Shedding Of Microparticles Research Articles

Uptake of tumor-derived microparticles induces metabolic reprogramming of macrophages in the early metastatic lung

Pre-metastatic niche formation is a critical step during the metastatic spread of cancer. One way by which primary tumors prime host cells at future metastatic sites is through the shedding of tumor-derived microparticles as a consequence of vascular sheer flow. However, it remains unclear how the uptake of such particles by resident immune cells affects their phenotype and function. Here, we show that ingestion of tumor-derived microparticles by macrophages induces a rapid metabolic and phenotypic switch that is characterized by enhanced mitochondrial mass and function, increased oxidative phosphorylation, and upregulation of adhesion molecules, resulting in reduced motility in the early metastatic lung. This reprogramming event is dependent on signaling through the mTORC1, but not the mTORC2, pathway and is induced by uptake of tumor-derived microparticles. Together, these data support a mechanism by which uptake of tumor-derived microparticles induces reprogramming of macrophages to shape their fate and function in the early metastatic lung.

Quantifying Stent Wall Shear Stress for Assessing Stent and Flow Diverter Performance for Treating Intracranial Aneurysms

AbstractAnalysis of flow diverting intracranial aneurysm repair devices has traditionally focused on reducing intrasaccular blood flow velocity and aneurysm wall shear stress (WSS) as the primary metrics for improved perceived device performance. However, the interpretation of this data has been debated, particularly with regard to the specific biological benefits of high or low aneurysm WSS. Therefore, this research proposes an additional parameter of WSS at the stent struts that could provide valuable insight regarding the device's potential to promote occlusion at the aneurysm neck by indicating locations of increased platelet activation and microparticle shedding. Fluid flow effects were evaluated for two flow diverters (Pipeline™ and FRED™) and three stents (Enterprise™, Atlas™, and LVIS™) using computational fluid dynamics (CFD) models developed from two patient-derived CTA datasets with aneurysms in the middle cerebral artery (MCA) and basilar artery (BA), respectively. The device WSS data provides an additional metric for evaluating the ability of the device to constrain the blood flow within the main vessel, as well as indicating potential locations of the initiation of aneurysm occlusion. It is hypothesized that high device WSS close to the aneurysm neck creates a higher likelihood of thrombus formation and aneurysm occlusion due to platelet activation and microparticle shedding, while high device WSS proximal or distal on the device would indicate a higher likelihood of undesirable daughter vessel occlusion. Conversely, low-to-moderate device WSS throughout the device length could be interpreted as a reduced likelihood of complete occlusion of the aneurysm over time, resulting in lesser device performance.

Microvascular Thrombosis as a Critical Factor in Severe COVID-19.

Platelet-endothelial interactions have a critical role in microcirculatory function, which maintains tissue homeostasis. The subtle equilibrium between platelets and the vessel wall is disturbed by the coronavirus disease 2019 (COVID-19), which affects all three components of Virchow's triad (endothelial injury, stasis and a hypercoagulable state). Endotheliitis, vasculitis, glycocalyx degradation, alterations in blood flow and viscosity, neutrophil extracellular trap formation and microparticle shedding are only few pathomechanisms contributing to endothelial damage and microthrombosis resulting in capillary plugging and tissue ischemia. In the following opinion paper, we discuss major pathological processes leading to microvascular endothelial activation and thrombosis formation as a possible major adverse factor driving the deterioration of patient disease course in severe COVID-19.

Vitreous M2 Macrophage-Derived Microparticles Promote RPE Cell Proliferation and Migration in Traumatic Proliferative Vitreoretinopathy.

