A potential mechanism for the cytoprotective effects of activated protein C-released endothelial extracellular vesicles.

ABSTRACT: Activated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.

Factor VIIa suppresses inflammation and barrier disruption through the release of EEVs and transfer of microRNA 10a.

Coagulation protease, factor VIIa (FVIIa), binds to endothelial cell protein C receptor (EPCR) and induces anti-inflammatory and endothelial barrier protective responses via protease-activated receptor-1 (PAR1)-mediated, biased signaling. Our recent studies had shown that the FVIIa-EPCR-PAR1 axis induces the release of extracellular vesicles (EVs) from endothelial cells. In the present study, we investigated the mechanism of FVIIa release of endothelial EVs (EEVs) and the contribution of FVIIa-released EEVs to anti-inflammatory and vascular barrier protective effects, in both in vitro and in vivo models. Multiple signaling pathways regulated FVIIa release of EVs from endothelial cells, but the ROCK-dependent pathway appeared to be a major mechanism. FVIIa-released EEVs were enriched with anti-inflammatory microRNAs (miRs), mostly miR10a. FVIIa-released EEVs were taken up readily by monocytes/macrophages and endothelial cells. The uptake of FVIIa-released EEVs by monocytes conferred anti-inflammatory phenotype to monocytes, whereas EEV uptake by endothelial cells resulted in barrier protection. In additional experiments, EEV-mediated delivery of miR10a to monocytes downregulated the expression of TAK1 and activation of the NF-κB-mediated inflammatory pathway. In in vivo experiments, administration of FVIIa-released EEVs to wild-type mice attenuated LPS-induced increased inflammatory cytokines in plasma and vascular leakage into vital tissues. The incorporation of anti-miR10a into FVIIa-released EEVs diminished the ability of FVIIa-released EEVs to confer cytoprotective effects. Administration of the ROCK inhibitor Y27632, which significantly inhibits FVIIa release of EEVs into the circulation, to mice attenuated the cytoprotective effects of FVIIa. Overall, our study revealed novel insights into how FVIIa induces cytoprotective effects and communicates with various cell types.

The Gab2-MALT1 axis regulates thromboinflammation and deep vein thrombosis.

Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1ß (IL-1ß) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]-BCL-10 [B-cell lymphoma 10]-MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1ß-induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1ß-induced NF-κB-dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2-MALT1 axis in thromboinflammation. Targeting the Gab2-MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.

Selective inhibition of activated protein C anticoagulant activity protects against hemophilic arthropathy in mice.

Recurrent spontaneous or trauma-related bleeding into joints in hemophilia leads to hemophilic arthropathy (HA), a debilitating joint disease. Treatment of HA consists of preventing joint bleeding by clotting factor replacement, and in extreme cases, orthopedic surgery. We recently showed that administration of endothelial cell protein C receptor (EPCR) blocking monoclonal antibodies (mAb) markedly reduced the severity of HA in factor VIII (FVIII)-/- mice. EPCR blocking inhibits activated protein C (APC) generation and EPCR-dependent APC signaling. The present study was aimed to define the role of inhibition of APC anticoagulant activity, APC signaling, or both in suppressing HA. FVIII-/- mice were treated with a single dose of isotype control mAb, MPC1609 mAb, that inhibits anticoagulant, and signaling properties of APC, or MAPC1591 mAb that only blocks the anticoagulant activity of APC. Joint bleeding was induced by needle puncture injury. HA was evaluated by monitoring joint bleeding, change in joint diameter, and histopathological analysis of joint tissue sections for synovial hypertrophy, macrophage infiltration, neoangiogenesis, cartilage degeneration, and chondrocyte apoptosis. No significant differences were observed between MPC1609 and MAPC1591 in inhibiting APC anticoagulant activity in vitro and equally effective in correcting acute bleeding induced by the saphenous vein incision in FVIII-/- mice. Administration of MAPC1591, and not MPC1609, markedly reduced the severity of HA. MAPC1591 inhibited joint bleed-induced inflammatory cytokine interleukin-6 expression and vascular leakage in joints, whereas MPC1609 had no significant effect. Our data show that an mAb that selectively inhibits APC's anticoagulant activity without compromising its cytoprotective signaling offers a therapeutic potential alternative to treat HA.

Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis.

BACKGROUND: Our recent studies suggest that sphingomyelin levels in the plasma membrane influence TF (tissue factor) procoagulant activity. The current study was performed to investigate how alterations to sphingomyelin metabolic pathway would affect TF procoagulant activity and thereby affect hemostatic and thrombotic processes. METHODS: Macrophages and endothelial cells were transfected with specific siRNAs or infected with adenoviral vectors to alter sphingomyelin levels in the membrane. TF activity was measured in factor X activation assay. Saphenous vein incision-induced bleeding and the inferior vena cava ligation-induced flow restriction mouse models were used to evaluate hemostasis and thrombosis, respectively. RESULTS: Overexpression of SMS (sphingomyelin synthase) 1 or SMS2 in human monocyte-derived macrophages suppresses ATP-stimulated TF procoagulant activity, whereas silencing SMS1 or SMS2 increases the basal cell surface TF activity to the same level as of ATP-decrypted TF activity. Consistent with the concept that sphingomyelin metabolism influences TF procoagulant activity, silencing of acid sphingomyelinase or neutral sphingomyelinase 2 or 3 attenuates ATP-induced enhanced TF procoagulant activity in macrophages and endothelial cells. Niemann-Pick disease fibroblasts with a higher concentration of sphingomyelin exhibited lower TF activity compared with wild-type fibroblasts. In vivo studies revealed that LPS+ATP-induced TF activity and thrombin generation were attenuated in ASMase-/- mice, while their levels were increased in SMS2-/- mice. Further studies revealed that acid sphingomyelinase deficiency leads to impaired hemostasis, whereas SMS2 deficiency increases thrombotic risk. CONCLUSIONS: Overall, our data indicate that alterations in sphingomyelin metabolism would influence TF procoagulant activity and affect hemostatic and thrombotic processes.

Elucidation of the signalling pathways for enhanced exosome release from Mycobacterium-infected macrophages and subsequent induction of differentiation.

Exosomes are extracellular vesicles released by all cell types; perform several important functions such as cell-to-cell communication, growth, differentiation and so on. Exosomes elicit several signalling mechanisms as they carry information in the form of DNA, RNA or protein docked on them. We show that exosomes released from Mycobacterium tuberculosis (Mtb)-infected macrophages not only induce differentiation of naïve monocytes but also generate functionally active macrophages via MAPK-dependent signalling mechanism through MK-2 and NF-κß activation which is completely different from the differentiation induced by exosomes from uninfected macrophages. Further, we elucidate unequivocally the signalling mechanism behind the enhanced release of exosome generation from infected macrophages driven by AKT phosphorylation involving Rab7a and Rab11a. Genes of both ESCRT-dependent and -independent pathways are found to be involved in enhanced exosomes release and are modulated by AKT. However, interestingly, the genes of the ESCRT-independent pathway are dependent on NF-κß activation while the genes of the dependent pathway are not, suggesting two parallel signalling cascades operating in tandem.

Factor VIIa induces extracellular vesicles from the endothelium: a potential mechanism for its hemostatic effect.

Recombinant factor FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type (WT), EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice. In vivo studies revealed that administration of FVIIa to WT, EPCR-overexpressing, and PAR1-R46Q-mutant mice, but not EPCR-deficient or PAR1-R41Q-mutant mice, increased the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

The Role of microRNAs in Inflammation.

