beta-Activated Kinase 1-Dependent Microglial and Macrophage Responses Aggravate Long-Term Outcomes After Ischemic Stroke.

Background and Purpose- Microglia/macrophages (Mi/MΦ) can profoundly influence stroke outcomes by acquiring functionally dominant phenotypes (proinflammatory or anti-inflammatory; deleterious or salutary). Identification of the molecular mechanisms that dictate the functional status of Mi/MΦ after brain ischemia/reperfusion may reveal novel therapeutic targets for stroke. We hypothesized that activation of TAK1 (transforming growth factor beta-activated kinase 1), a key MAP3K upstream of multiple inflammation-regulating pathways, drives Mi/MΦ toward a proinflammatory phenotype and potentiates ischemia/reperfusion brain injury. Methods- Young adult mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by reperfusion. TAK1 was targeted by tamoxifen-induced Mi/MΦ-specific knockout or administration of a selective inhibitor 5Z-7-Oxozeaenol after MCAO. Neurobehavioral deficits and long-term gray matter and white matter injury were assessed up to 35 days after MCAO. Mi/MΦ functional status and brain inflammatory profiles were assessed 3 days after MCAO by RNA-seq, flow cytometry, and immunohistochemistry. Results- TAK1 Mi/MΦ-specific knockout markedly ameliorated neurological deficits in the rotarod and cylinder tests for at least 35 days after MCAO. Mechanistically, RNA-seq of purified brain Mi/MΦ demonstrated that proinflammatory genes and their predicted biological functions were downregulated or inhibited in microglia and macrophages from TAK1 Mi/MΦ-specific knockout mice versus WT mice 3 days after MCAO. Consistent with the anti-inflammatory phenotype of Mi/MΦ-specific knockout, oxozeaenol treatment mitigated neuroinflammation 3 days after MCAO, manifested by less Iba1+/CD16+ proinflammatory Mi/MΦ and suppressed brain invasion of various peripheral immune cells. Oxozeaenol treatment beginning 2 hours after MCAO improved long-term sensorimotor and cognitive functions in the foot fault, rotarod, and water maze tests. Furthermore, Oxozeaenol promoted both gray matter and white matter integrity 35 days after MCAO. Conclusions- TAK1 promotes ischemia/reperfusion-induced inflammation, brain injury, and maladaptive behavior by enhancing proinflammatory and deleterious Mi/MΦ responses. Therefore, TAK1 inhibition is a promising therapy to improve long-term stroke outcomes.

HDAC2 (Histone deacetylase 2): A critical factor in environmental enrichment-mediated stroke recovery.

Environmental enrichment (EE) is a generally accepted strategy to promote stroke recovery and its beneficial effect is positively correlated with neuroplasticity. However, the mechanisms underlying it remain elusive. Histone deacetylase 2 (HDAC2), a negative regulator of neuroplasticity, is up-regulated after stroke. Thus, we hypothesized that HDAC2 may participate in EE-mediated stroke recovery. In this study, focal stroke was induced by photothrombosis in male mice exposing to EE or standard housing (SH) conditions. Recombinant virus vectors, including Ad-HDAC2-Flag, AAV-CAG-EGFP-Cre, LV-shHDAC2, or their controls were microinjected into the motor cortex at 3 days before stroke. Grid-walking and cylinder tasks were conducted to assess motor function. Western blot and immunostaining were used to uncover the mechanisms underlying EE-mediated stroke recovery. We found that EE exposure reversed stroke-induced HDAC2 up-regulation, implicating HDAC2 in EE-mediated functional recovery. Importantly, EE-dependent stroke recovery was counteracted by over-expressing HDAC2, and HDAC2 knockdown promoted functional recovery from stroke to the similar extent as EE exposure. Moreover, the knockdown of HDAC2 epigenetically enhanced expressions of neurotrophins and neuroplasticity-related proteins, with similar effects as EE, and consequently, whole brain and corticospinal tract (CST) rewiring. Together, our findings indicate that HDAC2 is critical for EE-dependent functional restoration. Precisely targeting HDAC2 may mimic EE and serve as a novel therapeutic strategy for stroke recovery.

Genetic Variations of CYP19A1 Gene and Stroke Susceptibility: A Case-Control Study in the Chinese Han Population.

