Apoptotic Vesicles Research Articles

Pattern of apoptotic endothelial cell-derived micro vesicles in patients with different phenotypes of chronic heart failure

Background: The aim of the study was to evaluate the associations between the signature of apoptotic endothelial cellderived microvesicles (MVs) and biomarkers of fibrosis, inflammation and cardiac remodeling in patients with different phenotypes of chronic Heart Failure (HF). Methods: The study cohort consisted of 388 prospectively involved subjects with established chronic HF. The phenotype of HF was determined according to left ventricular ejection fraction (LVEF) value per contemporary clinical guideline. HFrEF (LVEF ≤ 40%), HFmrEF (41-49%) and HFpEF (LVEF ≥ 50%) were determined. All biomarkers were measured at baseline. Results: The number of circulating CD31+/annexin V+MVs in HFpEF patients was significantly different from both HFrEF and HFmrEF individuals, but it was similar in HFrEF and HFmrEF patients. The number of circulating CD144+/ annexin V+MVs in HFrEF patients was significantly higher to HFmrEF and HFpEF. We determined that a combination of a number of circulating CD31+/annexin V+MVs and galectin-3 (AUC=0.68; 95% CI=0.61-0.77; P=0.001) was the best predictor of HFpEF. The predictive values of sST2 (AUC=0.65; 95% CI=0.60-0.69), number of circulating CD31+/annexin V+MVs (AUC=0.63; 95% CI=0.58-0.69) alone and their combination (AUC=0.65; 95% CI=0.59-0.70) for HFmrEF did not distinguished significantly (P=0.48). The double combinations of number of circulating CD144+/annexin V+MVs and sST2 (AUC=0.70; 95% CI=0.66-0.75) or number of circulating CD144+/annexin V+MVs and galectin-3 (AUC=0.71; 95% CI=0.650.76) were the best prognosticators for HFrEF. Conclusion: we found that the number of circulating CD31+/annexin V+MVs may improve a prediction of galectin-3 for HFpEF, and that number of circulating CD144+/annexin V+MVs is able to increase predictive capabilities of sST2 and galectin-3 for HFrEF.

Molecular mechanisms of biogenesis of apoptotic exosome-like vesicles and their roles as damage-associated molecular patterns

Recent research has led to contradictory notions regarding the conventional theory that apoptotic cell death can evoke inflammatory or immunogenic responses orchestrated by released damage-associated patterns (DAMPs). By inducing IL-1β from bone marrow-derived macrophages in an effort to determine the inflammatory mediators released from apoptotic cells, we found that exosomal fractions called "apoptotic exosome-like vesicles" (AEVs) prepared from apoptotic-conditioned medium were the main inflammatory factors. These AEVs showed characteristics of exosomes in their size, density, morphology, and protein expression but had unique marker proteins, sphingosine-1-phosphate receptors 1 and 3 (S1PR1 and 3). Their biogenesis was completely dependent on cellular sphingosine-1-phosphate (S1P)/S1PRs signaling from multiple fine spindles of plasma membrane accompanied by F-actin, S1PR1, S1PR3, and CD63 at the early apoptotic phase and progressing to the maturation of F-actin-guided multivesicular endosomes mediated by Gβγ subunits of S1PRs downstream. S1P-loaded S1PRs on AEVs were critical factors for inducing IL-1β via NF-κB transcriptional factor and p38 MAPK, possibly through the RHOA/NOD2 axis, in differentiating macrophages. The AEVs induced genes of proinflammatory cytokines, chemokines, and mediators in both in vitro and in vivo models. In conclusion, AEVs could be key inflammatory mediators, acting as DAMPs that could explain the pathogeneses of various chronic inflammations, autoimmune diseases, or cancers in the future.

Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis

Plant-derived anti-cancer agents have been of considerable interest due to their promising effectiveness with low side effects. Asiatic acid, the main constituent of the medicinal plant Centella asiatica (L.) Urban, has a wide range of biological properties such as antioxidant, anti-inflammatory and anti-cancer activities. Cholangiocarcinoma (CCA), which is a malignant tumor of bile duct epithelium, is one of the leading cancers in Southeast Asia, notably the northeast of Thailand where the liver fluke, Opisthorchis viverrini predominates. Many in vitro and in vivo studies have provided evidence supporting that oxidative stress induced by chronic inflammation is involved in CCA genesis with aggressive clinical outcomes. This study was performed to evaluate the cytotoxic effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213). Cell viability was determined by a sulforhodamine B (SRB) assay. Morphological changes of the cells were observed by microscopy. Cell apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) staining. Messenger RNA (mRNA) expression levels of BAX, BCL2 and Survivin/BIRC5 were analyzed by real-time polymerase chain reaction (PCR). It was found that asiatic acid efficiently suppressed CCA cellular viability via induction of apoptosis. In addition, the occurrence of asiatic acid-induced apoptosis was confirmed by microscopic observation of apoptotic vesicles, down-regulation of anti-apoptotic genes (BCL2 and Survivin/BIRC5) and increased early and late apoptotic cells. Our results showed the chemotherapeutic activities of asiatic acid, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival of CCA patients.

The Proteasome, Active within Apoptotic Exosome-like Vesicles Triggers γδT-17 Activation, Tertiary Lymphoid Structure Formation and Accelerate Rejection

Ischemia-reperfusion injury (IRI) is a major risk factor of progressive organ failure in solid organ transplant recipients. Our group recently identified exosome-like membrane vesicles (apoExo) released during IRI by apoptotic endothelial cells (apoEC) as an important trigger for the production of anti-perlecan/LG3 antibodies and acceleration of rejection. Here we aim to characterize the key molecular components controlling the autoimmune and inflammatory responses triggered by ApoExo. Naïve WT and aortic allografted WT and γδTcells KO mice were injected every other day with purified ApoExo, bortezomib treated ApoExo, Apoptotic bodies or vehicle until sacrifice at 3 weeks. Serum cytokine 36plex assay was performed at sacrifice. Graft histology was evaluated by immunohistochemistry. In all models, various autoantibodies productions were monitored by ELISA. In naive mice, injection of ApoExo (but not of ApoBodies) triggered an increase in circulating levels of IL-23, IL-17, CXCL-1 and TNF-alpha (p<0.01), hallmarks of the IL-23/IL-17 autoimmune axis. Strong anti-LG3 IgG (p<0.001) and ANA responses (p<0.001) were also noted while anti-vimentin, anti-AT1R and anti-fibronectin levels were not affected by ApoExo injection. Similarly, the injection of ApoExo (but not of ApoBodies) to allografted WT mice did not affect anti-vimentin, anti-AT1R and anti-fibronectin levels but increased anti-LG3 (p<0.01) and ANA (p<0.005) levels and heightened vascular remodelling (p<0.01). IL-17 rich lymphoid-like structures (TLS) formation was observed within vascular allografts in apoExo injected WT recipient but not in γδT-KO recipient or in WT recipient injected with Bortezo treated apoExo, showing that the proteasome, active within apoExo triggers γδT-17 dependent TLS formation. In conclusion, these results identify IRI derived apoExo as inducers of γδT-17 dependent autoimmune pathways leading to the formation of TLS and accelerated rejection. These results provide novel insights into the cellular and molecular pathways regulating autoimmune responses in association with chronic vascular injury. Canadian Institutes of Health Research (CIHR). Shire Chair in Nephrology, Transplantation and Renal Regeneration of the Université de Montréal. J.-L. Lévesque Foundation. Fonds de Recherche du Québec - Santé (FRQS).

Apoptotic Cell-Derived Extracellular Vesicles: More Than Just Debris.

The many functions of extracellular vesicles (EVs) like exosomes and microvesicles released from healthy cells have been well characterized, particularly in relation to their roles in immune modulation. Apoptotic bodies, a major class of EV released as a product of apoptotic cell disassembly, and other types of EVs released from dying cells are also becoming recognized as key players in this emerging field. There is now increasing evidence to suggest that EVs produced during apoptosis have important immune regulatory roles, a concept relevant across different disease settings including autoimmunity, cancer, and infection. Therefore, this review focuses on how the formation of EVs during apoptosis could be a key mechanism of immune modulation by dying cells.