Inflammation is a biological response of the immune system to various insults, such as pathogens, toxic compounds, damaged cells, and radiation. The complex network of pro- and anti-inflammatory factors and their direction towards inflammation often leads to the development and progression of various inflammation-associated diseases. The role of small non-coding RNAs (small ncRNAs) in inflammation has gained much attention in the past two decades for their regulation of inflammatory gene expression at multiple levels and their potential to serve as biomarkers and therapeutic targets in various diseases. One group of small ncRNAs, microRNAs (miRNAs), has become a key regulator in various inflammatory disease conditions. Their fine-tuning of target gene regulation often turns out to be an important factor in controlling aberrant inflammatory reactions in the system. This review summarizes the biogenesis of miRNA and the mechanisms of miRNA-mediated gene regulation. The review also briefly discusses various pro- and anti-inflammatory miRNAs, their targets and functions, and provides a detailed discussion on the role of miR-10a in inflammation.

Triple-negative breast cancer-derived microvesicles transfer microRNA221 to the recipient cells and thereby promote epithelial-to-mesenchymal transition.

The triple-negative phenotype is the most prevalent form of human breast cancer worldwide and is characterized by poor survival, high aggressiveness, and recurrence. Microvesicles (MV) are shredded plasma membrane components and critically mediate cell-cell communication, but can also induce cancer proliferation and metastasis. Previous studies have revealed that protease-activated receptor 2 (PAR2) contributes significantly to human triple-negative breast cancer (TNBC) progression by releasing nano-size MV and promoting cell proliferation, migration, and invasion. MV isolated from highly aggressive human TNBC cells impart metastatic potential to nonmetastatic cells. Over-expression of microRNA221 (miR221) has also been reported to enhance the metastatic potential of human TNBC, but miR221's relationship to PAR2-induced MV is unclear. Here, using isolated MV, immunoblotting, quantitative RT-PCR, FACS analysis, and enzymatic assays, we show that miR221 is translocated via human TNBC-derived MV, which upon fusion with recipient cells, enhance their proliferation, survival, and metastasis both in vitro and in vivo by inducing the epithelial-to-mesenchymal transition (EMT). Administration of anti-miR221 significantly impaired MV-induced expression of the mesenchymal markers Snail, Slug, N-cadherin, and vimentin in the recipient cells, whereas restoring expression of the epithelial marker E-cadherin. We also demonstrate that MV-associated miR221 targets phosphatase and tensin homolog (PTEN) in the recipient cells, followed by AKT Ser/Thr kinase (AKT)/NF-κB activation, which promotes EMT. Moreover, elevated miR221 levels in MV derived from human TNBC patients' blood could induce cell proliferation and metastasis in recipient cells. In summary, miR221 transfer from TNBC cells via PAR2-derived MV induces EMT and enhances the malignant potential of recipient cells.

Investigation of the Origin of Voltage Generation in Potentized Homeopathic Medicine through Raman Spectroscopy.

BACKGROUND: For the study of homeopathic medicines in proper perspective, emerging techniques in material science are being used. Vibrational spectroscopy is one such tool for providing information on different states of hydrogen bonding as an effect of potentization. The associated change in electrical properties is also correlated with this effect. OBJECTIVE: From the vibrational spectra, the changes in hydrogen bonding due to dilution followed by unidirectional vigorous shaking (together termed potentization) of 91% ethanol and two homeopathic medicines Chininum purum and Acidum benzoicum have been studied. The aim was to correlate the result with the change in the electrical properties of the system. METHODS: Raman spectroscopy was used to study the vibrational spectra. A U-shaped glass tube (electrochemical cell), where one arm contained bi-distilled water and the other arm alcohol/homeopathic medicine (the arms being separated by a platinum foil), was used to measure the voltage generated across two symmetrically placed platinum electrodes. RESULTS: For all samples, it was observed that potentization affected the intensity of OH stretching bands at the frequencies 3240 cm-1, 3420 cm-1 and 3620 cm-1, corresponding to strong hydrogen bond, weak hydrogen bond and broken hydrogen bond, respectively. With the increase in potency, in the presence and absence of the two medicines in ethanol, the number of OH groups linked by strong hydrogen bonds decreased, while the number of OH groups with weak hydrogen bonds increased. With the increase in potentization, the number of OH groups with broken hydrogen bonds showed a difference in the presence and absence of the medicine.The voltage measurements for ethanol show that, with succussion, the magnitude of voltage increased with the two medicines at lower potencies, but not at higher potency where the voltage is lower. Acidum benzoicum, which is acidic in nature, had higher voltage values (113mV, 130 mV and 118 mV at 6C, 30C and 200C, respectively), compared with Chininum purum, which is basic in nature (20 mV, 85 mV and 65 mV at 6C, 30C and 200C, respectively). CONCLUSION: The experimental results indicate a correlation between the vibrational and electrical properties of the homeopathic medicines Acidum benzoicum and Chininum purum at different potencies.

