Cell Surface Phosphatidylserine Research Articles

Platelet and endothelial cell responses under concurrent shear stress and tensile strain

Thrombosis can lead to significant mortality and morbidity. Both platelets and vascular endothelial cells play significant roles in thrombosis. Platelets' response to blood flow-induced shear stress can vary greatly depending on shear stress magnitude, pattern and shear exposure time. Endothelial cells are also sensitive to the biomechanical environment. Endothelial cell activation and dysfunction can occur under low oscillatory shear stress and low tensile strain. Platelet and endothelial cell interaction can also be affected by mechanical conditions. The goal of this study was to investigate how blood flow-induced shear stress, vascular wall tensile strain, platelet-endothelial cell stress history, and platelet-endothelial cell interaction affect platelet thrombogenicity. Platelets and human coronary artery endothelial cells were pretreated with physiological and pathological shear stress and/or tensile strain separately. The pretreated cells were then put together and exposed to pulsatile shear stress and cyclic tensile strain simultaneously in a shearing-stretching device. Following treatment, platelet thrombin generation rate, platelet and endothelial cell activation, and platelet adhesion to endothelial cells was measured. The results demonstrated that shear stress pretreatment of endothelial cells and platelets caused a significant increase in platelet thrombin generation rate, cell surface phosphatidylserine expression, and adhesion to endothelial cells. Shear stress pretreatment of platelets and endothelial cells attenuated endothelial cell ICAM-1 expression under stenosis conditions, as well as vWF expression under recirculation conditions. These results indicate that platelets are sensitized by prior shearing, while in comparison, the interaction with shear stress-pretreated platelets may reduce endothelial cell sensitivity to pathological shear stress and tensile strain.

Inhibition of Cell Proliferation and Induction of Cell Cycle Arrest in Colon Cancer Cells by Lyophilized Mango (Mangifera indica L.) Pulp Extract.

The present study evaluated the antiproliferative capacity and possible cell death mechanisms of lyophilized mango pulp extract (LMPE), applied to human colon cancer cells (SW480) and their metastasis-derived counterparts (SW620). The total phenolic content of LMPE was estimated by the Folin-Ciocalteu method. Three assays were employed to determine its antioxidant capacity: ferric-reducing antioxidant power, oxygen radical absorbance capacity, and 2,2-diphenyl-1-picrylhydrazyl. Furthermore, the antiproliferative activity of LMPE was assessed by sulforhodamine B, clonogenic, and Ki-67 assays. Flow cytometry was employed to examine the cell cycle, production of intracellular reactive oxygen species (ROS), cell-surface phosphatidylserine, and change in mitochondrial membrane potential. LMPE exhibited a high level of total phenolic content and antioxidant activity. The mean maximal inhibitory concentration values of LMPE at 48 h of exposure were 43 and 29 mg/mL for SW480 and SW620, respectively. In the SW480 and SW620 cell lines, LMPE at 50 mg/mL and 48 h of exposure induced an increase in intracellular ROS, cell cycle arrest in the G2/M phase, and probably, apoptotic processes without mitochondrial depolarization. LMPE had an antiproliferative capacity against the human colorectal cancer cell lines SW480 and SW620. These results highlight the chemopreventive potential of LMPE in colorectal cancer treatments.

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phaseI/II/III clinical trials. Erythrocytes do not have anucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin‑V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Involvement of phosphatidylserine receptors in the skeletal muscle regeneration: therapeutic implications.

