Microvesicles, blood cells, and endothelial cells mediate phosphatidylserine-related prothrombotic state in patients with periodontitis.

BACKGROUND: Phosphatidylserine (PS) is essential for inflammation-associated thrombogenesis, but the exact effect of PS on the prothrombotic state in periodontitis is uncertain. This study aimed to determine the PS-related procoagulant state in patients with periodontitis. METHODS: A total of 138 patients with periodontitis were examined compared with 42 healthy controls. PS-exposing cells and microvesicles in blood samples were detected by confocal microscopy and flow cytometry. The clotting time assay and prothrombinase complex formation assay were used to measure the procoagulant activity of microvesicles, blood cells and endothelial cells. Periodontal clinical parameters and laboratory characteristics of patients with severe periodontitis were recorded and analyzed at baseline and 6 months after non-surgical periodontal therapy. RESULTS: Total PS-positive (PS+ ) microvesicles and the percentage of PS+ blood cells increased in patients with severe periodontitis compared with patients with moderate/mild periodontitis or healthy controls. Endothelial cells cultured in serum from patients with severe periodontitis expressed more PS compared with those cultured in serum from healthy controls. Specifically, PS exposure on blood cells and endothelial cells significantly decreased after inhibiting the effect of inflammatory cytokines. The elevated levels of PS+ cells and microvesicles in severe periodontitis shortened clotting time and led to increased prothrombinase complex formation. Non-surgical periodontal therapy significantly attenuated the release of microvesicles and the PS exposure of blood cells in severe periodontitis. CONCLUSIONS: The prothrombotic state of patients with periodontitis is mediated by PS+ cells and microvesicles stimulated by elevated levels of inflammatory cytokines.

Phosphatidylserine-exposing tumor-derived microparticles exacerbate coagulation and cancer cell transendothelial migration in triple-negative breast cancer.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations. Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay. Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells. Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Endothelial damage and a thin intercellular fibrin network promote haemorrhage in acute promyelocytic leukaemia.

BACKGROUND: The role of vascular endothelium in acute promyelocytic leukaemia (APL) remains unknown. We aimed to investigate the mechanisms by which APL cells interact with endothelial cells (ECs) and to further explore how the endothelium affects bleeding as well as therapeutic interventions. METHOD: APL cells and an original APL cell line, NB4 cells, were used for experiments. The effects of leukaemic cells on ECs were analyzed in vitro and in vivo. Moreover, the endothelial barrier function and procoagulant activity were detected. An APL mouse model was established for in vivo studies. FINDINGS: APL cells interacted with ECs via ICAM-1 and VCAM-1 receptors to disrupt endothelial integrity. This binding activated MLCK signaling, resulting in the trans-endothelial passage of protein and red blood cells (RBCs). Combined treatment with asiatic acid or anti-adhesion receptor antibody inhibited the response of ECs to APL cells, thereby preventing APL-associated haemorrhage in vitro and in vivo. Activated ECs exhibited a procoagulant phenotype after phosphatidylserine exposure. Plasma from APL patients formed a thin fibrin network between procoagulant ECs, and this intercellular fibrin decreased the passage of albumin and RBCs. Ex vivo addition of fibrinogen further enhanced this barrier function in a dose-dependent manner. INTERPRETATION: Endothelial damage induced by leukaemic cell adherence promotes haemorrhaging in APL. Stabilization of ECs, decreasing adhesion receptor expression, and increasing fibrinogen transfusion levels may be a new therapeutic avenue to alleviate this fatal bleeding complication. FUNDING: National Science Foundation of China (81670128, 81873433).

Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.

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. FUNDING: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).

Phosphatidylserine-exposing blood cells, microparticles and neutrophil extracellular traps increase procoagulant activity in patients with pancreatic cancer.