OBJECTIVE: This study aimed to explore the association between genetic variations of CYP19A1 and stroke susceptibility in the Chinese Han population. METHODS: A total of 477 stroke patients and 480 healthy controls were recruited in this study. The genotyping of CYP19A1 polymorphisms (rs4646, rs6493487, rs1062033, rs17601876, and rs3751599) was performed by the Agena MassARRAY platform. Under logistic regression models, we evaluated the associations of CYP19A1 polymorphisms and stroke susceptibility by odds ratio and 95% confidence interval. RESULTS: Our study showed that rs4646 (codominant: P = 0.020; recessive: P = 0.016) and rs17601876 (allele: P = 0.044; codominant: P = 0.011; dominant: P = 0.009; recessive: P = 0.046) significantly decreased the risk of stroke. In the stratification analysis, rs4646 is associated with decreased stroke risk among the individuals older than 64 years (codominant: P = 0.028; recessive: P = 0.010) and women (codominant: P = 0.029; recessive: P = 0.029), whereas rs1062033 increased stroke risk in the subgroup of age 64 years and younger (recessive: P = 0.042). The rs17601876 polymorphism has a strong relationship with stroke susceptibility, which is age and gender dependent. In haplotype analysis, we found a block (rs17601876 and rs3751599), and Ars17601876Grs3751599 haplotype is related to an increased stroke risk (P < 0.05). In addition, CYP19A1 variations had effects on clinical characteristics. CONCLUSION: CYP19A1 polymorphisms were significantly associated with stroke susceptibility in the Chinese Han population.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Pharmacodynamic assessment of prasugrel and clopidogrel in patients with non-cardioembolic stroke: a multicenter, randomized, active-control clinical trial.

Prasugrel, a novel P2Y12 receptor antagonist, has been shown to be more effective than clopidogrel for preventing cardiovascular events in patients with acute coronary syndromes undergoing percutaneous coronary intervention. We investigated the dose-response antiplatelet effects of prasugrel compared with clopidogrel in Japanese patients with non-cardioembolic stroke. The influence of cytochrome P450 (CYP) polymorphisms on the antiplatelet effects of both drugs was also compared. In this multicenter randomized active-control comparative study, patients were randomized to receive prasugrel 2.5 mg, 5 mg, or 7.5 mg (double blind) or clopidogrel 75 mg (open label) once daily for 14 days. The primary endpoint was inhibition of platelet aggregation (IPA) in response to adenosine diphosphate 20 µM within 8 h of study drug administration on day 14. Of the 66 patients randomized, data from 63 (prasugrel 2.5 mg, 5 mg, and 7.5 mg groups, n = 14, 16, and 18, respectively; clopidogrel group, n = 15) were used in the pharmacodynamic assessment. IPA (arithmetic mean ± SD) after prasugrel administration increased dose-dependently (33 ± 9%, 44 ± 11%, and 53 ± 14%, at 2.5 mg, 5 mg, and 7.5 mg, respectively) and was higher in these groups than after clopidogrel (23 ± 16%). In a subgroup of CYP2C19 intermediate metabolizers, IPA was higher in the prasugrel 5 mg and 7.5 mg groups than in the clopidogrel group. No death or serious adverse events were reported. Prasugrel was well tolerated at doses up to 7.5 mg/day and had antiplatelet effects higher than those of clopidogrel 75 mg/day. CYP2C19 polymorphisms may have reduced clopidogrel-induced IPA.

Localization and Expression of Sirtuins 1, 2, 6 and Plasticity-Related Proteins in the Recovery Period after a Photothrombotic Stroke in Mice.

