Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.

Inflammatory cytokines enhance procoagulant activity of platelets and endothelial cells through phosphatidylserine exposure in patients with essential hypertension.

The exact mechanism of the prothrombotic state of essential hypertension (EH) patients remains elusive. Our objective was to assess whether phosphatidylserine (PS) exposure on endothelial cells (ECs), platelets, and microparticles (MPs) can account for the hypercoagulability in EH patients. PS exposure on cells and MPs, mainly from platelets and ECs was analyzed with flow cytometry. Procoagulant activity (PCA) was evaluated by purified coagulation complex assays, clotting time, and fibrin turbidity. We found that EH patients exhibited elevated levels of PS+ platelets, serum-cultured ECs, MPs, endothelial-derived MPs and platelet-derived MPs compared to the controls (all P < 0.05). Moreover, platelets and MPs from the patients and their sera-cultured ECs showed markedly enhanced intrinsic/extrinsic FXa, thrombin, and fibrin generation, and greatly shortened coagulation time. This PCA could be blocked approximately 80%, by the addition of lactadherin. Furthermore, we detected elevated levels of IL-8, IL-6, and TNF-α in EH patients could activate platelets/ECs and induce elevated PS exposure on their membranes. Our results suggest that inflammatory cytokines could enhance procoagulant activity of platelets and endothelial cells via their PS exposure in EH patients. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.

Phosphatidylserine-exposing blood and endothelial cells contribute to the hypercoagulable state in essential thrombocythemia patients.

The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P < 0.001)] than those in controls. Among ET patients, those with JAK2 mutations showed higher levels of PS-positive erythrocytes, platelets, neutrophils, and serum-cultured ECs than TN patients or those with CALR mutations, which show similar levels. Coagulation function assays showed that higher levels of PS-positive blood cells and serum-cultured ECs led to markedly shortened coagulation time and dramatically increased levels of FXa, thrombin, and fibrin production. This procoagulant activity could be largely blocked by addition of lactadherin (approx. 70% inhibition). Confocal microscopy showed that the FVa/FXa complex and fibrin fibrils colocalized with PS on ET serum-cultured ECs. Additionally, we found a relationship between D-dimer, prothrombin fragment F1 + 2, and PS exposure. Our study reveals a previously unrecognized link between hypercoagulability and exposed PS on cells, which might also be associated with distinct thrombotic risks among mutational subtypes in ET. Thus, blocking PS-binding sites may represent a new therapeutic target for preventing thrombosis in ET.

Inflammatory cytokines induce neutrophil extracellular traps interaction with activated platelets and endothelial cells exacerbate coagulation in moderate and severe essential hypertension.

BACKGROUND: Essential hypertension (EH) patients suffer from paradoxically thrombotic rather than haemorrhagic, although the exact mechanism remains elusive. Our aim is to explore whether and how neutrophil extracellular traps (NETs) play the procoagulant role in EH patients, as well as evaluated whether the NET releasing were triggered by inflammatory cytokines. METHODS: The concentration of plasma NETs components were detected by ELISA. The morphology of cells and NETs formation were analysed using immunofluorescence. Procoagulant activity was analysed by clotting time, purified coagulation complex and fibrin generation assays. Phosphatidylserine (PS) exposure on endothelial cells (ECs) was analysed with flow cytometry. RESULTS: Moderate to severe EH patients plasma NETs levels were significantly higher compared to mild EH patients or controls. Furthermore, inflammatory cytokines can induce NETs generation, depleting these patients plasma inflammatory cytokines led to a reduction in NET releasing. NETs from moderate to severe EH patients neutrophils led to significantly decreased clotting time (CT), increased potency to generate thrombin and fibrin (all P  < 0.05). These procoagulant effects were markedly attenuated by approximately 70% using DNase I. Additionally, high concentrations NETs exerted a strong cytotoxic effect on ECs, conferring them a procoagulant phenotype. CONCLUSION: Our study reveals that EH drives a systemic inflammatory environment, which, in turn, drives neutrophils to prime and NET releasing, and found a link between hypercoagulability and NETs levels in moderate to severe EH patients. Therefore, anti-inflammatory combined with block the generation of NETs may represent a new therapeutic target for preventing thrombosis in EH patients.

Neutrophil extracellular traps exacerbate coagulation and endothelial damage in patients with essential hypertension and hyperhomocysteinemia.