Sarcopenia is a progressive loss of muscle mass and strength with a risk of adverse outcomes such as disability, poor quality of life, and death. Increasing evidence indicates that diminished ability of the muscle to activate satellite cell‐dependent regeneration is one of the factors that might contribute to its development. Skeletal muscle regeneration following myogenic cell death results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibres. Satellite cell differentiation is not a satellite cell‐autonomous process but depends on signals provided by the surrounding cells. Infiltrating macrophages play a key role in the process partly by clearing the necrotic cell debris, partly by producing cytokines and growth factors that guide myogenesis. At the beginning of the muscle regeneration process, macrophages are pro‐inflammatory, and the cytokines produced by them trigger the proliferation and differentiation of satellite cells. Following the uptake of dead cells, however, a transcriptionally regulated phenotypic change (macrophage polarization) is induced in them resulting in their transformation into healing macrophages that guide resolution of inflammation, completion of myoblast differentiation, myoblast fusion and growth, and return to homeostasis. Impaired efferocytosis results in delayed cell death clearance, delayed macrophage polarization, prolonged inflammation, and impaired muscle regeneration. Thus, proper efferocytosis by macrophages is a determining factor during muscle repair. Here we review that both efferocytosis and myogenesis are dependent on the cell surface phosphatidylserine (PS), and surprisingly, these two processes share a number of common PS receptors and signalling pathways. Based on these findings, we propose that stimulating the function of PS receptors for facilitating muscle repair following injury could be a successful approach, as it would enhance efferocytosis and myogenesis simultaneously. Because increasing evidence indicates a pathophysiological role of impaired efferocytosis in the development of chronic inflammatory conditions, as well as in impaired muscle regeneration both contributing to the development of sarcopenia, improving efferocytosis should be considered also in its management. Again applying or combining those treatments that target PS receptors would be expected to be the most effective, because they would also promote myogenesis. A potential PS receptor‐triggering candidate molecule is milk fat globule‐EGF‐factor 8 (MFG‐E8), which not only stimulates PS‐dependent efferocytosis and myoblast fusion but also promotes extracellular signal‐regulated kinase (ERK) and Akt activation‐mediated cell proliferation and cell cycle progression in myoblasts.

Investigations of the effectiveness of heparin variants as inhibitors of histones

BackgroundExtracellular histones exert cytotoxic and procoagulant effects which contribute to immunothrombosis in vascular diseases such as sepsis. Heparin has been shown to neutralize the pathologic effects of histones in vitro and in animal models. ObjectivesTo compare the effectiveness of unfractionated heparin (UFH), low‐molecularweight heparin (LMWH), Vasoflux (lacks anticoagulant activity), and fondaparinux in neutralizing the cytotoxic and procoagulant activities of histones MethodsBinding affinities between heparin variants and histone subunits were determined by Bio‐layer Interferometry. The ability of heparin variants to diminish the cytotoxic and procoagulant effects of histones was studied by treating endothelial cells or monocytic THP‐1 cells with histones ± heparin variants. ResultsUnfractionated heparin, LMWH, and Vasoflux bind histone subunits with high affinities (Kd <1 pM–66.7 nM) whereas fondaparinux exhibited a low affinity (Kd of 3.06 µM–81.1 mM). UFH, LMWH, and Vasoflux neutralize histone‐mediated cytotoxicity as well as monocytic procoagulant activity (as assessed by cell surface tissue factor and phosphatidylserine). In contrast, fondaparinux has no effect on these activities. All four heparin variants reverse histone‐mediated impairment of APC generation in a dose‐dependent manner. ConclusionsThe ability of heparin to neutralize the cytotoxic and procoagulant effects of histones require heparin fragments >1.7 kDa and is independent of the antithrombin‐binding pentasaccharide. In contrast, the ability of heparin to neutralize histone‐mediated impairment of APC generation is independent of size and anticoagulant activity. These findings suggest that heparin variants may have differential therapeutic potential in vascular diseases associated with elevated levels of histones.

Axonal degeneration induces distinct patterns of phosphatidylserine and phosphatidylethanolamine externalization

Axonal degeneration is a common feature of multiple neurodegenerative diseases, yet the mechanisms underlying its various manifestations are incompletely understood. We previously demonstrated that axonal degeneration is associated with externalization of phosphatidylserine (PS), which precedes morphological evidence of degeneration, is redox-sensitive, and is delayed in Wallerian degeneration slow (WldS) mutant animals. Phosphatidylethanolamine (PE) is the other major membrane phospholipid in the inner leaflet of the cell membrane, and given that PS signals apoptosis, phagocytosis, and degeneration, we hypothesized that PS and PE membrane dynamics play distinct roles in axonal degeneration. To test this hypothesis, axonal degeneration was induced with calcium ionophores in postnatal rat retinal ganglion cells, and PS- and PE-specific fluorescent probes used to measure their externalization over time. In untreated cells, cell-surface PS was prominent in the cell body alone. Elevation of intracellular calcium with calcium ionophores resulted in significantly increased levels of PS externalization in the cell body, axon, and axon growth cone. Unlike PS, cell-surface PE was diffusely distributed in untreated cells, with comparable levels across the soma, axons, and axon terminals. After exposure to calcium ionophores, PE externalization significantly increased in the cell body and axon. Elevated intracellular calcium also resulted in the formation of axonal blebs which exclusively contained externalized PS, but not PE. Together, these results indicated distinct patterns of externalized PS and PE in normal and degenerating neurons, suggesting a differential role for these phospholipids in transducing neuronal injury.