PurposeTo characterize vitreous microparticles (MPs) in patients with traumatic proliferative vitreoretinopathy (PVR) and investigate their role in PVR pathogenesis.MethodsVitreous MPs were characterized in patients with traumatic PVR, patients with rhegmatogenous retinal detachment (RRD) complicated with PVR, and control subjects by flow cytometry. The presence of M2 macrophages in epiretinal membranes was measured by immunostaining. Vitreous cytokines were quantified by ELISA assay. For in vitro studies, MPs isolated from THP-1 cell differentiated M1 and M2 macrophages, termed M1-MPs and M2-MPs, were used. The effects and mechanisms of M1-MPs and M2-MPs on RPE cell proliferation, migration, and epithelial to mesenchymal transition were analyzed.ResultsVitreous MPs derived from photoreceptors, microglia, and macrophages were significantly increased in patients with traumatic PVR in comparison with control and patients with RRD (PVR), whereas no significance was identified between the two control groups. M2 macrophages were present in epiretinal membranes, and their signature cytokines were markedly elevated in the vitreous of patients with traumatic PVR. Moreover, MPs from M2 macrophages were increased in the vitreous of patients with traumatic PVR. In vitro analyses showed that M2-MPs promoted the proliferation and migration of RPE cells via activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. However, M2-MPs did not induce the expression of fibrotic proteins, including fibronectin, α-smooth muscle actin, and N-cadherin in RPE cells.ConclusionsThis study demonstrated increased MP shedding in the vitreous of patients with traumatic PVR; specifically, MPs derived from M2 polarized macrophages may contribute to PVR progression by stimulating RPE cell proliferation and migration.

Hyperuricemia enhances procoagulant activity of vascular endothelial cells through TMEM16F regulated phosphatidylserine exposure and microparticle release

The link between serum uric acid (SUA) and the risk of venous thromboembolism (VTE) is well established. Recent data suggested a causative role of UA in endothelial cells (ECs) dysfunction. However, the molecular mechanism of high UA on thrombogenesis is unknown. We investigate whether high UA induce phosphatidylserine (PS) externalization and microparticle (MP) shedding in cultured EC, and contribute to UA-induced hypercoagulable state. In the present study, we demonstrate that UA induces PS exposure and EMP release of EC in a concentration- and time-dependent manner, which enhances the procoagulant activity (PCA) of EC and inhibited over 90% by lactadherin in vitro. Furthermore, hyperuricemic rat model was used to evaluate the development of thrombi following by flow stasis in the inferior vena cava (IVC). Hyperuricemia group is more likely to form large and hard thrombi compared with control. Importantly, we found that TMEM16F expression is significantly upregulated in UA-treated EC, which is crucial for UA-induced PS exposure and MP formation. Additionally, UA increases the generation of reactive oxygen species (ROS), lipid peroxidation, and cytosolic Ca2+ concentration in EC, which might contribute to increased TMEM16F expression. Using confocal microscopy, we also observed disruption of the actin cytoskeleton, suggesting that depolymerization of actin filaments might be required for TMEM16F activation and followed by PS exposure and membrane blebbing in UA-treated EC. Our results demonstrate a thrombotic role of EC in hyperuricemia through TMEM16F-mediated PS exposure and MPs release.

Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells

BackgroundThrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs).MethodsWhole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer.ResultsExposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index).ConclusionsTaken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases.FKY5xz95WsHn6abiyZ6BGLVideo abstract

HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated.Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo.Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg).Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

Endoplasmic Reticulum (ER) stress‐generated microparticles self‐perpetuate ER stress and mediate endothelial cell dysfunction independently of cell survival

Microparticles (MPs) are nanosized membrane‐shed vesicles released from activated cells. MPs can induce endothelial dysfunction, the first step in atherosclerosis, through endoplasmic reticulum (ER) stress. However, it is not known whether ER stress can lead to MPs release and what biological messages these MPs would carry. Therefore, we aimed to assess the impact of ER stress on MPs shedding from endothelial cells, and to investigate their effects on endothelial function.Human umbilical vein endothelial cells (HUVECs) were treated for 24h with ER stress inducer, thapsigargin, in the presence or absence of 4‐Phenylbutyric acid (PBA), a chemical chaperone to inhibit ER stress. Then, MPs were isolated and used to treat naïve HUVECs (20 g/mL) for 24‐48h before assessing ER‐stress response, autophagy and apoptosis.MPs generated under ER stress conditions did not differ quantitatively from controls; however, they carried deleterious messages for endothelial function. The treatment of naïve HUVECs with ER stress‐generated MPs (48h) increased mRNA and protein expression of key ER stress markers [activating transcription factor (ATF)‐4, and Binding immunoglobulin protein (BiP)], indicating a vicious circle between MPs production and ER stress activation. ER stress‐generated MPs impaired the angiogenic capacity of HUVECs to form tube‐like structures, indicating an impaired endothelial function. However, ER stress‐generated MPs did not affect autophagy (no changes in protein expression of LC3‐II) or apoptosis in HUVECs.Our data show that ER stress activation caused the shedding of MPs, which were capable of activating ER stress in HUVECs in a vicious circle and impaired angiogenic capacity of these cells. These MPs impaired endothelial function in a mechanism independent from cell death and autophagy.Support or Funding InformationThis work was supported by awards from Qatar University (QUUG‐CPH‐CPH‐15/16‐6; QUST‐2‐CPH‐2017‐19; QUST‐1‐CPH‐2018‐1) to Dr Abdelali Agouni. The findings achieved herein are solely the responsibility of the authors.