Sirtuins, class III histone deacetylases, are involved in the regulation of tissue repair processes and brain functions after a stroke. The ability of some isoforms of sirtuins to circulate between the nucleus and cytoplasm may have various pathophysiological effects on the cells. In present work, we focused on the role of non-mitochondrial sirtuins SIRT1, SIRT2, and SIRT6 in the restoration of brain cells following ischemic stroke. Here, using a photothrombotic stroke (PTS) model in mice, we studied whether local stroke affects the level and intracellular localization of SIRT1, SIRT2, and SIRT6 in neurons and astrocytes of the intact cerebral cortex adjacent to the ischemic ipsilateral hemisphere and in the analogous region of the contralateral hemisphere at different time points during the recovery period after a stroke. We evaluated the co-localization of sirtuins with growth-associated protein-43 (GAP-43), the presynaptic marker synaptophysin (SYN) and acetylated α-tubulin (Ac-α-Tub), that are associated with brain plasticity and are known to be involved in brain repair after a stroke. The results show that during the recovery period, an increase in SIRT1 and SIRT2 levels occurred. The increase of SIRT1 level was associated with an increase in synaptic plasticity proteins, whereas the increase of SIRT2 level was associated with an acetylated of α-tubulin, that can reduce the mobility of neurites. SIRT6 co-localized with GAP-43, but not with SYN. Moreover, we showed that SIRT1, SIRT2, and SIRT6 are not involved in the PTS-induced apoptosis of penumbra cells. Taken together, our results suggest that sirtuins functions differ depending on cell type, intracellular localization, specificity of sirtuins isoforms to different substrates and nature of post-translational modifications of enzymes.

CXCL8 gene silencing promotes neuroglial cells activation while inhibiting neuroinflammation through the PI3K/Akt/NF-κB-signaling pathway in mice with ischemic stroke.

OBJECTIVE: Ischemic stroke is known as a neurodegenerative disorder, which induces long-period tissue damage. Chemokine (C-X-C motif) ligand 8 (CXCL8) is involved in acute inflammation and tumor progression through the phosphoinositide-3-kinase/protein kinase B/nuclear factor-κB (PI3K/Akt/NF-κB)-signaling pathway. In this study, we aimed to explore the mechanism of CXCL8 in ischemic stroke in relation to the PI3K/Akt/NF-κB-signaling pathway. METHODS: Microarray-based gene expression profiling of peripheral blood mononuclear cells was used to identify ischemic stroke-related differentially expressed genes and explore role of CXCL8 in ischemic stroke. Next, the ischemic mice model was successfully established, with transfection efficiency detected. After that, deflection index, recovery of nervous system, infarct sizes, ischemia-induced apoptosis, and neuroinflammatory response in ischemic stroke were measured. At last, the content of inflammatory factors as well as the expression of CXCL8, caspase-3, caspase-9, Bad, interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), Akt, PI3K, and NF-κB were determined. RESULTS: Comprehensive gene expression profiling analysis identified that CXCL8 might affect the development of ischemic stroke through regulating the PI3K/Akt/NF-κB-signaling pathway. CXCL8 silencing significantly reduced deflection index and infarct size, improved neurological function, and suppressed neuroglial cell loss and apoptosis index. In addition, glial fibrillary acidic portein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA-1) expressions were decreased following CXCL8 suppression, suggesting CXCL8 affected neuroglial activation. Importantly, we also found that CXCL8 silencing activated neuroglial cell and suppressed inflammatory cytokine production in ischemic stroke mice. CONCLUSION: Taken together, these findings highlight that functional suppression of CXCL8 promotes neuroglial activation and inhibits neuroinflammation by regulating the PI3K/Akt/NF-κB-signaling pathway in mice with ischemic stroke, which might provide new insight for ischemic stroke treatment.

Phenotypic Consequences of a Genetic Predisposition to Enhanced Nitric Oxide Signaling.