Coagulation factor VIIa-mediated protease-activated receptor 2 activation leads to ß-catenin accumulation via the AKT/GSK3ß pathway and contributes to breast cancer progression.

Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3ß inactivation is involved in these processes and that ß-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of ß-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. ß-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent ß-catenin accumulation may represent a potential therapeutic approach to control breast cancer.

Protease-activated receptor 2 promotes actomyosin dependent transforming microvesicles generation from human breast cancer.

Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter-cellular signaling, mediated predominantly by protease-activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease-mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor-FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK followed by MLC phosphorylation to alter myosin organization whereas, P38 reorganizes actin dynamics by the sequential activation of MK2 and HSP27. RhoA-dependent ROCK-II activation again contributes to remodeling myosin II activity. Further, both our in vitro and in vivo analyses showed that these MVs potentiate invasive and migratory property to the recipient cells. Breast cancer patients blood show an elevation of TF-bearing, pro-metastatic MVs than normal. These findings give an insight into the detailed signaling mechanism involved in the production of MVs with transforming ability from PAR2-activated human breast cancer cells. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in MVs-associated human breast cancer metastasis.

Does design matter? Cervical disc replacements under review.

The present article reviews the design rationale of currently available cervical disc replacements. Recent prospective randomized control trials comparing cervical disc replacement and anterior fusion have demonstrated safety as well as equal or superior clinical results. Increasingly, more devices are becoming available on the market. Understanding design rationale will provide context for the surgeon to optimize decision making for the most appropriate prosthesis. Cervical arthroplasty is a technique that is undergoing rapid design refinement and development. Further improvements in device design will enable patient-specific device selection. Understanding the design rationale and complication profile of each device will improve clinical and radiographic outcomes.

Evaluation of facial artery perforator-based flaps in reconstruction of facial defects.

INTRODUCTION: Several flaps have been described for reconstructing facial or oral defects. Flaps such as forehead and pectoralis major are often too bulky for small-to-moderate-sized defects, for which nasolabial flaps are often ideal. However, nasolabial flaps have limited mobility and reach and may need two stages, particularly for intraoral defects. According to recent literatures, facial artery provides numerous small cutaneous perforators, based on which skin flaps can be islanded, with greater mobility and reach for reconstruction of small-to-moderate-sized intraoral and facial defects in one stage. Our study aims to evaluate the reliability and versatility of facial artery perforator-based flaps in the reconstruction of such defects. MATERIALS AND METHODS: A ethical committee-approved retrospective study was conducted on data of the patients attending our outpatient department between February 2014 and October 2015 with small-to-moderate-sized facial/oral lesions. The total sample size was 23. We studied the relation of flap survival with size of flap, route of inset and neck dissection, functional and aesthetic outcomes and feasibility of adjuvant therapy in cases of malignancies. RESULTS AND ANALYSIS: A wide range of facial defects, especially intraoral defects, could be reconstructed in one stage using facial artery perforator-based flaps. The flaps were reliable. Complications included only partial skin loss of the flaps in a few cases. Complications were directly related to the length of the flaps and the route of inset. Functional and aesthetic outcomes were satisfactory and none of the flaps showed any significant post-radiotherapy changes. CONCLUSIONS: We concluded that facial artery perforator flap can be a simple, safe, versatile and one-stage alternative to the traditional flaps in the reconstruction of small-to-moderate-sized facial defects. Neck dissection can be safely done in the same sitting.