Microparticles as Potential Mediators of High Glucose-Induced Renal Cell Injury.

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease worldwide. Activation of signaling pathways such as the mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), endoplasmic reticulum (ER) stress, transforming growth factor-beta (TGF-β), and epithelial-mesenchymal transition (EMT), are thought to play a significant role in the etiology of DN. Microparticles (MPs), the small membrane vesicles containing bioactive signals shed by cells upon activation or during apoptosis, are elevated in diabetes and were identified as biomarkers in DN. However, their exact role in the pathophysiology of DN remains unclear. Here, we examined the effect of MPs shed from renal proximal tubular cells (RPTCs) exposed to high glucose conditions on naïve RPTCs in vitro. Our results showed significant increases in the levels of phosphorylated forms of 4E-binding protein 1 and ERK1/2 (the downstream targets of mTOR and ERK pathways), phosphorylated-eIF2α (an ER stress marker), alpha smooth muscle actin (an EMT marker), and phosphorylated-SMAD2 and nuclear translocation of SMAD4 (markers of TGF-β signaling). Together, our findings indicate that MPs activate key signaling pathways in RPTCs under high glucose conditions. Pharmacological interventions to inhibit shedding of MPs from RPTCs might serve as an effective strategy to prevent the progression of DN.

The effect of mild agonist stimulation on the platelet reactivity in patients with type 2 diabetes mellitus

BackgroundPatients with type 2 diabetes mellitus (T2DM) have accelerated atherosclerosis as a pro thrombotic state that is associated with the platelet activation priming. Platelets, which undergo the continuous mild stimulation, may lose their sensitivity to react to a strong stimulation. The present study aimed to investigate activation responses of platelets to mild and subsequent strong stimulations in patients with T2DM and healthy individuals.MethodsBlood samples, which were taken from 40 patients with T2DM and 35 healthy individuals, were collected into the citrate containing tubes. The samples were subjected to the soft centrifugation to prepare the platelet rich plasma (PRP). Platelets in PRP samples were treated at a low (1 μM) concentration and then at a high (10 μM) concentration of ADP. Before and after stimulation with different doses of ADP, levels of CD62P expression and formation of platelet micro particles (PMPs) were measured using a flow cytometry method.ResultsThe platelets from patients with T2DM had higher levels of CD62P expression before any stimulation (P = 0.003) than control samples. Platelets, which underwent the mild stimulation, indicated lower responses to CD62P expression, but higher PMPs formation after stimulation with high dose of ADP. Patients with T2DM had higher platelet micro particles in all states with the ADP stimulation. (P = 0.004, SD: ±74.52).ConclusionsThe flow cytometry data indicated that platelets were pre-active and associated with metabolic conditions in patients with type 2 diabetes mellitus. The induction of desensitization state helped platelets to reduce the platelet activation and sensitivity to ADP in a diabetic environment. Furthermore, the production of platelets micro-particles was high in the patients; and desensitized platelets were more susceptible to shedding of micro-particles.

Microparticle Responses to Aerobic Exercise and Meal Consumption in Healthy Men.