Patients with pancreatic cancer (PC) are at increased risk of venous thrombosis, but the precise mechanisms of hypercoagulable state in PC remain unclear. We aimed to identify how phosphatidylserine positive (PS+) blood cells (BCs), PS+ microparticles (MPs) and neutrophil extracellular traps (NETs) regulate procoagulant activity (PCA) in PC, and to assess the relationship between PCA and PC staging. A total of 83 PC patients with different stages of disease were compared to 30 healthy controls, with confocal microscopy and flow cytometry used to assess MP and cellular PS exposure. MP and cell PCA was determined using both fibrin production assays and procoagulant enzyme complex analyses, and coagulation time was further measured. Patients with stage I PC and healthy controls exhibited significantly lower frequencies of PS+ MPs and BCs relative to those with more advanced disease, which may partly due to the increased levels of inflammation cytokines in advanced disease. Functional coagulation assays indicated that PS+ MPs and BCs derived from patients with stage II/III/IV PC directly contribute to elevated FXa, thrombin, and fibrin formation, and to more rapid coagulation relative to healthy control samples. In inhibition assays, lactadherin, which antagonizes PS, led to a roughly 80% inhibition of PCA. We further used isolated NETs to stimulate endothelial cells, revealing that this led to morphological changes including retraction from cell-cell junctions and a more pro-coagulative phenotype, with DNase I and activated protein C treatment reversing these changes. In patients with stage III PC, curative resection surgery significantly reduced PCA, whereas non-curative surgery did not have a marked impact based on studies of pre- and post-operative samples. These results highlight the pathogenic activity of PS+ cells, MPs, and NETs in promoting a prothrombotic environment within individuals suffering from advanced PC. Targeting PS and NETs in these patients may thus be a viable means of preventing pathological thrombosis.

Neutrophil Extracellular Traps Induce Intestinal Damage and Thrombotic Tendency in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Despite the presence of neutrophil extracellular traps [NETs] in inflamed colon having been confirmed, the role of NETs, especially the circulating NETs, in the progression and thrombotic tendency of inflammatory bowel disease [IBD] remains elusive. We extended our previous study to prove that NETs constitute a central component in the progression and prothrombotic state of IBD. METHODS: In all 48 consecutive patients with IBD were studied. Acute colitis was induced by the treatment of C57BL/6 mice with 3.5% dextran sulphate sodium [DSS] in drinking water for 6 days. Peripheral blood neutrophils and sera were collected from IBD patients and murine colitis models. Exposed phosphatidylserine [PS] was analysed with flow cytometry and confocal microscopy. Procoagulant activity was evaluated using clotting time, purified coagulation complex, and fibrin formation assays. RESULTS: We observed higher plasma NET levels and presence of NETs in colon tissue in patients with active IBD. More importantly, NETs were induced in mice with DSS colitis, and inhibition of NET release attenuated colitis as well as colitis-associated tumorigenesis. NET degradation through DNase administration decreased cytokine levels during DSS-induced colitis. In addition, DNase treatment also significantly attenuated the accelerated thrombus formation and platelet activation observed in DSS-induced colitis. NETs triggered PS-positive microparticle release and PS exposure on platelets and endothelial cells partially through TLR2 and TLR4, converting them to a procoagulant phenotype. CONCLUSIONS: NETs exacerbate colon tissue damage and drive thrombotic tendency during active IBD. Strategies directed against NET formation may offer a potential therapeutic approach for the treatment of IBD.

No Association between Estrogen Receptor-Β Rs4986938 and Cancer Risk: A Systematic Review and Meta-Analysis.

BACKGROUND: The association between estrogen receptor-ß (ESR2) rs4986938 polymorphism and the risk of various types of cancer have been investigated in previous studies. However, the results remained disputable. Here, we conducted a meta-analysis to investigate the association between ESR2 rs4986938 polymorphism and the risk of cancer. METHODS: We searched for relevant articles collected by the PubMed, EMBASE, and Cochrane library up to March 30, 2018. The association was assessed using Odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: The meta-analysis involved a total of 23 studies in 20 papers, including 24,334 cases and 31,707 controls. No significant association was detected between the rs4986938 polymorphism and cancer risk in the additive model (A compared with G: OR=0.97, 95% CI=0.92-1.02, P=0.20), dominant model (AA+AG compared with GG: OR=0.96, 95% CI=0.93-1.03, P=1.00), recessive model (AA compared with AG + GG: OR=0.94, 95% CI=0.86-1.03, P=0.18), heterozygous model (AG compared with GG: OR=0.97, 95% CI=0.94-1.01, P=0.14), and homozygous model (AA compared with GG: OR=0.96, 95% CI=0.87-1.06, P=0.39). Results of subgroup analysis stratified by ethnicity and cancer types further validated the results. CONCLUSION: We found no evidence of an association between rs4986938 and the risk of overall cancer.

