Appearance Of Phosphatidylserine Research Articles

Subtle membrane changes in cryopreserved bull spermatozoa when modified temperature drop rates are used during the first phase of freezing

BACKGROUND: Cryopreservation of spermatozoa involves reduction of temperature to a subzero level, leading to increased longevity. However, temperature reduction has a significant effect on sperm membranes. OBJECTIVE: To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa. MATERIALS AND METHODS: Thirty-two ejaculates from four bulls (eight ejaculates/bull) were collected using artificial vagina while keeping a 3 to 4 days gap between two collections. Diluted semen samples were equilibrated at 5°C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5°C till −10°C was subjected to three different temperature drop rates: accelerated (F1), moderate (F2), and slow (F3), at 20°C per min, 10°C per min and 5°C per min, respectively. After thawing, spermatozoa were assessed for percentage live, plasma, and acrosomal membrane integrity, along with the external appearance of phosphatidyl serine, indicating apoptosis. RESULTS: A significant difference (p<0.05) in viability, plasma membrane integrity (HOS test), and acrosome membrane integrity (PSA test) was observed between F3 and the other groups. However, the parameters did not significantly differ between F1 and F2. The annexin V-PI assay (AN/PI) categorized four types of sperm populations: non-apoptotic and viable (AN−/PI−), apoptotic and viable (AN+/PI−), non-apoptotic and non-viable (AN−/PI+), and apoptotic and non-viable (AN+/PI+). The proportion of spermatozoa with (AN−/PI−) and (AN+/PI+) differed significantly (p<0.05) between F3 and the other groups. The values for apoptotic and viable (AN+/PI−) and non-apoptotic and non-viable (AN−/PI+) sperm were not significantly different among all freezing categories. CONCLUSION: A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes.

Effects of progesterone and selective ligands of membrane progesterone receptors in HepG2 cells of human hepatocellular carcinoma

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors of the adiponectin and progestin receptor (mPRs) family. The effect of progesterone on cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of a high level of mRNA and mPRβ protein in HepG2 tumor cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. May be mPRβ has an important function in HepG2 cells. The main goal of the work was to study the function of this protein and the mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. The work studied the effects of progesterone, LS-01 and LS-02 on the proliferation and death of HepG2 cells, as well as the activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 hours of incubation with cells suppressed their viability and stimulated the appearance of phosphatidylserine on the outer surface of membranes, which was detected by binding to V-FITC annexin, but they did not affect DNA fragmentation of cell nuclei. Progesterone significantly reduced the expression of proliferation marker genes and stimulated the expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRβ. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

Effects of Progesterone and Selective Ligands of Membrane Progesterone Receptors in HepG2 Cells of Human Hepatocellular Carcinoma.

Progesterone exerts multiple effects in different tissues through nuclear receptors (nPRs) and through membrane receptors (mPRs) of adiponectin and progestin receptor families. The effect of progesterone on the cells through different types of receptors can vary significantly. At the same time, it affects the processes of proliferation and apoptosis in normal and tumor tissues in a dual way, stimulating proliferation and carcinogenesis in some tissues, suppressing them and stimulating cell death in others. In this study, we have shown the presence of high level of mPRβ mRNA and protein in the HepG2 cells of human hepatocellular carcinoma. Expression of other membrane and classical nuclear receptors was not detected. It could imply that mPRβ has an important function in the HepG2 cells. The main goal of the work was to study functions of this protein and mechanisms of its action in human hepatocellular carcinoma cells. Previously, we have identified selective mPRs ligands, compounds LS-01 and LS-02, which do not interact with nuclear receptors. Their employment allows differentiating the effects of progestins mediated by different types of receptors. Effects of progesterone, LS-01, and LS-02 on proliferation and death of HepG2 cells were studied in this work, as well as activating phosphorylation of two kinases, p38 MAPK and JNK, under the action of three steroids. It was shown that all three progestins after 72 h of incubation with the cells suppressed their viability and stimulated appearance of phosphatidylserine on the outer surface of the membranes, which was detected by binding of annexin V, but they did not affect DNA fragmentation of the cell nuclei. Progesterone significantly reduced expression of the proliferation marker genes and stimulated expression of the p21 protein gene, but had a suppressive effect on the expression of some proapoptotic factor genes. All three steroids activated JNK in these cells, but had no effect on the p38 MAPK activity. The effects of progesterone and selective mPRs ligands in HepG2 cells were the same in terms of suppression of proliferation and stimulation of apoptotic changes in outer membranes, therefore, they were mediated through interaction with mPRβ. JNK is a member of the signaling cascade activated in these cells by the studied steroids.