Apoptotic Cell-Derived Extracellular Vesicles Promote Malignancy of Glioblastoma Via Intercellular Transfer of Splicing Factors

Aggressive cancers such as glioblastoma (GBM) contain intermingled apoptotic cells adjacent to proliferating tumor cells. Nonetheless, intercellular signaling between apoptotic and surviving cancer cells remain elusive. In this study, we demonstrate that apoptotic GBM cells paradoxically promote proliferation and therapy resistance of surviving tumor cells by secreting apoptotic extracellular vesicles (apoEVs) enriched with various components of spliceosomes. apoEVs alter RNA splicing in recipient cells, thereby promoting their therapy resistance and aggressive migratory phenotype. Mechanistically, we identified RBM11 as a representative splicing factor that is upregulated in tumors after therapy and shed in extracellular vesicles upon induction of apoptosis. Once internalized in recipient cells, exogenous RBM11 switches splicing of MDM4 and Cyclin D1 toward the expression of more oncogenic isoforms.

Apoptotic vesicles: deathly players in cancer-associated coagulation.

Although cancer is associated with coagulation disorders, it is still unclear how the combination of tumor cell and host factors enhance the hypercoagulable state of cancer patients. Emerging evidence points to a central role for tumor endosomal and plasma membrane-derived vesicular components in the pathogenesis of cancer-related thrombosis. In particular, tumor cell membranes and extracellular vesicles (EV) harbor lipids and proteinaceous coagulation factors able to initiate multiple points within the coagulation matrix. The impact of chemotherapy upon a host already burdened with a hypercoagulable state increases the risk of pathological coagulation. We argue that chemotherapy-induced EV harbor the most active components for cancer related thrombosis and discuss how membrane components of the host and tumor act to initiate coagulation to enhance thrombotic risk in cancer patients.

Apoptotic Tumor Cell-Derived Extracellular Vesicles as Important Regulators of the Onco-Regenerative Niche.

Cells undergoing apoptosis produce heterogeneous populations of membrane delimited extracellular vesicles (Apo-EVs) which vary not only in size—from tens of nanometers to several microns—but also in molecular composition and cargo. Apo-EVs carry a variety of potentially biologically active components, including small molecules, proteins, and nucleic acids. Larger forms of Apo-EVs, commonly termed “apoptotic bodies,” can carry organelles, such as mitochondria and nuclear fragments. Molecules displayed on the surface of extracellular vesicles (EVs) can contribute substantially to their size, as well as their functions. Thus far, relatively little is known of the functional significance of Apo-EVs apart from their roles in fragmentation of dying cells and indicated immunomodulatory activities. Here, we discuss EV production by dying tumor cells and consider the possible roles of Apo-EVs in a cell death-driven sector of the tumor microenvironment known as the onco-regenerative niche (ORN). We propose that tumor-derived Apo-EVs are significant vehicles of the ORN, functioning as critical intercellular communicators that activate oncogenic tissue repair and regeneration pathways. We highlight important outstanding questions and suggest that Apo-EVs may harbor novel therapeutic targets.

Endothelial Dysfunction in Kidney Transplantation.

Kidney transplantation entails a high likelihood of endothelial injury. The endothelium is a target of choice for injury by ischemia-reperfusion, alloantibodies, and autoantibodies. A certain degree of ischemia-reperfusion injury inevitably occurs in the immediate posttransplant setting and can manifest as delayed graft function. Acute rejection episodes, whether T-cell or antibody-mediated, can involve the graft micro- and macrovasculature, leading to endothelial injury and adverse long-term consequences on graft function and survival. In turn, caspase-3 activation in injured and dying endothelial cells favors the release of extracellular vesicles (apoptotic bodies and apoptotic exosome-like vesicles) that further enhance autoantibody production, complement deposition, and microvascular rarefaction. In this review, we present the evidence for endothelial injury, its causes and long-term consequences on graft outcomes in the field of kidney transplantation.

Communicating with the dead: lipids, lipid mediators and extracellular vesicles.

Apoptosis is a key event in the control of inflammation. However, for this to be successful, dying cells must efficiently and effectively communicate their presence to phagocytes to ensure timely removal of dying cells. Here, we consider apoptotic cell-derived extracellular vesicles and the role of contained lipids and lipid mediators in ensuring effective control of inflammation. We discuss key outstanding issues in the study of cell death and cell communication, and introduce the concept of the 'active extracellular vesicle' as a metabolically active and potentially changing intercellular communicator.

Tumor-Derived Apoptotic Vesicles: With Death They Do Part.