Pathway-based network analyses and candidate genes associated with Kashin-Beck disease.

To perform a comprehensive analysis focusing on the biological functions and interactions of Kashin-Beck disease (KBD)-related genes to provide information towards understanding the pathogenesis of KBD.A retrospective, integrated bioinformatics analysis was designed and conducted. First, by reviewing the literature deposited in PubMed, we identified 922 genes genetically associated with KBD. Then, biological function and network analyses were conducted with Cytoscape software. Moreover, KBD specific molecular network analysis was conducted by Cytocluster using the Molecular Complex Detection Algorithm (MCODE).The biological function enrichment analysis suggested that collagen catabolic process, protein activation cascade, cellular response to growth factor stimulus, skeletal system development, and extrinsic apoptosis played important roles in KBD development. The apoptosis pathway, NF-kappa B signaling pathway, and the glutathione metabolism pathway were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway network, suggesting that these pathways may play key roles in KBD occurrence and development. MCODE clusters showed that in top 3 clusters, 54 of KBD-related genes were included in the network and 110 candidate genes were discovered might be potentially related to KBD.The 110 candidate genes discovered in the current study may be related to the development of KBD. The expression changes of apoptosis and oxidative stress-related genes might serve as biomarkers for early diagnosis and treatment of KBD.

Neutrophil extracellular traps enhance procoagulant activity in patients with oral squamous cell carcinoma.

BACKGROUND: Hypercoagulability is a major cancer-associated complication linked to poor patient prognosis. The production of neutrophil extracellular traps (NETs) is increasingly found to be linked with the development and metastasis of cancer, as well as with thrombi formation in cancer patients. We hypothesized that the neutrophil NET release may be triggered by specific cytokines in oral squamous cell carcinoma (OSCC) patients, thereby predisposing them to a hypercoagulable state. Moreover, we have evaluated the interaction between NETs and endothelial cells (ECs). METHODS: NET procoagulant activity was assessed based on fibrin and purified coagulation complex production assays, as well as by measuring coagulation time (CT). We further used confocal microscopy to quantify the exposure of phosphatidylserine (PS), fibrin strands, and cell FVa/Xa binding. RESULTS: OSCC patients with stage III/IV exhibited elevated plasma NET levels compared to stage I/II or CTR (all P < 0.05). Neutrophils from OSCC patients are predisposed to amplified NET release compared to those from CTR. Furthermore, depleting IL-8, IL-6, and TNF-α led to a reduction in NET release in the plasma. OSCC NETs increased thrombin and fibrin generation and decreased CT significantly (P < 0.05). When NETs were isolated and used to treat ECs, these cells exhibited disrupted morphology by retracting from their cell-cell junctions and convert to a procoagulant phenotype. These effects could be attenuated by approximately 70% using DNase I. CONCLUSIONS: Our findings are consistent with a model wherein OSCC drives a systemic inflammatory state, which, in turn, drives neutrophils to prime and release NETs, which drive the development of a hypercoagulable state. Intervening in this process may be a viable means of disrupting these undesirable coagulation dynamics in stage III/IV OSCC patients.

Potential candidate biomarkers associated with osteoarthritis: Evidence from a comprehensive network and pathway analysis.