Nuclear matrix protein SMAR1 represses c-Fos-mediated HPV18 E6 transcription through alteration of chromatin histone deacetylation.

Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.

Coagulation Protease-Driven Cancer Immune Evasion: Potential Targets for Cancer Immunotherapy.

Blood coagulation and cancer are intrinsically connected, hypercoagulation-associated thrombotic complications are commonly observed in certain types of cancer, often leading to decreased survival in cancer patients. Apart from the common role in coagulation, coagulation proteases often trigger intracellular signaling in various cancers via the activation of a G protein-coupled receptor superfamily protease: protease-activated receptors (PARs). Although the role of PARs is well-established in the development and progression of certain types of cancer, their impact on cancer immune response is only just emerging. The present review highlights how coagulation protease-driven PAR signaling plays a key role in modulating innate and adaptive immune responses. This is followed by a detailed discussion on the contribution of coagulation protease-induced signaling in cancer immune evasion, thereby supporting the growth and development of certain tumors. A special section of the review demonstrates the role of coagulation proteases, thrombin, factor VIIa, and factor Xa in cancer immune evasion. Targeting coagulation protease-induced signaling might be a potential therapeutic strategy to boost the immune surveillance mechanism of a host fighting against cancer, thereby augmenting the clinical consequences of targeted immunotherapeutic regimens.

MicroRNAs in extracellular vesicles: A potential role in cancer progression.

Intercellular communication, an essential biological process in multicellular organisms, is mediated by direct cell-to-cell contact and cell secretary molecules. Emerging evidence identifies a third mechanism of intercellular communication- the release of extracellular vesicles (EVs). EVs are membrane-enclosed nanosized bodies, released from cells into the extracellular environment, often found in all biofluids. The growing body of research indicates that EVs carry bioactive molecules in the form of proteins, DNA, RNAs, microRNAs (miRNAs), lipids, metabolites, etc., and upon transferring them, alter the phenotypes of the target recipient cells. Interestingly, the abundance of EVs is found to be significantly higher in different diseased conditions, most importantly cancer. In the past few decades, numerous studies have identified EV miRNAs as an important contributor in the pathogenesis of different types of cancer. However, the underlying mechanism behind EV miRNA-associated cancer progression and how it could be used as a targeted therapy remain ill-defined. The present review highlights how EV miRNAs influence essential processes in cancer, such as growth, proliferation, metastasis, angiogenesis, apoptosis, stemness, immune evasion, resistance to therapy, etc. A special emphasis has been given to the potential role of EV miRNAs as cancer biomarkers. The final section of the review delineates the ongoing clinical trials on the role of miRNAs in the progression of different types of cancer. Targeting EV miRNAs could be a potential therapeutic means in the treatment of different forms of cancer alongside conventional therapeutic approaches.

MicroRNA-10a enrichment in factor VIIa-released endothelial extracellular vesicles: potential mechanisms.

BACKGROUND: Factor VIIa induces the release of extracellular vesicles (EVs) from endothelial cells (EEVs). Factor VIIa-released EEVs are enriched with microRNA-10a (miR10a) and elicit miR10a-dependent cytoprotective responses. OBJECTIVES: To investigate mechanisms by which FVIIa induces miR10a expression in endothelial cells and sorts miR10a into the EVs. METHODS: Activation of Elk-1 and TWIST1 expression was analyzed by immunofluorescence microscopy and immunoblot analysis. Small interfering RNA silencing approach was used to knock down the expression of specific genes in endothelial cells. EVs secreted from endothelial cells or released into circulation in mice were isolated by centrifugation and quantified by nanoparticle tracking analysis. Factor VIIa or EVs were injected into mice; mice were challenged with lipopolysaccharides to assess the cytoprotective effects of FVIIa or EVs. RESULTS: FVIIa activation of ERK1/2 triggered the activation of Elk-1, which led to the induction of TWIST1, a key transcription factor involved in miR10a expression. Factor VIIa also induced the expression of La, a small RNA-binding protein. Factor VIIa-driven acid sphingomyelinase (ASM) activation and the subsequent activation of the S1P receptor pathway were responsible for the induction of La. Silencing of ASM or La significantly reduced miR10a levels in FVIIa-released EEVs without affecting the cellular expression of miR10a. Factor VIIa-EEVs from ASM knocked-down cells failed to provide cytoprotective responses in cell and murine model systems. Administration of FVIIa protected wild-type but not ASM-/- mice against lipopolysaccharide-induced inflammation and vascular leakage. CONCLUSION: Our data suggest that enhanced cellular expression of miR10a coupled with La-dependent sorting of miR10a is responsible for enriching FVIIa-released EVs with miR10a.