ΔM4: Membrane-Active Peptide with Antitumoral Potential against Human Skin Cancer Cells.

Peptides have become attractive potential agents due to their affinity to cancer cells. In this work, the biological activity of the peptide ΔM4 against melanoma cancer cell line A375, epidermoid carcinoma cell line A431, and non-tumoral HaCaT cells was evaluated. The cytotoxic MTT assay demonstrates that ΔM4 show five times more activity against cancer than non-cancer cells. The potential membrane effect of ΔM4 was evaluated through lactate dehydrogenase release and Sytox uptake experiments. The results show a higher membrane activity of ΔM4 against A431 in comparison with the A375 cell line at a level of 12.5 µM. The Sytox experiments show that ΔM4 has a direct effect on the permeability of cancer cells in comparison with control cells. Infrared spectroscopy was used to study the affinity of the peptide to membranes resembling the composition of tumoral and non-tumoral cells. The results show that ΔM4 induces a fluidization effect on the tumoral lipid system over 5% molar concentration. Finally, to determine the appearance of phosphatidylserine on the surface of the cell, flow cytometry analyses were performed employing an annexin V-PE conjugate. The results suggest that 12.5 µM of ΔM4 induces phosphatidylserine translocation in A375 and A431 cancer cells. The findings of this study support the potential of ΔM4 as a selective agent for targeting cancer cells. Its mechanism of action demonstrated selectivity, membrane-disrupting effects, and induction of phosphatidylserine translocation.

Association of Circulating Procoagulant Microvesicles with Painful Vaso-Occlusive Crisis in Sickle Cell Disease

Introduction: Thrombotic complication is one of the features of sickle cell disease (SCD), characterized by appearance of phosphatidylserine on the outer membrane of sickle-shaped red blood cells and most abundantly on membrane protrusions called microvesicles (MVs). However, the exact mechanism by which MVs may enhance coagulant activity in SCD patients has not been fully addressed. The aim of this study was to further investigate the procoagulant activity of circulating MVs in sickle cell crises. Materials and Methods: Subjects included in this cross-sectional study were 47 patients with SCD and 25 normal subjects with written informed consent obtained from all the participants. MV analysis was conducted by using CD61, CD235α, and Annexin-V monoclonal antibodies. The coagulant activity of MVs was determined by an ELISA-based procoagulant activity assay. Results: The majority of MVs were originated from platelets (CD61+) and erythrocytes (CD235+). These MVs demonstrated significantly enhanced levels during the painful crisis when compared with the steady-state period (p < 0.001) and controls (p < 0.001). Also, the procoagulant activity of MVs was significantly higher in crisis compared to those of steady state (p < 0.001) and positively correlated with the number of Annexin-V+ MVs (p < 0.001). Significant correlations were found between erythrocyte-derived MVs with hemolysis marker (r = 0.51, p < 0.001) and the hemoglobin level (r = −0.63, p < 0.001). Conclusion: The numbers of platelet- and erythrocyte-derived MVs are related to painful crisis, and their quantification in SCD may be helpful for identifying cases at increased risk of thrombotic complications.

Pro-apoptotic properties of (1,3)(1,4)-β-d-glucan from Avena sativa on human melanoma HTB-140 cells in vitro

In this study, the growth-inhibitory effect of polysaccharide (1,3)(1,4)-β-d-glucan from oat, Avena sativa L. grains was explored on the human skin melanoma HTB-140 cells in vitro. The oat β-d-glucan (OBG) exerted cytotoxic action on HTB-140 cells. After 24h of incubation, LD50 (concentration at which 50% of the cells were found dead) was obtained of 194.6±9.8μg/mL. The oat β-d-glucan caused a concentration-dependent increase of caspase-3/-7 activation and appearance of phosphatidylserine on the external surface of cellular membranes where it was bound to annexin V-FITC, demonstrating the induction of apoptosis. Intracellular ATP level decreased along with the mitochondrial potential, which suggested a mitochondrial pathway of apoptosis. A cell cycle analysis showed increase in the number of apoptotic cells, increase in the number of cells in G1 phase and decrease in the number of cells in G2/M. Although the detailed mechanism for the anti-tumor activity of the oat β-d-glucan still needs further investigation, this study provides preliminary insights into this direction along with perspectives of developing it as an anti-tumor agent.

