Increased intracellular Cl- concentration mediates neutrophil extracellular traps formation in atherosclerotic cardiovascular diseases.

Neutrophil extracellular traps (NETs) play crucial roles in atherosclerotic cardiovascular diseases such as acute coronary syndrome (ACS). Our preliminary study shows that oxidized low-density lipoprotein (oxLDL)-induced NET formation is accompanied by an elevated intracellular Cl- concentration ([Cl-]i) and reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression in freshly isolated human blood neutrophils. Herein we investigated whether and how [Cl-]i regulated NET formation in vitro and in vivo. We showed that neutrophil [Cl-]i and NET levels were increased in global CFTR null (Cftr-/-) mice in the resting state, which was mimicked by intravenous injection of the CFTR inhibitor, CFTRinh-172, in wild-type mice. OxLDL-induced NET formation was aggravated by defective CFTR function. Clamping [Cl-]i at high levels directly triggered NET formation. Furthermore, we demonstrated that increased [Cl-]i by CFTRinh-172 or CFTR knockout increased the phosphorylation of serum- and glucocorticoid-inducible protein kinase 1 (SGK1) and generation of intracellular reactive oxygen species in neutrophils, and promoted oxLDL-induced NET formation and pro-inflammatory cytokine production. Consistently, peripheral blood samples obtained from atherosclerotic ApoE-/- mice or stable angina (SA) and ST-elevation ACS (STE-ACS) patients exhibited increased neutrophil [Cl-]i and SGK1 activity, decreased CFTR expression, and elevated NET levels. VX-661, a CFTR corrector, reduced the NET formation in the peripheral blood sample obtained from oxLDL-injected mice, ApoE-/- atherosclerotic mice or patients with STE-ACS by lowering neutrophil [Cl-]i. These results demonstrate that elevated neutrophil [Cl-]i during the development of atherosclerosis and ACS contributes to increased NET formation via Cl--sensitive SGK1 signaling, suggesting that defective CFTR function might be a novel therapeutic target for atherosclerotic cardiovascular diseases.

Platelet CFTR inhibition enhances arterial thrombosis via increasing intracellular Cl- concentration and activation of SGK1 signaling pathway.

Platelet hyperactivity is essential for thrombus formation in coronary artery diseases (CAD). Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients with cystic fibrosis elevates intracellular Cl- levels ([Cl-]i) and enhanced platelet hyperactivity. In this study, we explored whether alteration of [Cl-]i has a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl-]i was increased in platelets from CAD patients. In a FeCl3-induced mouse mesenteric arteriole thrombosis model, platelet-specific Cftr-knockout and/or pre-administration of ion channel inhibitor CFTRinh-172 increased platelet [Cl-]i, which accelerated thrombus formation, enhanced platelet aggregation and ATP release, and increased P2Y12 and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl-]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl-]i at high levels or Cftr deficiency-induced [Cl-]i increasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12 and PAR4 expression via NF-κB signaling. Constitutively active mutant S422D SGK1 markedly increased P2Y12 and PAR4 expression. The specific SGK1 inhibitor GSK-650394 decreased platelet aggregation in wildtype and platelet-specific Cftr knockout mice, and platelet SGK1 phosphorylation was observed in line with increased [Cl-]i and decreased CFTR expression in CAD patients. Co-transfection of S422D SGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl-]i is a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl-]i-sensitive SGK1 signaling pathway. Therefore, [Cl-]i in platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.

Precise determination of U isotopic compositions in low concentration carbonate samples by MC-ICP-MS.

We developed a fast and simple analytical procedure for precise determination of U isotopic compositions in low concentration natural samples. The main advantage of the new method is that it requires only 12ng U and can obtain all U isotopic ratios without using spike. Five carbonate reference materials (JCp-1, RKM-4, RKM-5, GBW04412 and GBW04413) and 3 international standards with different matrices (IAPSO, IRMM-3184 and CRM-U010) were analyzed for ((234)U/(238)U) and (238)U/(235)U ratios by MC-ICPMS. Using our method, the results for these standards are in close agreement with the certified values, 1.144 ± 0.004, 0.966 ± 0.004 and 0.990 ± 0.003 for ((234)U/(238)U) and 137.72 ± 0.13, 137.64 ± 0.15 and 98.63 ± 0.04 for (238)U/(235)U, in IAPSO, IRMM-3184 and CRM-U010, respectively. The long-term reproducibility of ((234)U/(238)U) and (238)U/(235)U is 0.970 ± 0.002 and 137.56 ± 0.09; 1.144 ± 0.004 and 137.72 ± 0.13, respectively, for in-house U solution and IAPSO. The new ((234)U/(238)U) results for carbonates show much better precision than previous studies and also reflect their age variability. The obtained (238)U/(235)U ratios, representing the first measurements in these carbonate specimens, are rather constant. The method described here requires only 12 ng of U for analysis and can be completed in 5.2 min. The approach provides a fast method to measure ((234)U/(238)U) and (238)U/(235)U ratios in sample matrices commonly encountered in studies of chemical weathering, oceanography and paleoclimatology.