Osteoarthritis (OA) is one of the most common forms of arthritis world widely. Some key genes and diagnostic markers have been reported due to the development of modern molecular biology technologies. However, the etiology and pathogenesis of OA remains unknown. In this study, an integrated network and pathway analysis towards the biological function of OA-associated genes was conducted to provide valuable information to further explore the etiology and pathogenesis of OA. A total of 2,548 genes which reported a statistically significant association with OA were screened. An integrated network and pathway analysis was performed to identify the pathways and genes most associated to OA. Moreover, OA-specific protein-protein interaction (PPI) network was constructed by cytocluster based on the Molecular Complex Detection Algorithm (MCODE) to screen its candidate biomarkers. Quantitative real-time polymerase chain reaction was used to confirm the expression levels and to validate the results of MCODE cluster analysis by six genes. The pathway networks suggested that extracellular matrix (ECM) organization, collagen degradation and collagen formation showed important associations with OA. In top two PPI clusters, 61 of the OA-associated genes were included in the OA-specific PPI network, which also included 23 candidate genes that are likely to be highly associated with OA based on MCODE clusters. Analysis of mRNA showed that the expression levels of COL9A1, COL9A2, ITGA3, COL9A3, ITGA2, and LAMA1 in the peripheral blood mononuclear cells of OA patients were significantly lower than those of the normal controls (p<0.005). To our knowledge, this is the first comprehensive and systematic report based on OA-related genes demonstrating that the functional destruction of collagen in cartilage may be a very important contributing factor to OA. Quantitative detection of collagen synthesis may be of great help in early identification and prediction of OA. Maintaining the quality and quantity of collagen can be a potential target for clinical treatment of OA in the future practice.

Identification of potential biomarkers for differential diagnosis between rheumatoid arthritis and osteoarthritis via integrative genome­wide gene expression profiling analysis.

The present study aimed to identify potential novel biomarkers in synovial tissue obtained from patients with Rheumatoid Arthritis (RA) and Osteoarthritis (OA) for differential diagnosis. The genome­wide expression profiling datasets of synovial tissues from RA and OA cohorts, including GSE55235, GSE55457 and GSE55584 datasets, were retrieved and used to identify differentially expressed genes (DEGs; P<0.05; false discovery rate <0.05 and Fold Change >2) between RA and OA using R software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of DEGs were performed to determine molecular and biochemical pathways associated with the identified DEGs, and a protein­protein interaction (PPI) network of the DEGs was constructed using Cytoscape software. Significant modules in the PPI network and candidate driver genes were screened using the Molecular Complex Detection Algorithm. Potential biomarkers were evaluated by receiver operating characteristic and logistic regression analyses. Large numbers of DEGs were detected, including 273, 205 and 179 DEGs in the GSE55235, GSE55457 and GSE55584 datasets, respectively. Among them, 80 DEGs exhibited identical expression trends in all the three datasets, including 49 upregulated and 31 downregulated genes in patients with RA. DEGs in patients suffering from RA compared with patients suffering from OA were predominantly associated with the primary immunodeficiency pathway, including interleukin 7 receptor (IL7R) and signal transducer activator of transcription 1 (STAT1). The sensitivity of IL7R + STAT1 to differentiate RA from OA was 93.94% with a specificity of 80.77%. The results generated from analyses of the GSE36700 dataset were closely associated with results generated from analyses of GSE55235, GSE55457 and GSE55584 datasets, which further verified the reliability of the aforementioned results. The results of the present study suggested that increased expression of IL7R and STAT1 in synovial tissue as well as in the primary immunodeficiency may be associated with RA occurrence. These identified novel biomarkers may be used to predict disease occurrence and clinically differentiate RA from OA.

Analysis of gene expression and methylation datasets identified ADAMTS9, FKBP5, and PFKBF3 as biomarkers for osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a kind of chronic osteoarthropathy and degenerative joint disease. Epigenetic regulation in the gene expression dynamics has become increasingly important in OA. We performed a combined analysis of two types of microarray datasets (gene expression and DNA methylation) to identify methylation-based key biomarkers to provide a better understanding of molecular biological mechanisms of OA. METHODS: We obtained two expression profiling datasets (GSE55235, GSE55457) and one DNA methylation profiling data set (GSE63695) from the Gene Expression Omnibus. First, differentially expressed genes (DEGs) between patients with OA and controls were identified using the Limma package in R(v3.4.4). Then, function enrichment analysis of DEGs was performed using a DAVID database. For DNA methylation datasets, ChAMP methylation analysis package was used to identify differential methylation genes (DMGs). Finally, a comprehensive analysis of DEGs and DMGs was conducted to identify genes that exhibited differential expression and methylation simultaneously. RESULTS: We identified 112 DEGs and 2,896 DMGs in patients with OA compared with controls. Functional analysis of DEGs obtained that inflammatory responses, immune responses, and positive regulation of apoptosis, tumor necrosis factor (TNF) signaling pathway, and osteoclast differentiation may be involved in the pathogenesis of OA. Cross-analysis revealed 26 genes that exhibited differential expression and methylation in OA. Among them, ADAMTS9, FKBP5, and PFKBF3 were identified as valuable methylation-based biomarkers for OA. CONCLUSION: In summary, our study identified different molecular features between patients with OA and controls. This may provide new clues for clarifying the pathogenetic mechanisms of OA.