BACKGROUND: Nitric oxide signaling plays a key role in the regulation of vascular tone and platelet activation. Here, we seek to understand the impact of a genetic predisposition to enhanced nitric oxide signaling on risk for cardiovascular diseases, thus informing the potential utility of pharmacological stimulation of the nitric oxide pathway as a therapeutic strategy. METHODS: We analyzed the association of common and rare genetic variants in 2 genes that mediate nitric oxide signaling (Nitric Oxide Synthase 3 [NOS3] and Guanylate Cyclase 1, Soluble, Alpha 3 [GUCY1A3]) with a range of human phenotypes. We selected 2 common variants (rs3918226 in NOS3 and rs7692387 in GUCY1A3) known to associate with increased NOS3 and GUCY1A3 expression and reduced mean arterial pressure, combined them into a genetic score, and standardized this exposure to a 5 mm Hg reduction in mean arterial pressure. Using individual-level data from 335 464 participants in the UK Biobank and summary association results from 7 large-scale genome-wide association studies, we examined the effect of this nitric oxide signaling score on cardiometabolic and other diseases. We also examined whether rare loss-of-function mutations in NOS3 and GUCY1A3 were associated with coronary heart disease using gene sequencing data from the Myocardial Infarction Genetics Consortium (n=27 815). RESULTS: A genetic predisposition to enhanced nitric oxide signaling was associated with reduced risks of coronary heart disease (odds ratio, 0.37; 95% confidence interval [CI], 0.31-0.45; P=5.5*10-26], peripheral arterial disease (odds ratio 0.42; 95% CI, 0.26-0.68; P=0.0005), and stroke (odds ratio, 0.53; 95% CI, 0.37-0.76; P=0.0006). In a mediation analysis, the effect of the genetic score on decreased coronary heart disease risk extended beyond its effect on blood pressure. Conversely, rare variants that inactivate the NOS3 or GUCY1A3 genes were associated with a 23 mm Hg higher systolic blood pressure (95% CI, 12-34; P=5.6*10-5) and a 3-fold higher risk of coronary heart disease (odds ratio, 3.03; 95% CI, 1.29-7.12; P=0.01). CONCLUSIONS: A genetic predisposition to enhanced nitric oxide signaling is associated with reduced risks of coronary heart disease, peripheral arterial disease, and stroke. Pharmacological stimulation of nitric oxide signaling may prove useful in the prevention or treatment of cardiovascular disease.

Inhibition of calcium/calmodulin-dependent protein kinase kinase (CaMKK) exacerbates impairment of endothelial cell and blood-brain barrier after stroke.

Brain microvascular endothelial cells play an essential role in maintaining blood-brain barrier (BBB) integrity, and disruption of the BBB aggravates the ischemic injury. CaMKK (α and ß) is a major kinase activated by elevated intracellular calcium. Previously, we demonstrated that inhibition of CaMKK exacerbated outcomes, conversely, overexpression reduced brain injury after stroke in mice. Interestingly, CaMKK has been shown to activate a key endothelial protector, sirtuin 1 (SIRT1). We hypothesized that CaMKK protects brain endothelial cells via SIRT1 activation after stroke. In this study, Oxygen-Glucose Deprivation (OGD) was performed in human brain microvascular endothelial cells. Stroke was induced by middle cerebral artery occlusion (MCAO) in male mice. Knockdown of CaMKK ß using siRNA increased cell death following OGD. Inhibition of CaMKK ß by STO-609 significantly and selectively down-regulated levels of phosphorylated SIRT1 after OGD. Changes in the downstream targets of SIRT1 were observed following STO-609 treatment. The effect of STO-609 on cell viability after OGD was absent, when SIRT1 was concurrently inhibited. We also demonstrated that STO-609 increased endothelial expression of the pro-inflammatory proteins ICAM-1 and VCAM-1 and inhibition of CaMKK exacerbated OGD-induced leukocyte-endothelial adhesion. Finally, intracerebroventricular injection of STO-609 exacerbated endothelial apoptosis and reduced BBB integrity after 24-hr reperfusion following MCAO in vivo. Collectively, these results demonstrated that CaMKK inhibition reduced endothelial cell viability, exacerbated inflammatory responses and aggravated BBB impairment after ischemia. CaMKK activation may attenuate ischemic brain injury via protection of the microvascular system and a reduction in the infiltration of pro-inflammatory factors.

High-risk screening for Anderson-Fabry disease in patients with cardiac, renal, or neurological manifestations.

Anderson-Fabry disease (AFD) is an X-linked lysosomal storage disorder caused by abnormalities in the α-galactosidase (Gal) A gene (GLA; MIM:300644). The reduced activity of the lysosomal enzyme, α-galactosidase A (α-Gal A) leads to classic early manifestations and vascular disease of the heart, kidneys, and brain. As a high-risk screening for symptomatic AFD using an enzymatic assay on dried blood spot samples, we enrolled 2325 individuals (803 females and 1522 males; median age: 66 years) with cardiac, renal, or neurological manifestations that met at least one of the following criteria: (a) family history of early-onset cardiovascular diseases; (b) typical classic manifestations, such as acroparesthesias, clustered angiokeratoma, cornea verticillata, and hypo-anhidrosis; (c) proteinuria; (d) receiving dialysis; (e) left ventricular hypertrophy on electrocardiography or echocardiography; or (f) history of stroke. Ninety-two patients displayed low α-Gal A activity. Four males and two females had different pathogenic GLA mutations (0.26%) including a novel mutation c.908-928del21. Four males (0.17%) harbored the GLA c.196G>C (p.E66Q) variant. This simple screening protocol using dried blood spot samples is useful for early diagnosis of AFD in high-risk and underdiagnosed patients suffering from various cardiac, renal, or neurological manifestations.