Patients with essential hypertension (EH) and hyperhomocysteinemia (HHCY) suffer from more increased thrombotic events than those in EH alone. However, the underlying mechanisms for this effect are not well understood. This study hypothesized that neutrophil extracellular trap (NET) releasing may be triggered by HHCY in patients in EH, thereby predisposing them to a more hypercoagulable state. Using a modified-capture enzyme-linked immunosorbent assay (ELISA) method, we observed that cell-free DNA (CF-DNA) and myeloperoxidase DNA (MPO-DNA) in patients With EH and HHCY were significantly higher. The NET formation was also positively correlated with homocysteine levels, neutrophil-lymphocyte ratio (NLR), and hypercoagulable markers (thrombin-antithrombin complex, D-dimers). Furthermore, neutrophils from patients in EH with HHCY were found to be predisposed to amplified NET release when compared to patients in EH without HHCY or CTR. Coagulation function assays showed that NETs in patients With EH and HHCY resulted in a significantly increased ability to generate thrombin and fibrin than in those in EH without HHCY or CTR. These procoagulant effects of NETs in patients With EH and HHCY were markedly inhibited (approximately 70%) by the cleavage of NETs with DNase I. Isolated NETs from patients With EH and HHCY neutrophils also exerted a strong cytotoxic effect on endothelial cells (ECs), converted them to apoptosis. This study revealed a previously unrecognized association between the hypercoagulable state and neutrophils in patients With EH and HHCY. Therefore, blocking NETs may represent a new therapeutic objective for preventing thrombosis in these patients.

Cortistatin ameliorates Ang II-induced proliferation of vascular smooth muscle cells by inhibiting autophagy through SSTR3 and SSTR5.

AIMS: Vascular smooth muscle cell (VSMC) proliferation plays a significant role in the development of various vascular disorders. However, the effect of cortistatin (CST) on VSMC proliferation remains unclear. Therefore, the purpose of our research aimed to study whether CST protected VSMCs from angiotensin II (Ang II)-induced proliferation and which mechanisms participated in the process. MAIN METHODS: Cultured rat VSMCs were treated with Ang II with or without CST for 24 h. Cell proliferation rate was measured by cell counting kit-8 (CCK8) assay. The expressions of CST and its receptors were assessed by quantitative real-time PCR (qRT-PCR). The protein expression levels were analyzed by western blots. Immunofluorescence and transmission electron microscopy (TEM) were used to observe autophagy. KEY FINDINGS: Our results showed that different concentrations of CST alleviated the Ang II-induced VSMC proliferation. The autophagy and reactive oxygen species (ROS) stimulated by Ang II were attenuated by CST. Furthermore, when the autophagy inhibitor 3-methyladenine (3-MA) was added, it exerted similar inhibition effects like CST, but didn't augment the protective role of CST on Ang II-induced VSMC autophagy and proliferation. Moreover, blocking somatostatin receptor 3 and 5 (SSTR3 and SSTR5) partially abrogated the suppressive effect of CST on Ang II-stimulated VSMC proliferation and autophagy. SIGNIFICANCE: This study indicated that CST could ameliorate Ang II-stimulated VSMC proliferation by inhibiting autophagy partially through its receptors SSTR3 and SSTR5, providing a reasonable evidence for CST as a novel perspective therapeutic target of vascular diseases.

LINC00958 regulated miR-627-5p/YBX2 axis to facilitate cell proliferation and migration in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC), the subtype of head and neck cancers, is notorious for its high incidence and death rate. The role of long non-coding RNAs (lncRNAs) is discovered to be significant for the canceration and cancer progression. Long intergenic non-protein coding RNA 958 (LINC00958) is discovered as a carcinogene in multiple cancers, such as gastric cancer, pancreatic cancer, and glioma, but there has been no report about how LINC00958 functions in OSCC. The objective of our study is to unfold function and mechanism investigation on LINC00958 in OSCC. First, TCGA database showed the upregulation and prognostic significance of LINC00958 in head and neck squamous carcinoma. Then, we discovered in OSCC clinical samples that LINC00958 presented high expression and predicted poor prognosis. Also, LINC00958 was elevated in OSCC cells. In vitro gain- and loss-function experiments proved that LINC00958 facilitated cell growth, retarded apoptosis, accelerated migration, and epithelial-to-mesenchymal transition (EMT) in OSCC. Mechanistically, we confirmed the cytoplasmic expression of LINC00958 in OSCC cells, and revealed that LINC00958 sequestered miR-627-5p to upregulate YBX2 expression. Rescue assays indicated that LINC00958 regulated OSCC cell proliferation, motility and EMT through YBX2. Together, we showed that LINC00958 promoted OSCC progression through miR-627-5p/YBX2 axis, indicating LINC00958 as a new prognostic marker, and provided new perspectives for molecular targeted treatment for OSCC.

Poor Sleep Quality Associated With High Risk Of Ventricular Tachycardia After Acute Myocardial Infarction.