Microparticles (MP) are shed extracellular vesicles that express the prothrombotic tissue factor (TF). Aerobic exercise may reduce MP count and TF expression. This study investigated the impact of acute running or rest followed by standardized meal consumption on MP phenotypes and TF expression. Fifteen males (age, 22.9 ± 3.3 yr; body mass, 81.9 ± 11.4 kg; V˙O2max, 54.9 ± 6.5 mL·kg·min; mean ± SD) completed 1 h of running (70% V˙O2max) or rest at 9:00 AM and consumed a standardized meal (1170 kcal, 43% CHO, 17% PRO, 40% fat) at 10:45 AM. Venous blood samples were taken at 9:00 AM, 10:00 AM, and 11:30 AM. The MP concentration, diameter, phenotypes, and TF expression were assessed using nanoparticle tracking analysis and flow cytometry. Nanoparticle tracking analysis identified no changes in MP concentration or diameter in response to time or trial. Flow cytometry revealed total MP count increased from 9:00 AM to 10:00 AM (1.62 ± 2.28 to 1.74 ± 2.61 × 10 L, P = 0.016, effect size (η) = 0.105), but was unaffected by trial. TF platelet-derived MP % reduced from 9:00 AM to 10:00 AM (44.0% ± 21.2% to 21.5% ± 9.3%, P = 0.001, η = 0.582) after exercise only (control, 36.8% ± 18.2% to 34.9% ± 11.9%; P = 0.972). TF neutrophil-derived MP percentage reduced from 9:00 AM to 11:30 AM (42.3% ± 17.2% to 25.1% ± 14.9%; P = 0.048, η = 0.801) in the exercise trial only (control, 28.5% ± 15.7% to 32.2% ± 9.6%; P = 0.508). Running induced a significant reduction in %TF platelet and neutrophil MP, suggesting a transient reduction in cardiovascular risk via reduced TF-stimulated thrombosis. This requires further investigation over longer periods in cardiovascular disease populations.

Circulating microparticles as biomarkers of stroke: A focus on the value of endothelial- and platelet-derived microparticles.

Stroke is one of the leading causes of mortality anddisability worldwide. Numerouspathophysiological mechanisms involving blood vessels, coagulation and inflammation contribute to the vascular occlusion. Perturbations in these pathways can be detected by numerous methods including changes in endoplasmic membrane remodeling and rearrangement leading to the shedding of microparticles (MPs) from various cellular origins in the blood. MPs are small membrane-derived vesicles that are shed from nearly all cells in the body in resting state or upon stimulation. MPs act as biological messengers to transfer information to adjacent and distant cells thus regulating various biological processes. MPs may be important biomarkers and tools for the identification of the risk and diagnosis of cerebrovascular diseases. Endothelial activation and dysfunction and altered thrombotic responses are two of the main features predisposing to stroke. Endothelial MPs (EMPs) have been recognized as both biomarkers and effectors of endothelial cell activation and injury while platelet-derived MPs (PMPs) carry a strong procoagulant potential and are activated in thrombotic states. Therefore, we reviewed here the role of EMPs and PMPs as biomarkers of stroke. Most studies reported high circulating levels of EMPs and PMPs in addition to other cell origins in stroke patients and have been linked to stroke severity, the size of infarction, and prognosis. The identification and quantification of EMPs and PMPs may thus be useful for the diagnosis and management of stroke.

Microparticulate P2X7 and GSDM-D mediated regulation of functional IL-1β release.

The pro-inflammatory cytokine IL-1β is a secreted protein that is cleaved by caspase-1 during inflammasome activation upon recognition of internal and external insults to cells. Purinergic receptor P2X7 has been described to be involved in the release pathway of bioactive mature IL-1β by activated immune cells. Microparticle (MP) shedding has also been recently recognized as a manner of cytokine IL-1β release. However, the understanding of purinergic receptor roles in the MP-mediated IL-1β release process is still rudimentary. Gasdermin-D (GSDM-D), a protein involved in pyroptosis and inflammasome activation, has been recently described to be involved in the release of microparticles by virtue of its pore-forming ability. Hence, our current work is aimed to study the role of P2X7 in regulating GSDM-D-mediated microparticles and thereby bioactive IL-1β release. We provide evidence that cleaved functional IL-1β release in microparticles upon LPS stimulation is regulated by GSDM-D and P2X7 in a two-step fashion. GSDM-D activation first regulates release of IL-1β and P2X7 into microparticles. Then, microparticulate active P2X7 receptor then regulates the release of bioactive IL-1β encapsulated in microparticles to be able to target other cells inducing IL-8. Using an ATP model of stimulation, we further demonstrated that extracellular ATP stimulation to IL-1β containing LPS microparticles induces release of its content, which when subjected to epithelial cells induced IL-8. This effect was blocked by P2X7 inhibitor, KN62, as well as by IL-1RA. Taken together, our findings demonstrate for the first time the synergistic critical roles of GSDM-D and purinergic receptors in the regulation of microparticulate bioactive IL-1β release and induction of target cell responses.