Tumor cells release lipid particles known as extracellular vesicles (EV) that contribute to cancer metastasis, to the immune response, and to thrombosis. When tumors are exposed to radiation or chemotherapy, apoptotic vesicles (ApoVs) are released in abundance as the plasma membrane delaminates from the cytoskeleton. Recent studies have suggested that ApoVs are distinct from the EVs released from living cells, such as exosomes or microvesicles. Depending on their treatment conditions, tumor-released ApoV have been suggested to either enhance or suppress anti-cancer immunity. In addition, tumor-derived ApoV possess procoagulant activity that could increase the thrombotic state in cancer patients undergoing chemotherapy or radiotherapy. Since ApoVs are one of the least appreciated type of EVs, we focus in this review on the distinctive characterization of tumor ApoVs and their proposed mechanistic effects on cancer immunity, coagulation, and metastasis.

Lysosome-Mediated Plasma Membrane Repair Is Dependent on the Small GTPase Arl8b and Determines Cell Death Type in Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis is an extremely successful pathogen, and its success is widely attributed to its ability to manipulate the intracellular environment of macrophages. A central phenomenon of tuberculosis pathology enabling immune evasion is the capacity of virulent M. tuberculosis (H37Rv) to induce macrophage necrosis, which facilitates the escape of the mycobacteria from the macrophage and spread of infection. In contrast, avirulent M. tuberculosis (H37Ra) induces macrophage apoptosis, which permits Ag presentation and activation of adaptive immunity. Previously, we found that H37Rv induces plasma membrane microdisruptions, leading to necrosis in the absence of plasma membrane repair. In contrast, H37Ra permits plasma membrane repair, which changes the host cell death modality to apoptosis, suggesting that membrane repair is critical for sequestering the pathogen in apoptotic vesicles. However, mechanisms of plasma membrane repair induced in response to M. tuberculosis infection remain unknown. Plasma membrane repair is known to induce a Ca2+-mediated signaling, which recruits lysosomes to the area of damaged plasma membrane sites for its resealing. In this study, we found that the small GTPase Arl8b is required for plasma membrane repair by controlling the exocytosis of lysosomes in cell lines and in human primary macrophages. Importantly, we found that the Arl8b secretion pathway is crucial to control the type of cell death of the M. tuberculosis-infected macrophages. Indeed, Arl8b-depleted macrophages infected with avirulent H37Ra undergo necrotic instead of apoptotic cell death. These findings suggest that membrane repair mediated by Arl8b may be an important mechanism distinguishing avirulent from virulent M. tuberculosis-induced necrotic cell death.

An apoptosis-driven 'onco-regenerative niche': roles of tumour-associated macrophages and extracellular vesicles.

The cell-death programme, apoptosis, is well established as a tumour suppressor mechanism. Paradoxically, high levels of apoptosis in tumours are closely coupled with poor prognosis. Indeed, where it has been studied, cell loss is a striking feature of high-grade cancers, illustrating the importance of considering malignant disease as an imbalance between cell gain and cell loss that favours cell gain rather than as a unidirectional disorder of cell gain alone. In addition to orchestrating cell loss, apoptosis can signal regenerative responses—for example compensatory proliferation—in neighbouring cells. Accumulating evidence suggests that normal tissue repair and regenerative processes are hijacked in the malignant tissue microenvironment such that cancer may be likened to a ‘wound that fails to stop repairing’. We have proposed that a critical requirement for the successful growth, progression and re-growth of malignant tumours is a complex milieu, conceptually termed the ‘onco-regenerative niche’, which is composed, in addition to transformed neoplastic cells, of a network of normal cells and factors activated as if in tissue repair and regeneration. Our work is based around the hypothesis that tumour cell apoptosis, macrophage activation and endothelial activation are key, interlinked elements of the onco-regenerative niche and that apoptotic tumour cell–derived extracellular vesicles provide critical intercellular communication vehicles of the niche. In aggressive B-cell lymphoma, tumour cell apoptosis promotes both angiogenesis and the accumulation of pro-tumour macrophages in the lymphoma microenvironment. Furthermore, apoptotic lymphoma-derived extracellular vesicles have potent pro-tumour potential. These findings have important implications for the roles of apoptosis in regulation of malignant diseases and for the efficacy of apoptosis-inducing anti-cancer therapies.This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’.

Phosphatidylserine-mediated platelet clearance by endothelium decreases platelet aggregates and procoagulant activity in sepsis

The mechanisms that eliminate activated platelets in inflammation-induced disseminated intravascular coagulation (DIC) in micro-capillary circulation are poorly understood. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered were and ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or αvβ3 integrin on ECs attenuated platelet clearance resulting in increased platelet count in a mouse model of sepsis. Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets. Pretreatment with lactadherin significantly increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contribute to maintaining platelet homeostasis during acute inflammation. These results suggest a new therapeutic target for impeding the development of DIC.