BACKGROUND: Sleep disorders (SDs) are usually associated with an increase in frequency of ventricular tachycardia (VT). However, the relationship between SDs and the prevalence of VT within the first week of acute myocardial infarction (AMI) remains unclear. This study aimed to evaluate their associations and potential mechanisms. METHODS: This structured questionnaire-based cross-sectional study enrolled 303 patients with AMI from a hospital in northern China. Pittsburgh Sleep Quality Index (PSQI) was used to determine sleep quality of subjects. Heart rate variability (HRV) of patients was investigated by ambulatory electrocardiography recorders. Enzyme-linked immunosorbent assay was used to measure the plasma levels of catecholamine in a subgroup including 80 patients with AMI. RESULTS: After adjusting to basic cardiovascular characteristics, results of multivariate logistic regression demonstrated that the global PSQI score and its main components were positively associated with VT prevalence in inpatients with AMI. There were significantly different HRV parameters interpreted as autonomic nerve activity in two groups of AMI patients with different sleep quality. In addition, we found the influence of sleep quality on plasma concentrations of adrenaline and norepinephrine in AMI patients. CONCLUSION: Sleep status was significantly associated with the initiation of VT within the first week of AMI, probably due to the effect of SDs on sympathetic nerve activity. Amelioration of sleep quality and sympathetic hyperactivity may be prospective strategy to curb arrhythmias after AMI.

Cortistatin exerts antiproliferation and antimigration effects in vascular smooth muscle cells stimulated by Ang II through suppressing ERK1/2, p38 MAPK, JNK and ERK5 signaling pathways.

BACKGROUND: Vascular remodeling, that contributes to cardiovascular diseases such as hypertension develops by anomalous proliferation and migration of vascular smooth muscle cells (VSMCs). Cortistatin (CST), a newly discovered biological peptide, has been acknowledged for its protective effects against cardiovascular diseases. Whether CST has an inhibitory regulation role in angiotensin II (Ang II)-induced proliferation and migration of VSMCs and what molecular mechanisms may participate in the CST inhibition process are still unknown. METHODS: VSMCs were divided into control group, Ang II (10-7 M) group, Ang II + PD98059 (5×10-5 M) group, Ang II + SB203580 (10-5 M) group, Ang II + SP600125 (10-5 M) group, Ang II + XMD17-109 (10-6 M) group, Ang II + CST (10-8 M) group and Ang II + CST (10-7 M) group. Cell proliferation was detected by western blotting and cell counting kit-8 (CCK8) analysis. Migration of VSMCs was measured by Transwell assay. RESULTS: Compared with control group, Ang II upregulated the expression levels of proliferating cell nuclear antigen (PCNA) and osteopontin (OPN) and downregulated that of α-smooth muscle actin (α-SMA), increased the proliferation rate as shown by CCK8 and VSMC migration as shown by Transwell assay in cultured VSMCs of the Ang II group. Meanwhile, in Ang II-cultured VSMCs, we found activation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and ERK5 pathways by western blotting at different time points. However, the proliferation and migration stimulated by Ang II were partly reversed by drug inhibitors of the four pathways, namely, PD98059, SB203580, SP600125 and XMD17-109. When Ang II-stimulated VSMCs were cultured with CST pretreatment, we found that proliferation and migration were greatly suppressed as well as that the ERK1/2, p38 MAPK, JNK and ERK5 pathways were deactivated by CST. CONCLUSIONS: The accumulated data suggest that CST may play a protective role in Ang II-promoted proliferation and migration of VSMCs via inhibiting the mitogen-activated protein kinase (MAPK) family pathways, providing a new orientation of CST in protecting against cardiovascular diseases.

Different Na+/K+-ATPase signal pathways was involved in the increase of [Ca2+]i induced by strophanthidin in normal and failing isolated guinea pig ventricular myocytes.

AIM: To determine whether different Na+/K+-ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). METHODS: A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 micromol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 micromol/L Str-only group by a video-based, motion-edge detection system. RESULTS: Str at 1, 10, and 25 micromol/L in NC and Str at 0.1, 1, 10, and 25 micromol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. CONCLUSION: The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different.

Osteopontin RNA aptamer can prevent and reverse pressure overload-induced heart failure.