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner.

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

Effect of Daunorubicin on Release and Procoagulant Activity of Microparticles Derived from Acute Promyelocytic Leukemia Cells

To investigate the effect of daunorubicin on the number and procoagulant activity of Microparticles derived from acute promyelocytic leukemia(APL) cells. APL cells were isolated from bone Marrow of 5 newly diagnosed APL patients, the bone marrow mononuclear cells were collected from 5 patients with iron deficiency anemia as control.APL cells were treated with different concentration of daunorubicin(0.1,0.5,1.0 and 2.0µmol/L) for 24 h. Microparticles were extracted from the cell culture medium for qualitative anaysis of the extracted microparticles.The morphologic features of the microparticles were observed by transmission electron microscopy.The number of microparticles was detected by flow cytometry.The procoagulant activity of microparticles was measured by recalcification time assays. Under a transmission electron microscope, theextracted microparticles took a round or oval morphology with a transparent center,and their diameters were arund 100nm, consistent with the morphological characteristics of microparticles. Compared with bone marrow mononuclear cells-derived microparticles,the counts of the bone marrow APL cells-derived microparticles significantly increased(P<0.05).Daunorubicin increased the shedding of microparticles in a dose-dependent manner(r=0.73,P<0.01).Compared with normal bone marrow mononuclear cells-derived microparticles,bone marrow APL cells-derived microparticles showed higher procoagulant activity(P<0.05).Daunorubicin treatment enhanced the prccoagulant activity of APL cells-derived microparticles which paralleled the increasing drug concentrations(r=-0.78,P<0.01). Daunorubicin can promote the release of APL cells-derived microparticles and enhance their related procoagulan activity.

Red Blood Cell Homeostasis: Mechanisms and Effects of Microvesicle Generation in Health and Disease.

Red blood cells (RBCs) generate microvesicles to remove damaged cell constituents such as oxidized hemoglobin and damaged membrane constituents, and thereby prolong their lifespan. Damage to hemoglobin, in combination with altered phosphorylation of membrane proteins such as band 3, lead to a weakening of the binding between the lipid bilayer and the cytoskeleton, and thereby to membrane budding and microparticle shedding. Microvesicle generation is disturbed in patients with RBC-centered diseases, such as sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, spherocytosis or malaria. A disturbance of the membrane-cytoskeleton interaction is likely to be the main underlying mechanism, as is supported by data obtained from RBCs stored in blood bank conditions. A detailed proteomic, lipidomic and immunogenic comparison of microvesicles derived from different sources is essential in the identification of the processes that trigger vesicle generation. The contribution of RBC-derived microvesicles to inflammation, thrombosis and autoimmune reactions emphasizes the need for a better understanding of the mechanisms and consequences of microvesicle generation.

The intersection of protein disulfide isomerase and cancer associated thrombosis

The mechanisms underlying the hypercoagulability of cancer are complex and include the upregulation coagulation factors or procoagulant proteins, shedding of microparticles, and direct activation of vascular cells. Protein disulfide isomerase (PDI) is a thiol isomerase secreted from activated platelets and endothelial cells and plays a critical role in both platelet aggregation and fibrin generation. A number of potential intravascular targets of PDI have been identified including cell surface receptors (e.g. β-integrins and glycoprotein Ib), receptor ligands (e.g. fibrinogen and von Willebrand factor), serine proteases (e.g. cathepsin G and kallekrein-14), and coagulation factors (e.g. factor XI and factor V). Recent clinical studies demonstrated that a small molecule inhibitor of PDI, isoquercetin, decreases platelet-dependent thrombin generation and PDI activity in plasma following oral administration. This review explores the mechanistic overlap between the molecular drivers of cancer associated thrombosis and the potential roles PDI plays in mediating thrombosis. These molecular insights provide rationale for clinical trials targeting PDI to prevent thrombosis in cancer patients.

