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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−]ihas a pathological role in regulating platelet hyperactivity and arterial thrombosis formation. CFTR expression was significantly decreased, while [Cl−]iwas 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 P2Y12and PAR4 expression in platelets. Conversely, Cftr-overexpressing platelets resulted in subnormal [Cl−]i, thereby decreasing thrombosis formation. Our results showed that clamping [Cl−]iat high levels or Cftr deficiency-induced [Cl−]iincreasement dramatically augmented phosphorylation (Ser422) of serum and glucocorticoid-regulated kinase (SGK1), subsequently upregulated P2Y12and PAR4 expression via NF-κB signaling. Constitutively active mutant S422DSGK1 markedly increased P2Y12and 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−]iand decreased CFTR expression in CAD patients. Co-transfection of S422DSGK1 and adenovirus-induced CFTR overexpression in MEG-01 cells restored platelet activation signaling cascade. Our results suggest that [Cl−]iis a novel positive regulator of platelet activation and arterial thrombus formation via the activation of a [Cl−]i-sensitive SGK1 signaling pathway. Therefore, [Cl−]iin platelets is a novel potential biomarker for platelet hyperactivity, and CFTR may be a potential therapeutic target for platelet activation in CAD.

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−]iregulated NET formation in vitro and in vivo. We showed that neutrophil [Cl−]iand NET levels were increased in global CFTRnull (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−]iat high levels directly triggered NET formation. Furthermore, we demonstrated that increased [Cl−]iby CFTRinh-172 or CFTRknockout 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−]iand SGK1 activity, decreased CFTRexpression, 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−]iduring 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.

Haynaldia villosa(2n=14, VV), a wild grass of the subtribe Triticeae,serves as potential gene resources for wheat genetic improvement. In this study, the proteome characterization during grain development of Yangmai 5 and Yangmai 5-H. villosa6VS/6AL translocation line was investigated by a comparative proteomic approach. Two-dimensional electrophoresis identified 81 differentially accumulated proteins (DAPs) during five grain developmental stages in wheat-H. villosatranslocation line. These proteins were mainly involved in stress defense, storage protein, energy metabolism, protein metabolism and folding, carbon metabolism, nitrogen metabolism, and starch metabolism. In particular, 6VS/6AL translocation led to significant upregulation of 36 DAPs and specific expression of 11 DAPs such as chitinase, thaumatin-like proteins, glutathione transferase, α-amylase inhibitor, heat shock proteins, and betaine aldehyde dehydrogenase. These proteins mainly involved in biotic and abiotic stress responses. Further analysis found that the upstream 1 500 promoter regions of these stress-responsive DAP genes contained multiple high-frequency cis-acting elements related to stress defense such as abscisic acid response element ABRE, methyl jasmonate (MeJA)-response element TGACG-motif and CGTCA-motif involved in oxidative stress and antioxidant response element (ARE). RNA-seq and RT-qPCR analyses revealed the high expression of these stress-defensive DAP genes in the developing grains, particularly at the early-middle grain filling stages. Our results demonstrated that 6VS chromosome of H. villosacontains abundant stress-defensive proteins that have potential values for wheat genetic improvement.