AIMS: Cardiac myocyte hypertrophy, the main compensatory response to chronic stress in the heart often progresses to a state of decompensation that can lead to heart failure. Osteopontin (OPN) is an effector for extracellular signalling that induces myocyte growth and fibrosis. Although increased OPN activity has been observed in stressed myocytes and fibroblasts, the detailed and long term effects of blocking OPN signalling on the heart remain poorly defined. Targeting cardiac OPN protein by an RNA aptamer may be beneficial for tuning down OPN pathologic signalling. We aimed to demonstrate the therapeutic effects of an OPN RNA aptamer on cardiac dysfunction. METHODS AND RESULTS: In vivo, we show that in a mouse model of pressure overload, treating at the time of surgeries with an OPN aptamer prevented cardiomyocyte hypertrophy and cardiac fibrosis, blocked OPN downstream signalling (PI3K and Akt phosphorylation), reduced expression of extracellular matrix (Lum, Col3a1, Fn1) and hypertrophy (Nppa, Nppb) genes, and prevented cardiac dysfunction. Treating at two months post-surgeries with the OPN aptamer reversed cardiac dysfunction and fibrosis and myocyte hypertrophy. While genetic homozygous deletion of OPN reduced myocardial wall thickness, surprisingly cardiac function and myocardial fibrosis, specifically collagen deposition and myofibroblast infiltration, were worse compared with wild type mice at three months of pressure overload. CONCLUSION: Taken together, these data demonstrate that tuning down cardiac OPN signalling by an OPN RNA aptamer is a novel and effective approach for preventing cardiac hypertrophy and fibrosis, improving cardiac function, and reversing pressure overload-induced heart failure.

Oroxylin A attenuates cigarette smoke-induced lung inflammation by activating Nrf2.

Oroxylin A, a natural flavonoid isolated from the medicinal herb Scutellaria baicalensis Georgi, has been reported to have anti-inflammatory and antioxidant properties. However, the effect of oroxylin A on cigarette smoke (CS)-induced lung inflammation remains unclear. In this study, the ability of oroxylin A to protect against CS-induced lung inflammation was detected in vivo and in vitro. Oroxylin A was administered intraperitoneally to mice 2h prior CS exposure every day for five consecutive days. BEAS-2B bronchial epithelial cells and RAW264.7 cells were used to investigate the molecular mechanism of oroxylin A in vitro. In vivo, the results showed that oroxylin A dose-dependently attenuated CS-induced lung histopathologic changes, expression of cytokines TNF-α, IL-1ß, and MCP-1, and levels of oxidative biomarkers 3-nitrotyrosine and 8-isoprostane. Meanwhile, oroxylin A up-regulated GSH level and glutathione reductase (GR) activity in lung tissues. In vitro, oroxylin A significantly up-regulated Nrf2 expression and total cellular glutathione level in cigarette smoke extract (CSE)-stimulated cells. In addition, oroxylin A promoted Nrf2 binding to antioxidant response element (ARE) and up-regulated ARE-regulated gene such as heme oxygenase-1 (HO-1), GPx, and GR in CSE-stimulated cells. Oroxylin A could protect both epithelial cells and macrophages from damage by cigarette smoke in vitro. Taken together, these data indicated that oroxylin A attenuated oxidative stress and lung inflammation induced by CS via activating Nrf2 signaling pathway. Oroxylin A may be a protective agent against CS-induced lung inflammation and chronic obstructive pulmonary disease.

miR-30e targets IGF2-regulated osteogenesis in bone marrow-derived mesenchymal stem cells, aortic smooth muscle cells, and ApoE-/- mice.

AIMS: Activation of an osteogenic transcriptional program contributes to the initiation of aortic calcification in atherosclerosis. The role of microRNAs in regulating aortic calcification is understudied. We tested the hypothesis that miR-30e regulates an osteogenic program in bone marrow-derived mesenchymal stem cells (MSCs), aortic smooth muscle cells (SMCs), and ApoE(-/-) mice. METHODS AND RESULTS: In aortas of wild-type mice, we found that miR-30e is highly expressed in medial SMCs. In aortas of old ApoE(-/-) mice, we found that miR-30e transcripts are down-regulated in an inverse relation to the osteogenic markers Runx2, Opn, and Igf2. In vitro, miR-30e over-expression reduced the proliferation of MSCs and SMCs while increasing adipogenic differentiation of MSCs and smooth muscle differentiation of SMCs. In MSCs and SMCs over-expressing miR-30e, microarrays and qPCR showed repression of an osteogenic gene panel including Igf2. Inhibiting miR-30e in MSCs increased Igf2 transcripts. In SMCs, IGF2 recombinant protein rescued miR-30e-repressed osteogenic differentiation. Luciferase and mutagenesis assays showed binding of miR-30e to a novel and essential site at the 3'UTR of Igf2. In ApoE(-/-) mice, injections of antimiR-30e oligos increased Igf2 expression in the aortas and livers and significantly enhanced OPN protein expression and calcium deposition in aortic valves. CONCLUSION: miR-30e represses the osteogenic program in MSCs and SMCs by targeting IGF2 and drives their differentiation into adipogenic or smooth muscle lineage, respectively. Our data suggest that down-regulation of miR-30e in aortas with age and atherosclerosis triggers vascular calcification. The miR-30e pathway plays an important regulatory role in vascular diseases.

Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.

Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.