Pathophysiologic Implications of Type-2 Phosphatidylserine-Positive Erythrocytes in Hemoglobin SS Disease (sickle cell anemia) and β-Thalassemia

Background: Phosphatidylserine (PS), a cell membrane phospholipid present in the inner layer of the plasma membrane, is expressed on the surface of red blood cells (RBCs) from individuals with congenital and acquired hemoglobinopathies including sickle cell disease (SCD) and thalassemia. These cells are involved in the genesis of several pathobiologic processes including perturbed hemostasis. Based on the amount of cell surface PS, Yasin et al (Blood, 2003) have grouped PS+ RBCs into low level PS type-1 (PS1) and high level PS type-2 (PS2) cells. In SCD, the majority of transferrin receptor-positive (CD71+) stress reticulocytes (retics) are PS1+, whereas the majority of PS+ dense cells are PS2+and are specific to SCD. PS exposure on retics in subjects with Hemoglobinopathies appears to be physiologically relevant and may play a role in retic maturation. In contrast, dense cells may play a role in the pathogenesis of several SCD-related complications including skin ulcers, pulmonary hypertension and renal disease.Aim: To assess whether PS2+ red cells are present in other hemoglobinopathies besides SCD, and to evaluate which PS fraction associates with hemolysis and hemostatic activation.Methods: Sixty-one children with SCD (38 HbSS and 23 HbSC, age range 1.5 to 22 years), 23 age- and race-matched controls, and 11 with β-thalassemia were evaluated for the presence of PS+ RBCs by flow cytometry using RBCs labeled with annexin V-FITC (PS marker) and anti-glycophorin A-PE (red cell marker). The data was analyzed for PS1+ and PS2+ RBCs, and also for PS per RBC assessed as mean cell fluorescence (MCF). PS levels among groups and between groups were compared for statistical differences. Representative markers of hemostatic perturbation (D-dimer) and hemolysis (LDH) were measured, and relationships with PS1+ and PS2+ RBCs evaluated. In in vitro assays, osmotic fragility of PS2+ RBCs was tested. In a subset of SCD and controls, PS+ RBCs were further characterized using red cells labeled with annexin V-FITC and anti-CD71-PE (a stress retic marker) to delineate PS1+ and PS2+ stress retics and non-retics. Data was analyzed in HbSS based on stress retic levels (low vs high).Results: In contrast to Yasin, we found that PS2 cells were not specific for HbSS, since they were present at similar levels in subjects with β-thalassemia (Figure 1). Both PS1+ and PS2+ red cells (percent and absolute number) as also PS/cell were significantly increased in HbSS compared to HbSC and controls (Table 1). In HbSS, PS2+ RBCs correlated positively with D-dimer (p<0.02). No D-dimer correlation was found with PS1+ RBCs. Neither PS1+ nor PS2+ RBCs in HbSC correlated with D-dimer. Additional analysis demonstrated that PS2+ RBCs in HbSS correlated with the hemolytic marker LDH (p<0.001). In a complementary in vitro assay, we found that PS2+ RBCs were osmotically fragile and readily hemolyze. To confirm the previous demonstration by Yasin of minimal to no PS2+ stress retics in HbSS, we further undertook an analysis of this biomarker in HbSS subjects with low vs high stress retics. We demonstrate that while PS2+ stress retics were present minimally in subjects with low stress retics, they were significantly elevated in individuals with high stress retics (median PS2+ stress retics of 827 and 3269 /μl blood, p<0.03). A positive correlation was also noted between PS2+ stress retics and CD71+ RBCs (r=0.50, p<0.01, n=26).Conclusion: We demonstrate that PS2+ RBCs, which are elevated in HbSS compared to HbSC and controls, are not specific to SCD as they were also present at similarly elevated levels in β-thalassemia. We also demonstrate the presence of PS2+ stress retics in HbSS subjects with high retic levels. Previous studies in HbSS implicated PS+ RBCs in modulating hemostasis. Our results delineate that PS2+ red cells are the PS sub-group involved in this activation. We further demonstrate that PS2+ red cells are associated with elevated LDH levels. These cells are osmotically fragile releasing multiple inflammatory mediators including heme, which is a modulator of thrombin generation. These findings taken together suggest a pathophysiologic role for PS2+ red cells linking hemolysis with hemostasis in hemoglobinopathies including HbSS and β-thalassemia.Acknowledgments: supported by NIH grants P60HL62148, U54HL70585 and P20GM109021Data was collected at St Christopher's Hospital for Children and Thomas Jefferson University, Philadelphia PA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting of elevated cell surface phosphatidylserine with saposin C-dioleoylphosphatidylserine nanodrug as individual or combination therapy for pancreatic cancer.