Abstract 5609: Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that shows striking resistance to standard therapies. Immunotherapy is an exciting new treatment strategy for PDAC, but has yet to reproducibly demonstrate clinical efficacy. This finding may reflect poor effector T cell infiltration into PDAC or mechanisms of peripheral T cell tolerance. We hypothesize that successful immunotherapy in PDAC will require strategies that reverse elements of innate immunity that regulate T cell tolerance. This hypothesis is based on our prior studies in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model of PDAC where macrophages residing outside of the tumor microenvironment were found to regulate the efficacy of a vaccine strategy involving the combination of chemotherapy and a CD40 agonist (FGK45). Specifically, we found that depletion of peripheral phagocytes using clodronate-encapsulated liposomes (CEL) was necessary for gemcitabine chemotherapy and a CD40 agonist to induce productive anti-tumor T cell immunity against spontaneously arising PDAC tumors in the KPC model. We now investigate the mechanism by which CEL-depleted phagocytes regulate T cell tolerance in PDAC using the KPC mouse model and an orthotopic implantation model. We found that CEL depletes macrophages in the spleen and abdominal lymph nodes, thereby reversing the pathologic accumulation of macrophages in these secondary lymphoid organs that is induced by PDAC tumors. CEL targeted two macrophage subsets in the lymph node, CD11b+CD169+F4/80neg subcapsular sinus macrophages (SSM) and CD11b+CD169+F4/80+ medullary sinus macrophages (MSM). These macrophage populations may be exposed to tumor-derived antigens and apoptotic cell vesicles entering the lymph node, and have been previously identified as central orchestrators of tolerance in autoimmune models. We found that both fluorescently-labeled liposomes and OVA-FITC peptide injected into the peritoneal cavity were specifically targeted to SSM and MSM with little uptake by dendritic cells (DC). In contrast, depletion of these first-line phagocytes by CEL shifted the cellular uptake of these model antigens increasing uptake by CD11b+CD11c+ DC which are more efficient in antigen presentation and show increased expression of co-stimulatory molecules after FGK45 treatment. Using an orthotopic model of PDAC, we then determined that CEL can enhance the T cell stimulatory capacity of gemcitabine chemotherapy in combination with FGK45 as seen by a significant increase in CD38, CD44, CD69 and PD-1 expression on CD4 and CD8 T cells in secondary lymphoid organs. Together, our findings suggest that tumor-derived antigens may be sequestered by tolerogenic macrophages in peritumoral lymph nodes that act to impair optimal induction of T cell responses. Thus, strategies aimed at disrupting the tolerogenic functions of these macrophages may offer a novel approach for improving immunotherapy in PDAC. Citation Format: Fee Bengsch, Anni Liu, Kathleen Graham, Gregory L. Beatty. Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5609. doi:10.1158/1538-7445.AM2017-5609

Cancer-associated fibroblasts induce cancer cell apoptosis that regulates invasion mode of tumours.

In the process of cancer spreading, different modes of invasion exist. One is expansive invasion, in which a group of cancer cells gradually expands along with cancer cell proliferation. Invasion of cancer cells is also modified by their interaction with stromal cells including cancer-associated fibroblasts (CAFs). Cancer cells co-invade with CAFs, and invasion by CAFs frequently precede invasion by cancer cells, which indicates CAF-led cancer cell invasion. Here, we show that CAFs induce apoptosis in gastric cancer cells, which prevented expansive invasion by cancer cells and instead facilitated CAF-led invasion. Death receptor 4 and activation of caspase-8 in cancer cells mediated cancer cell apoptosis induced by CAFs, which was dependent on contact between cancer cells and CAFs. Apoptotic cancer cells in turn released apoptotic vesicles and stimulated invasion of CAFs. Accordingly, cancer cells followed the migrating CAFs. Treatment with a caspase inhibitor, ZVAD, or forced expression of a death domain fragment in cancer cells prevented cancer cell apoptosis induced by CAFs and increased expansive invasion by cancer cells in extracellular gel invasion assays, while the rate of cancer cell invasion led by CAFs was decreased. Death domain-fragment expression also prevented intramural invasion by gastric cancer cells in the stomach. Because CAF-led invasion is characterized by the movement of individual cancer cells away from the tumour, adequate cancer cell apoptosis may promote cancer dissemination.