The Role of Extracellular Vesicles in the Pathogenesis of Hematological Malignancies: Interaction with Tumor Microenvironment; a Potential Biomarker and Targeted Therapy.

The tumor microenvironment (TME) plays an important role in the development and progression of hematological malignancies. In recent years, studies have focused on understanding how tumor cells communicate within the TME. In addition to several factors, such as growth factors, cytokines, extracellular matrix (ECM) molecules, etc., a growing body of evidence has indicated that extracellular vesicles (EVs) play a crucial role in the communication of tumor cells within the TME, thereby contributing to the pathogenesis of hematological malignancies. The present review focuses on how EVs derived from tumor cells interact with the cells in the TME, such as immune cells, stromal cells, endothelial cells, and ECM components, and vice versa, in the context of various hematological malignancies. EVs recovered from the body fluids of cancer patients often carry the bioactive molecules of the originating cells and hence can be considered new predictive biomarkers for specific types of cancer, thereby also acting as potential therapeutic targets. Here, we discuss how EVs influence hematological tumor progression via tumor-host crosstalk and their use as biomarkers for hematological malignancies, thereby benefiting the development of potential therapeutic targets.

Coagulation factor VIIa enhances programmed death-ligand 1 expression and its stability in breast cancer cells to promote breast cancer immune evasion.

BACKGROUND: Immunotherapy for breast cancer has not gained significant success. Coagulation factor VIIa (FVIIa)-tissue factor (TF) mediated activation of protease-activated receptor 2 (PAR2) is shown to promote metastasis and secretion of the immune-modulatory cytokines but the role of FVIIa in cancer immunology is still not well understood. OBJECTIVES: Here, we aim to investigate whether FVIIa protects breast cancer cells from CD8 T-cell-mediated killing. METHODS: Peripheral blood mononuclear cell-derived CD8 T cells were cocultured with vehicle or FVIIa pretreated MDAMB468 cells. The proliferation and activity of CD8 T cells were measured by flow cytometry and ELISA. An allograft model, using wild-type or TF/PAR2-deleted 4T1 cells, was employed to determine the effect of FVIIa on breast cancer immune evasion in vivo. RESULTS: Here, we demonstrate that TF-FVIIa induces programmed death-ligand 1 (PD-L1) in breast cancer cells by activating PAR2. PAR2 activation triggers large tumor suppressor kinase 1 (LATS1) inactivation leading to loss of yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) phosphorylation and subsequent nuclear localization of YAP/TAZ. YAP/TAZ inhibition reduces PD-L1 expression and increases CD8 T-cell activity. We further demonstrate that, apart from transcriptional induction of PD-L1, PAR2 activation also increases PD-L1 stability by enhancing its glycosylation through N-glycosyltransferases STT3A and STT3B. CONCLUSION: In a mouse model of breast cancer, tumor cell-specific PAR2 depletion leads to PD-L1 downregulation and increases anti-PD-1 immunotherapy efficacy. In conclusion, we showed that FVIIa-mediated signaling cascade in cancer cells serves as a tumor intrinsic mechanism of immunosuppression to promote cancer immune evasion.