Pancreatic cancer is one of the deadliest of cancers with a five-year survival of roughly 8%. Current therapies are: surgery, radiation and chemotherapy. Surgery is curative only if the cancer is caught very early, which is rare, and the latter two modalities are only marginally effective and have significant side effects. We have developed a nanosome comprised of the lysosomal protein, saposin C (SapC) and the acidic phospholipid, dioleoylphosphatidylserine (DOPS). In the acidic tumor microenvironment, this molecule, SapC-DOPS, targets the phosphatidylserine cancer-biomarker which is predominantly elevated on the surface of cancer cells. Importantly, SapC-DOPS can selectively target pancreatic tumors and metastases. Furthermore, SapC-DOPS has exhibited an impressive safety profile with only a few minor side effects in both preclinical experiments and in phase I clinical trials. With the dismal outcomes for pancreatic cancer there is an urgent need for better treatments and SapC-DOPS is a good candidate for addition to the oncologist’s toolbox.

Simultaneous Monitoring Cytoplasmic Calcium Ion and Cell Surface Phosphatidylserine in the Necrotic Touch Neurons of Caenorhabditis elegans.

Calcium ions trigger many cellular events, including the release of neurotransmitters at the synaptic terminal and excitotoxic cell death. Recently, we have discovered that a transient increase in the level of cytoplasmic Ca2+ triggers the exposure of phosphatidylserine (PS) on the surfaces of necrotic cells in the nematode Caenorhabditis elegans. PS serves as an "eat me" signal that attracts engulfing cells to engulf and degrade necrotic cells. During the above study, we developed a microscopic imaging protocol for real-time monitoring the levels of cytoplasmic Ca2+ and cell surface PS in Caenorhabditis elegans touch neurons. Previously, Ca2+ dynamics was monitored in neurons in Caenorhabditis elegans larvae in time periods ranging from milliseconds to seconds. Methods for monitoring Ca2+ dynamics for a relatively long period of time during embryonic development were not available, let alone for simultaneous monitoring Ca2+ and PS dynamics. The protocol reported here utilizes a deconvolution imaging system with an optimized experimental setting that reduces photo-damage and allows the proper development of embryos during the real-time imaging process. This protocol enables the simultaneous measurement of cytosolic Ca2+ and cell surface PS levels in necrotic touch neurons during embryonic development in a period longer than six hours. Our method provides an easy and sensitive approach to perform long-time Ca2+ and PS recording in living animals, simultaneously or individually. This protocol can be applied to study various cellular and developmental events that involve the dynamic regulation of Ca2+ and/or PS.

Detection of cell-surface phosphatidylserine using the fluorogenic probe P-IID.

The fluorogenic probe P-IID enables the detection of cell-surface phosphatidylserine (PS) using both fluorescence imaging and flow cytometry. Here we provide a detailed protocol for the use of P-IID for the qualitative detection of externalized PS in apoptotic cells using confocal microscopy, including the real-time imaging of apoptosis upon drug treatment. We also provide a detailed method for the quantitative analysis of cell death by flow cytometry, using P-IID in conjunction with the nuclear stain propidium iodide. P-IID is superior to commonly used Annexin-V fluorophore conjugates for PS detection as it provides a "turn-on" fluorescence response, displays rapid binding kinetics and can be used at low temperature (4°C), without washing and in the absence of Ca2+ ions.