Brain Microvascular Endothelial Derived Microparticles as a Non‐Invasive Diagnostic Tool for Multiple Sclerosis

BackgroundMultiple sclerosis (MS), an autoimmune‐neurodegenerative disease, exhibits features of neurovascular inflammation including endothelial activation and shedding of microparticles (MPs). While the brain lacks ‘classical’ lymphatics, we previously reported expression of venous‐lymphatic endothelial markers (LYVE‐1, podoplanin, prox‐1, VEGFR‐3, VEGF‐C, VEGF‐D) in both brain tissue and sera, with altered expression of these proteins in MS. Based on differential expression of these markers in brain tissue, we propose that these markers may originate within brain microvascular endothelium and undergo changes in disease which can be analyzed diagnostically.ObjectivesTo validate the neurolymphatic biomarker matrix as a novel, non‐invasive and cost‐effective tool for diagnosing MS and discriminating MS subtypes.MethodsNeurolymphatic biomarker expression was determined in human cerebral microvascular endothelial cells (hCMECs) following 24–48 hours of TNF‐α (20 ng/ml) exposure +/− IFN‐γ (1000 U/ml) using western blotting. MPs from these cells were visualized using scanning electron microscopy. Human sera were analyzed by flow cytometry, western and dot blotting to characterize relative partitioning of neurolymphatic biomarkers in MP.ResultshCMECs released MPs that contain lymphatic and junctional markers as well as caveolin‐1. Plasma MPs were seen to liberate soluble (non‐particulate) neurolymphatic biomarkers upon storage, indicating that serum represents a more stable MP‐derived pool of these markers. MP flow analysis of intracellular actin revealed that permeabilization destabilizes phosphatidylserine‐Annexin V interaction which is used to designate MP identity, suggesting that flow cytometry of MPs may require more judicious interpretation. All neurolymphatic markers so far tested were able to distinguish MS from healthy control serum samples; FOXC2 expression can further distinguish subtypes of MS: RRMS from SPMS.ConclusionLymphatic biomarkers in plasma and sera are expressed by brain microvascular endothelial cells and exhibit changes in expression in response to inflammatory stimuli (cytokines). Such alterations parallel transfer of these markers into caveolin‐1 ‐enriched microparticles, which represent a circulating ‘snapshot’ of the vascular surface that can be analyzed by several immune approaches, particularly following storage‐dependent solubilization of these biomarkers.Support or Funding InformationSupported by the Annette Funicello Research Fund for Neurological Diseases and the Malcolm Feist Predoctoral Fellowship from Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center‐ShreveportThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Atrial endothelial cells senescence promotes thrombogenicity, inflammation and extracellular matrix remodeling: Role of the local Ang II/AT1 receptor pathway

Ageing promotes atrial remodeling that paves way to atrial fibrillation and thrombogenicity. Preclinical studies on endothelial atrial cells are lacking. This study aims to characterize phenotypical changes associated with atrial endothelial cells senescence and to decipher the link between ageing and thrombogenicity. Atrial endothelial cells (AEC) were obtained from freshly harvested porcine left atria. Endothelial senescence was assessed by senescence-associated beta-galactosidase activity (SA-β-gal), using flow cytometry, protein expression by Western blot analysis and platelet aggregation using an aggregometer. Replicative senescence was induced by passaging AEC from passage P1 to P4, and premature endothelial cell senescence by exposing AEC to L-NAME, an endothelial NO synthase (eNOS) inhibitor or H 2 O 2 . AEC senescence was characterized by an increase in SA-β-gal activity and an up-regulation of p53, a key regulator of cellular senescence, and of p21 and p16, key cyclin-dependent kinase inhibitors. Senescent AEC phenotype was characterized by: – cell thrombogenicity through an up-regulation of tissue factor expression, shedding of procoagulant microparticles, eNOS down-regulation and reduced NO-mediated inhibition of platelet aggregation; – cell adhesion through up-regulation of ICAM-1; – proteolysis and fibrosis remodeling through MMP-2, 9 and TGF-β1 expression; – up-regulation of the local Ang II system through enhanced AT1 receptors (AT1R) and angiotensin-converting enzyme (ACE) expression. Losartan, an AT1 receptor antagonist, and Perindoprilat, an ACE inhibitor, prevented atrial endothelial cell senescence. Thus atrial endothelial senescence promotes thrombogenicity, inflammation, matrix remodeling and the up-regulation of the local Ang II system. They further suggest that targeting the Ang II/AT1R pathway may be a promising therapeutic strategy to delay atrial endothelial ageing.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function.10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays.Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%.Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function.