Gamma-glutamyl transferase predicts future stroke: A Korean nationwide study.

OBJECTIVE: Although gamma-glutamyl transferase (GGT) is generally regarded as an alternative biomarker for alcohol consumption, its independent role in vascular diseases emerged recently. However, its role in stroke remains unknown. The aim of this study is to clarify the impact of GGT on stroke in a large-sized, national, standardized population cohort in Korea. METHODS: In Korea, the National Health Insurance Service (NHIS) provides full-coverage health insurance service for all citizens. Using data from the NHIS, the NHIS-National Sample Cohort was designed by randomly selecting 2% of Koreans, carefully considering demographic characteristics. We analyzed eligible individuals from this standardized cohort. The Cox proportional hazards model was used for the study investigating the relationship between GGT and stroke. RESULTS: Among the 456,100 eligible participants, 7,459 patients (1.64%) developed stroke as follows: 5,789 ischemic strokes, 1,046 intracerebral hemorrhages (ICHs), and 624 subarachnoid hemorrhages. GGT was independently correlated with increased risk of stroke after adjustment for alcohol consumption and stroke risk factors (hazard ratio [HR] = 1.39, 95% confidence interval [CI] = 1.29-1.51). The risks of both ischemic stroke (HR = 1.45, 95% CI = 1.32-1.58) and ICH (HR = 1.46, 95% CI = 1.18-1.80) were significantly elevated with increasing GGT. Despite some effect modifications by sex, age, and alcohol, the risk of total stroke and ischemic stroke in association with GGT remained significant in all subgroups. INTERPRETATION: In a standard Korean population, GGT was a novel biomarker predicting stroke risk, independently from alcohol consumption and other risk factors. Ann Neurol 2018;83:375-386.

Visfatin Level and The Risk of Hypertension and Cerebrovascular Accident: A Systematic Review and Meta-Analysis.

High blood pressure is related with increased cerebrovascular accident. High visfatin / NAMPT(nicotinamide phosphoribosyltransferase) plasma levels may promote vascular inflammation and atherosclerotic plaque destabilization and have been evaluated as a marker for identifying stages of essential hypertension. However, its role in the pathogenesis of hypertension and cerebrovascular accident (CVA) is still uncertain. In order to review and meta-analyze observational studies investigating visfatin concentration and the risk for hypertension or CVA, a systematic search of PubMed, ovid EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) until December 07, 2016 was performed. After data extraction and quality assessment, a meta-analysis was performed using RevMan 5.3 and STATA 14.0. A total of 1693 adults from 8 studies for hypertension (974 with hypertension) and 1696 adults from 7 CVA studies (957 with CVA) were enrolled in the current meta-analysis. Cochran's Q-statistic and I2 test were applied to estimate the heterogeneity of the studies. The fixed-effects were used to compute the weighted mean difference in visfatin levels. Plasma visfatin concentration was much higher in hypertension and CVA patients than in healthy individuals. These evidences suggested the association of hypertension and CVA with higher plasma visfatin level.

Interaction between COX-1 and COX-2 increases susceptibility to ischemic stroke in a Chinese population.

BACKGROUND: Mutations of cyclooxygenase gene (COX gene) may increase the susceptibility of ischemic stroke. We investigated five variants (rs5788, rs1330344, rs3842788, rs20417, and rs689466) of two COX genes in order to explaining the association between these polymorphisms and we also investigated the association between these variants and ischemic stroke risk to determine whether gene-gene interaction between these genes increases the susceptibility of ischemic stroke or its subtypes. METHODS: A total of 1981 study subjects (1078 cases and 903 control subjects) were recruited. The interaction of multiple factors was investigated using Multifactor Dimensionality Reduction. The additive effect of single nucleotide polymorphisms on ischemic stroke or its subtypes were analyzed by multiple factor logistic regression. RESULTS: At COX-1(rs1330344), AA genotype carriers had a lower susceptibility of ischemic stroke (OR = 0.657, 95%CI = 0.437-0.988, P = 0.044), and A allele carriers had a lower susceptibility of ischemic stroke (OR = 0.812, 95%CI = 0.657-0.978, P = 0.029). At COX-1(rs3842788), AA genotype carriers had a higher susceptibility of ischemic stroke (OR = 5.203, 95% CI = 1.519-5.159, P = 0.016). At COX-2 (rs689466), AA genotype carriers had a higher susceptibility of large-artery atherosclerosis (OR = 1.404, 95% CI = 1.019-1.934, P = 0.038). COX-1(rs1330344, rs3842788) and COX-2 rs689466 interacted in SVO, but had no additive effect with ischemic stroke and other subtypes. CONCLUSIONS: At rs1330344, AA genotype may reduce the susceptibility of ischemic stroke. At rs3842788, AA genotype may increase the susceptibility of ischemic stroke. At rs689466, AA genotype may increase the susceptibility of large-artery atherosclerosis (LAA). COX - 1(rs1330344, rs3842788) and COX-2 rs689466 interacted in small vessel occlusion (SVO), but had no additive effect with ischemic stroke and other subtypes.

Association of CYP2C19 Polymorphisms With Clopidogrel Reactivity and Clinical Outcomes in Chronic Ischemic Stroke.

BACKGROUND: CYP2C19variants are associated with the antiplatelet effects of clopidogrel against recurrent cardiovascular events. However, it remains unknown whether the elapsed time from stroke onset affects the relationship between the genetic variants and such events. To address this, we conducted a prospective cohort study to determine the effect ofCYP2C19variants on clinical outcomes in the chronic phase.Methods and Results:In total, 518 Japanese non-acute stroke patients treated with clopidogrel were registered at 14 institutions. Patients were classified into 3 clopidogrel-metabolizing groups according toCYP2C19genotype: extensive metabolizer (EM:*1/*1), intermediate metabolizer (IM:*1/*2or*1/*3), and poor metabolizer (PM:*2/*2,*2/*3, or*3/*3). Antiplatelet effects of clopidogrel were assessed by adenosine diphosphate (ADP)-induced platelet aggregation and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. The endpoint was composite cerebrocardiovascular events (CVEs). In 501 successfully followed-up patients, the median time from index stroke to enrollment was 181 days. There were 28 cardiovascular and 2 major bleeding events. There were no significant differences in the rates of cardiovascular events among the groups. CONCLUSIONS: Despite associations betweenCYP2C19variants and on-clopidogrel platelet reactivity, there was no significant difference in rates of CVEs in the chronic stroke phase among the 3 clopidogrel-metabolizing groups ofCYP2C19variants.

Increased Serum Alkaline Phosphatase in Patients with Acute Ischemic Stroke.

BACKGROUND: Stroke is one of the most common causes of disability and death. Higher alkaline phosphatase (ALP) levels have been associated with poor functional outcomes and mortality in previous studies. We investigated alterations in serum ALP concentrations and functional outcomes in patients with acute ischemic stroke (AIS). METHODS: Patients with first-ever AIS were recruited to participate in the study. Serum ALP levels were measured using a Cobas Integra 400 Plus automatic biochemical analyzer, and severity of stroke was evaluated using the National Institutes of Health Stroke Scale (NIHSS) score on admission. Functional outcome was measured using the modified Rankin scale 1 year after admission. RESULTS: Serum ALP concentration was increased in patients with AIS (81.75 ± 20.49 versus 69.93 ± 16.12 U/L, P = .000) and the optimal ALP cutoff point for diagnosing patients with AIS was 81.50 U/L, with a sensitivity of 49.5% and specificity of 78.9%. However, there was no significant correlation between ALP and NIHSS scores (r = .170, P = .085) and ALP was not significantly different between favorable and unfavorable functional outcomes (81.76 ± .60 versus 81.70 ± 20.54 U/L, P = .802). CONCLUSIONS: Serum ALP concentration, which was increased in patients with AIS, might represent a low-potency biomarker for the diagnosis of AIS. However, this was not significantly correlated with NIHSS scores or the functional outcome after 1 year.