Apoptosis-Inducing Effect of Pogostemin A Isolated from the Aerial Parts of Pogostemon auricularius Against the Human Lung Cancer Cells

The extracts or compounds isolated from Pogostemon spp. (Lamiaceae) showed various potential biological activities such as antimicrobial, analgesic, anti-inflammatory, antioxidant, as well as cytotoxicity. In our previous studies, pogostemin A (new compound), isolated from the aerial of Pogostemon auricularius, showed strong cytotoxic activities. Herein, our aim of the study was to investigate apoptosis induction activity of pogostemin A on SK-LU-1 human lung cancer cells. Pogostemin A was isolated from the aerial parts of P. auricularius growing in Vietnam. Apoptosis, as the type of cell death, was assessed morphologically by Hoechst staining activity and evaluation of the cell surface phosphatidylserine (PS) expression through annexin V/PI staining. Overall, the obtained data suggests that pogostemin A induced apoptosis in lung cancer cells at a concentration of 20 μg/mL, with the percentages of both early and late apoptotic cells found to be 14.9 % and 12.6 %, respectively, meanwhile, the negative control showed that only 9.8 % early apoptotic cells and 5.5 % late apoptotic cells. Pogostemin A, a new meroterpenoid with pyronesesquiterpenoid hybrid skeletons induced apoptotic cell death in lung cancer cells by activation of caspase 3.

Synthesis of a Fluorescent Probe for Cell Surface Phosphatidylserine

Synthesis of a Fluorescent Probe for Cell Surface Phosphatidylserine

Katrina (Kate) Jolliffe

Angewandte Chemie International EditionVolume 58, Issue 10 p. 2939-2939 Author ProfileFree Access Katrina (Kate) Jolliffe First published: 18 January 2019 https://doi.org/10.1002/anie.201814455AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract “When I was eighteen I wanted to be a chef. My favorite musicians are Nick Cave and PJ Harvey …” Find out more about Katrina A. Jolliffe in her Author Profile. Katrina (Kate) Jolliffe Katrina A. Jolliffe was recently honored with the RACI H. G. Smith Memorial Award. Her most recent contribution to Angewandte Chemie is published in this issue: “A Fluorogenic Probe for Cell Surface Phosphatidylserine Using an Intramolecular Indicator Displacement Sensing Mechanism”: V. E. Zwicker, B. L. Oliveira, J. H. Yeo, S. T. Fraser, G. J. L. Bernardes, E. J. New, K. A. Jolliffe, Angew. Chem. Int. Ed. 2019, 58, 3087; Angew. Chem. 2019, 131, 3119. Date of birth: April 10, 1970 Position: Professor of Chemistry, The University of Sydney E-mail: kate.jolliffe@sydney.edu.au Homepage: http://sydney.edu.au/science/people/kate.jolliffe.php ORCID: 0000-0003-1100-4544 Education: 1992 BSc, University of New South Wales (NSW) 1996 PhD (supervised by Prof. Michael Paddon-Row), University of NSW 1996–1998 Postdoctoral fellow with Prof. David Reinhoudt, Twente Universiteit 1998–2000 Postdoctoral fellow with Prof. Gerald Pattenden, University of Nottingham 2000–2002 Postdoctoral fellow with Prof. Martin G. Banwell, Australian National University Awards: 2004 Beckwith Lectureship for Organic Chemistry (Royal Australian Chemical Institute; RACI); 2006 Biota Award for Medicinal Chemistry (RACI); 2007 Young Tall Poppy Science Award; 2017 A. J. Birch Medal (RACI); 2018 H. G. Smith Memorial Award Research: Anion recognition, self-assembly, hydrogen bonding, cyclic peptides Hobbies: Cooking (and eating good food!), gardening, reading fiction When I was eighteen I wanted to be a chef—but synthetic chemistry can sometimes be a lot like cooking so that worked out. My favorite musicians are Nick Cave and PJ Harvey—I can't choose between them. I am waiting for the day when someone will discover a good cloning method, so one of me can be at work while the other one is on the beach. Of course, we'd need to be able to sync our clone minds so the beach me didn't get bored and the work me didn't get too stressed. Last time I went to the pub I was celebrating acceptance of my Angewandte Chemie manuscript on phosphatidylserine sensing. My favorite drink is gin, no need for tonic. My first experiment was in primary school. I tried to measure how things appear to increase in size when viewed through a drop of water. If I could be any age I would be my current age, but would have spent more time at the gym when I was younger! I advise my students to gain good time management skills and work smarter not longer hours. My favorite way to spend a holiday is reading a novel on a sunny beach. Preferably the beach is in Spain so I can go out for tapas afterwards. The most important thing I learned from my students is that most of them are smarter than me. What I appreciate most about my friends is their ability to make me laugh and forget about work for a while. My 5 top papers: 1“Macrocyclic squaramides: anion receptors with high sulfate binding affinity and selectivity in aqueous media”: L. Qin, A. Hartley, P. Turner, R. B. P. Elmes, K. A. Jolliffe, Chem. Sci. 2016, 7, 4563. (Anion-binding macrocycles that are able to discriminate sulfate from chromate with better selectivity than the sulfate-binding protein.) 2“Janus Cyclic Peptide—Polymer Nanotubes”: M. Danial, C. M.-N. Tran, P. G. Young, S. Perrier, K. A. Jolliffe, Nature Commun. 2013, 2780. (Self-assembled polymer-coated cyclic peptide nanotubes that cluster together to poke holes in membranes.) 3“Selective Pyrophosphate Recognition by Cyclic Peptide Receptors in Physiological Saline”: S. J. Butler, K. A. Jolliffe, Chem. Asian J. 2012, 7, 2621. (Counterintuitively, binding selectivity by chemosensing ensembles can be improved in more complex environments.) 4“Pseudo-prolines as Removable Turn Inducers: Tools for the Cyclisation of Small Peptides”: D. Skropeta, K. A. Jolliffe, P. Turner, J. Org. Chem. 2004, 69, 8804. (My first publication as an independent researcher, describing a useful methodology for the improved synthesis of head-to-tail cyclic peptides.) 5“Noncovalent Assembly of a Fifteen-Component Hydrogen-Bonded Nanostructure”: K. A. Jolliffe, P. Timmerman, D. N. Reinhoudt, Angew. Chem. Int. Ed. 1999, 38, 933; Angew. Chem. 1999, 111, 983. (My first foray into this exciting area of chemistry.) Volume58, Issue10Special Issue: Women in ChemistryMarch 4, 2019Pages 2939-2939 ReferencesRelatedInformation