Phosphatidylserine-Mediated Platelet Clearance By Endothelium Decreases Platelet Aggregates and Procoagulant Activity in Sepsis

Introduction: Disorders of coagulation are common in sepsis, with disseminated intravascular coagulation (DIC) occurring in approximately 35 % of severe cases, contributing to microvascular dysfunction and death. Intensive platelet activation in sepsis facilitates platelet aggregation, leading to the formation of microthrombi and platelet depletion. This results in the development of DIC and sepsis-associated thrombocytopenia. Therefore, platelets must be cleared locally and quickly in the early phase of activation. Previous studies mainly focused on the clearance of activated cold-stored and aging platelets as well as platelets in immune-mediated thrombocytopenia. However, platelet activation and their clearance in sepsis are poorly understood. Platelets can form aggregates with leukocytes resulting in leukocyte death, the release of extracellular traps (ETs), increased endothelial permeability, and aggravated thrombosis. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis.Methods: The subjects were septic patients (n=48) and healthy controls (n=48). Platelet engulfment by ECs was observed by electron microscopy, immunofluorescence, or immunochemistry both in vitro and in animal models. The PCA of platelets was measured by clotting time, purified coagulation complex assays, and fibrin formation.Results: Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered to and were ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or integrin on ECs attenuated platelet clearance, resulting in increased platelet count in a mouse model of sepsis (p<0.05). Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets (p<0.01). Pretreatment with lactadherin increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. A large decline in PS exposure on platelets, associated platelet PCA, and PLA formation is seen in patients in remission, which could be attributed to the elimination of abnormal platelets.Conclusions: Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contributes to maintaining platelet homeostasis during acute inflammation. Antiplatelet treatment has been suggested as a novel strategy in sepsis, and we speculate that promoting efficient removal of activated and apoptotic platelets could further improve patient outcomes. Therefore, clearance of activated platelets earlier in the disease process could hasten recovery of homeostasis in circulation by eliminating catalytic platforms for the coagulation pathway, protecting blood cells from excessive activation, and restoring their normal function. Endothelium, at least in part, contributes to platelet disposal and may further improve the hypercoagulable status in inflammation. It is noteworthy that PS-mediated and lactadherin-strengthened platelet engulfment may modify coagulopathy, and thus provide a new modality for treatment of septic clotting disorders. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mechanistic insight into the procoagulant activity of tumor-derived apoptotic vesicles

BackgroundChemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation. MethodsWe utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay. ResultsApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV. ConclusionsApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation. General significanceThis study clarifies the mechanisms of procoagulant activity of vesicles released from dying tumor cells.

Vitamin E (α-Tocopherol) Exhibits Antitumour Activity on Oral Squamous Carcinoma Cells ORL-48.

Cancers involving the oral cavity, head, and neck regions are often treated with cisplatin. In cancer therapy, the main target is to eliminate unwanted cancerous cells. However, reports on the nonselective nature of this drug have raised few concerns. Incorrect nutritional habits and lifestyle practices have been directly linked to cancer incidence. Nutrients with antioxidant activity inhibit cancer cells development, destroying them through oxidative stress and apoptosis. α-tocopherol, the potent antioxidant form of vitamin E is a known scavenger of free radicals. In vitro study exhibited effective antitumor activity of α-tocopherol on ORL-48 at 2.5 ± 0.42 µg/mL. Cisplatin exhibited stronger activity at 1.0 ± 0.15 µg/mL, but unlike α-tocopherol it exhibited cytotoxicity on normal human epidermal keratinocytes at very low concentration (<0.1 µg/mL). Despite the lower potency of α-tocopherol, signs of apoptosis such as the shrinkage of cells and appearance of apoptotic bodies were observed much earlier than cisplatin in time lapse microscopy. No apoptotic vesicles were formed with cisplatin, instead an increased population of cells in the holoclone form which may suggest different induction mechanisms between both agents. High accumulation of cells in the G0/G1 phase were observed through TUNEL and annexin V-biotin assays, while the exhibition of ultrastructural changes of the cellular structures verified the apoptotic mode of cell death by both agents. Both cisplatin and α-tocopherol displayed cell cycle arrest at the Sub G0 phase. α-tocopherol thus, showed potential as an antitumour agent for the treatment of oral cancer and merits further research.

Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles.

Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity.