Background: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. Methods: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. Findings: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. Interpretation: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions.

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Sodium-hydrogen exchanger isoform 1 (NHE1) plays a role in survival and migration/invasion of several cancers and is an emerging new therapeutic target. However, the role of NHE1 in glioblastoma and the interaction of NHE1 expression and function in glioblastoma cells with cytotoxic temozolomide (TMZ) therapy remain unknown. In this study, we detected high levels of NHE1 protein only in primary human glioma cells (GC), glioma xenografts and glioblastoma, but not in human neural stem cells or astrocytes. GC exhibited an alkaline resting pH i (7.46 ± 0.04) maintained by robust NHE1-mediated H + extrusion. GC treatment with TMZ for 2-24 h triggered a transient decrease in pH i , which recovered by 48 h and correlated with concurrent upregulation of NHE1 protein expression. NHE1 protein was colocalized with ezrin at lamellipodia and probably involved in GC migration. The TMZ-treated GC exhibited increased migration and invasion, which was attenuated by addition of NHE1 inhibitor HOE-642. Most importantly, NHE1 inhibition prevented prosurvival extracellular signal-regulated kinase activation and accelerated TMZ-induced apoptosis. Taken together, our study provides the first evidence that GC upregulate NHE1 protein to maintain alkaline pH i . Combining TMZ therapy with NHE1 inhibition suppresses GC migration and invasion, and also augments TMZ-induced apoptosis. These findings strongly suggest that NHE1 is an important cytoprotective mechanism in GC and presents a new therapeutic strategy of combining NHE1 inhibition and TMZ chemotherapy.

m6A RNA methylation is an emerging epigenetic modification, and its potential role in immunity and stemness remains unknown. Based on 17 widely recognized m6A regulators, the m6A modification patterns and corresponding characteristics of immune infiltration and stemness of 1152 low-grade glioma samples were comprehensively analyzed. Machine-learning strategies for constructing m6AScores were trained to quantify the m6A modification patterns of individual samples. Here, we reveal a significant correlation between the multi-omics data of regulators and clinicopathological parameters. We identified two distinct m6A modification patterns (an immune-activated differentiation pattern and an immune-desert dedifferentiation pattern) and four regulatory patterns of m6A methylation on immunity and stemness. We show that the m6AScores can predict the molecular subtype of low-grade glioma, the abundance of immune infiltration, the enrichment of signaling pathways, gene variation and prognosis. The concentration of high immunogenicity and clinical benefits in the low-m6AScore group confirmed the sensitive response to radio-chemotherapy and immunotherapy in patients with high-m6AScore. The results of the pan-cancer analyses illustrate the significant correlation between m6AScore and clinical outcome, the burden of neoepitope, immune infiltration and stemness. The assessment of individual tumor m6A modification patterns will guide us in improving treatment strategies and developing objective diagnostic tools.

The WNK-SPAK kinase signaling pathway controls renal NaCl reabsorption and systemic blood pressure by regulating ion transporters and channels. A WNK3-SPAK complex is highly expressed in brain, but its function in this organ remains unclear. Here, we investigated the role of this kinase complex in brain edema and white matter injury after ischemic stroke. Wild-type, WNK3 knockout, and SPAK heterozygous or knockout mice underwent transient middle cerebral artery occlusion. One cohort of mice underwent magnetic resonance imaging. Ex-vivo brains three days postischemia were imaged by slice-selective spin-echo diffusion tensor imaging magnetic resonance imaging, after which the same brain tissues were subjected to immunofluorescence staining. A second cohort of mice underwent neurological deficit analysis up to 14 days post-transient middle cerebral artery occlusion. Relative to wild-type mice, WNK3 knockout, SPAK heterozygous, and SPAK knockout mice each exhibited a >50% reduction in infarct size and associated cerebral edema, significantly less demyelination, and improved neurological outcomes. We conclude that WNK3-SPAK signaling regulates brain swelling, gray matter injury, and demyelination after ischemic stroke, and that WNK3-SPAK inhibition has therapeutic potential for treating malignant cerebral edema in the setting of middle cerebral artery stroke.