A Fluorogenic Probe for Cell Surface Phosphatidylserine Using an Intramolecular Indicator Displacement Sensing Mechanism

AbstractThe detection of externalized phosphatidylserine (PS) on the cell surface is commonly used to distinguish between living, apoptotic, and necrotic cells. The tools of choice for many researchers to study apoptosis are annexin V‐fluorophore conjugates. However, the use of this 35 kDa protein is associated with several drawbacks, including temperature sensitivity, Ca2+ dependence, and slow binding kinetics. Herein, a fluorogenic probe for cell surface PS, P‐IID, is described, which operates by an intramolecular indicator displacement (IID) mechanism. An intramolecularly bound coumarin indicator is released in the presence of cell surface PS, leading to a fluorescence “turn‐on” response. P‐IID demonstrates superior performance when compared to annexin V, for both fluorescence imaging and flow cytometry. This allows P‐IID to be used in time‐lapse imaging of apoptosis using confocal laser scanning microscopy and demonstrates the utility of the IID mechanism in live cells.

A Fluorogenic Probe for Cell Surface Phosphatidylserine Using an Intramolecular Indicator Displacement Sensing Mechanism.

The detection of externalized phosphatidylserine (PS) on the cell surface is commonly used to distinguish between living, apoptotic, and necrotic cells. The tools of choice for many researchers to study apoptosis are annexin V-fluorophore conjugates. However, the use of this 35 kDa protein is associated with several drawbacks, including temperature sensitivity, Ca2+ dependence, and slow binding kinetics. Herein, a fluorogenic probe for cell surface PS, P-IID, is described, which operates by an intramolecular indicator displacement (IID) mechanism. An intramolecularly bound coumarin indicator is released in the presence of cell surface PS, leading to a fluorescence "turn-on" response. P-IID demonstrates superior performance when compared to annexin V, for both fluorescence imaging and flow cytometry. This allows P-IID to be used in time-lapse imaging of apoptosis using confocal laser scanning microscopy and demonstrates the utility of the IID mechanism in live cells.

Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

Abstract 3999: Cross talk between tumor cell derived HMGB1 and adaptive B cells in the tumor microenvironment of esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is the most aggressive type of cancer among gastrointestinal cancers. The lack of effective anti-tumor immunity strategy for ESCC patients imposes urgent demand for new treatment paradigm. Tumor microenvironment (TME) is characterized by diverse cell populations (cancerous, immune cells, and pleiotropic nature of cytokines and inhibitory receptors) whose alternations are closely related with tumor progression. High mobility group box chromosomal protein 1 (HMGB1) has been recognized to be expressed in various tumor cells, and importantly it has also been identified as one of the key factors regulating inflammatory reactions. By comparing differentially expressed genes between ESCC tumors (T) and their corresponding non-tumorous (NT) tissues high-throughput transcriptome sequencing system (RNA-Seq, Illumina HiSeq 2500), we found that the ligands of TIM-3 (an inhibitory receptor), HMGB1 and galectin-9, but not, cell-surface phosphatidylserine, were overexpressed in all ESCC samples when compared to their NT counterparts. We further characterized HMGB1 and galectin-9 gene expression changes in 37 matched primary ESCC T and NT control samples by qRT-PCR. We found that HMGB-1 and galectin-9 mRNA is constitutively expressed in all samples, while ESCC T tissue significantly expressed ~2fold higher of HMGB-1, but not galectin-9, mRNA as compared to paired NT tissue. Histopathological characterization of ESCC tissue by sequential single staining for B-cells (CD20) and plasma cells (CD138), demonstrating that B cell infiltrate was dominated by CD20+ B cells lacking of terminal differentiation into plasma cells. Additionally, CD20+ B cells infiltrates were negative for CD27 (marker for memory B cells) and IgD (marker for naïve B cells). Additionally, we isolated CD20+ B cells from healthy peripheral blood mononuclear cells (PBMC), and stimulated with/without HMGB-1/IgM. Conditional medium (CM) were collected for foci formation and we found that ESCC cell lines KYSE30 and KYSE180 have better capacity for proliferation when culture with activated B cells CM as compared to unstimulated B cells. Collectively, we believe that the suboptimal clinical outcomes relies on CD20+HMGB1 receptor+ B cells that are exposed to high HMGB1 concentrations in the ESCC microenvironment may eventually become pro-tumor B cells subset. Citation Format: Ngar Woon Kam, Xin-Yuan Guan, Dora Lai-Wan Kwong. Cross talk between tumor cell derived HMGB1 and adaptive B cells in the tumor microenvironment of esophageal squamous cell carcinoma [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 3999. doi:10.1158/1538-7445.AM2017-3999

Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca2+ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cellswith low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling.

The cationic small molecule GW4869 is cytotoxic to high phosphatidylserine-expressing myeloma cells.

We have discovered that a small cationic molecule, GW4869, is cytotoxic to a subset of myeloma cell lines and primary myeloma plasma cells. Biochemical analysis revealed that GW4869 binds to anionic phospholipids such as phosphatidylserine - a lipid normally confined to the intracellular side of the cell membrane. However, interestingly, phosphatidylserine was expressed on the surface of all myeloma cell lines tested (n=12) and 9/15 primary myeloma samples. Notably, the level of phosphatidylserine expression correlated well with sensitivity to GW4869. Inhibition of cell surface phosphatidylserine exposure with brefeldin A resulted in resistance to GW4869. Finally, GW4869 was shown to delay the growth of phosphatidylserine-high myeloma cells invivo. To the best of our knowledge, this is the first example of using a small molecule to target phosphatidylserine on malignant cells. This study may provide the rationale for the development of phosphatidylserine-targeting small molecules for the treatment of surface phosphatidylserine-expressing cancers.

Biophysical and Biochemical Markers of Red Blood Cell Fragility

Background: Red blood cells (RBCs) undergo a natural aging process occurring in the blood circulation throughout the RBC lifespan or during routine cold storage in the blood bank. The aging of RBCs is associated with the elevation of mechanical fragility (MF) or osmotic fragility (OF) of RBCs, which can lead to cell lysis. The present study was undertaken to identify RBC properties that characterize their susceptibility to destruction under osmotic/mechanical stress. Methods: RBCs were isolated from freshly donated blood or units of packed RBCs (PRBCs) and suspended in albumin-supplemented phosphate-buffered saline (PBS). In addition, PRBCs were separated by filtration through a microsphere column into two fractions: enriched with rigid (R-fraction) and deformable (D-fraction) cells. The RBCs were subjected to determination of deformability, MF and OF, moreover, the level of cell surface phosphatidylserine (PS) and the stomatin level in isolated RBC membranes were measured. Results: In the RBC population, the cells that were susceptible to mechanical and osmotic stress were characterized by low deformability and increased level of surface PS. The OF/MF was higher in the R-fraction than in the D-fraction. Stomatin was depleted in destroyed cells and in the R-fraction. Conclusion: RBC deformability, the levels of surface PS, and membrane stomatin can be used as markers of RBC fragility.