The study of GPX3 methylation in patients with Kashin-Beck Disease and its mechanism in chondrocyte apoptosis.

OBJECTIVE: Selenium deficiency is a risk factor for Kashin-Beck Disease (KBD), an endemic osteoarthropathy. Although promoter hypermethylation of glutathione peroxidase 3 (GPX3) (a selenoprotein) has been identified in several cancers, little is known about promoter methylation and expression of GPX3 and their relation to selenium in KBD. The present study was thus conducted to investigate this research question. METHODS: Methylation and expressions of GPX3 in whole blood drawn from 288 KBD patients and 362 healthy controls and in chondrocyte cell line were evaluated using methylation-specific PCR and qRT-PCR, respectively. The protein levels of PI3K/Akt/c-fos signaling in the whole blood and chondrocyte cell line were determined with Western blotting. Chondrocytes apoptosis were detected by Hoechst 33342 and Annexin V-FITC/PI staining. RESULTS: GPX3 methylation was increased, GPX3 mRNA was decreased, and protein levels in the PI3K/Akt/c-fos signaling pathway were up-regulated in the whole blood collected from KBD patients as compared with healthy controls. Similar results were obtained for chondrocytes injured by oxidative stress. There was a significant, decreasing trend in GPX3 expression across groups of unmethylation, partial methylation, and complete methylation for GPX3, in sequence. Compared with unmethylation group, protein levels in PI3K/Akt/c-fos pathway were enhanced in partial and complete methylation groups. Treatment of chondrocytes with sodium selenite resulted in reduced methylation and increased expression of GPX3 as well as down-regulated level of PI3K/Akt/c-fos proteins. CONCLUSIONS: The methylation and expression of GPX3 and expression of PI3K/Akt/c-fos pathway are altered in KBD and these changes are reversible by selenium supplementation.

Phosphatidylserine-exposing cells contribute to the hypercoagulable state in patients with multiple myeloma.

Multiple myeloma (MM) is characterized by an increased incidence of thromboembolic events, particularly when treated with immunomodulatory drugs (IMiDs) in combination with dexamethasone. The optimal prophylactic strategy to prevent the hypercoagulable state of patients with MM is still debated. The aim of the current study was to investigate the definitive role of phosphatidylserine (PS) in supporting procoagulant activity (PCA) in patients with MM. Patients with MM (n=20) and healthy subjects (n=15) were recruited for the present study. PS analyses were performed by flow cytometry and confocal microscopy. The PCA was evaluated by clotting time, purified coagulation complex assays and fibrin production assays. The percentage of PS+ blood cells was significantly higher in patients with MM than in healthy subjects. Additionally, the patient serum induced more PS exposure on endothelial cells (ECs) in vitro than serum from healthy subjects. Isolated blood cells from patients with MM and ECs cultured with patient serum in vitro demonstrated significantly shortened coagulation time, greatly intrinsic/extrinsic factor Xa generation and increased thrombin formation. In addition, the levels of PS+ erythrocytes, platelets, leukocytes, and ECs incubated with IMiDs and dexamethasone were higher than with IMiDs alone. The findings support the hypothesis that increased PS exposure on blood cells and ECs participates in the hypercoagulable state in patients with MM. Thus, blocking PS may be a novel therapeutic target for the prevention of thrombosis in these patients.