Tannic Acid Induced Suicidal Erythrocyte Death

Background: The polyphenol tannic acid with antioxidant and antimicrobial potency may trigger suicidal death of nucleated cells or apoptosis and thus may counteract tumor growth. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with appearance of phosphatidylserine at the erythrocyte surface. A major trigger of eryptosis is increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). Erythrocytes could be sensitized to the eryptotic effect of cytosolic Ca²⁺ by ceramide. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fuorescence and ceramide utilizing fluorescent antibodies. Results: A 48 h treatment with tannic acid was followed by significant decrease of forward scatter (≥ 1 µg/ml) and significant increase of annexin-V-binding (≥ 10 µg/ml). Tannic acid did not significantly modify [Ca²⁺]_i (up to 50 µM) but significantly increased ceramide formation (50 µM). The annexin-V-binding following tannic acid treatment (50 µM) was significantly blunted in the nominal absence of extracellular Ca²⁺. Conclusions: Tannic acid stimulates eryptosis, an effect at least partially due to ceramide formation with subsequent sensitization of erythrocytes to cytosolic Ca²⁺.

Sensing Phosphatidylserine in Cellular Membranes

Phosphatidylserine, a phospholipid with a negatively charged head-group, is an important constituent of eukaryotic cellular membranes. On the plasma membrane, rather than being evenly distributed, phosphatidylserine is found preferentially in the inner leaflet. Disruption of this asymmetry, leading to the appearance of phosphatidylserine on the surface of the cell, is known to play a central role in both apoptosis and blood clotting. Despite its importance, comparatively little is known about phosphatidylserine in cells: its precise subcellular localization, transmembrane topology and intracellular dynamics are poorly characterized. The recent development of new, genetically-encoded probes able to detect phosphatidylserine within live cells, however, is leading to a more in-depth understanding of the biology of this phospholipid. This review aims to give an overview of the current methods for phosphatidylserine detection within cells, and some of the recent realizations derived from their use.

Clearance of dying ARPE-19 cells by professional and nonprofessional phagocytesin vitro- implications for age-related macular degeneration (AMD)

Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age-related macular degeneration (AMD). The dynamics and influence of different treatments on this clearance process can be studied in vitro using human ARPE-19 cells and macrophages as phagocytes modelling dry and wet type of AMD, respectively. Death through extracellular matrix detachment using polyHEMA-coated surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE-19 cells. Two-coloured phagocytic assays were performed to quantify the amount of dying cells phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning electron microscopy). The effect of phosphatidylserine inhibition with recombinant annexin-V and glucocorticoid (triamcinolone) treatment on the phagocytic process was tested. The clearance of anoikic and apoptotic cells by nondying ARPE-19 cells over 8 hr of co-incubation increased over time (at 8 hr, over 53% and 35% of the phagocytes contained engulfed dying cells, respectively). The human macrophages engulfed the anoikic and apoptotic ARPE-19 cells with seven and four times lower capacity, respectively. Phosphatidylserine appearance on the dying cells did not affect, but triamcinolone treatment enhanced the phagocytosis of the dying cells by macrophages. ARPE-19 cells are more efficient in clearing anoikic than UV-induced apoptotic cells. Macrophages are less efficient in the clearance process than ARPE-19 cells. The present model can be used for studying both dry and wet type of AMD in vitro and for testing different pharmacological aspects affecting this disease.

Pro-Oxidative and Pro-Apoptotic Action of Defatted Seeds ofOenothera paradoxaon Human Skin Melanoma Cells

Three extracts of defatted seeds of Oenothera paradoxa Hudziok, aqueous extract, 60% ethanolic extract, and 30% isopropanolic extract, differing by their total content of phenolic compounds and by their contents of individual polyphenols, were investigated in this study. The extracts exerted cytotoxic action on HTB-140 human skin melanoma cells. After 24 h of incubation, IC(50) values of 169.7 +/- 5.9 micog/mL, 72.4 +/- 3.8 microg/mL, and 155.3 +/- 6.3 microg/mL were obtained for HTB-140 cells with the aqueous extract, 60% ethanolic extract, and 30% isopropanolic extract at the tested concentrations (5-200 microg/mL), respectively, while IC(50) for normal fibroblast cells NHDFs was not attained. Moreover, for HTB-140 cells, LD(50) (concentration at which 50% of cells were dead) of 89.2 +/- 4.3 microg/mL and 181.4 +/- 6.5 microg/mL were obtained with 60% ethanolic extract and 30% isopropanolic extract, respectively. In melanoma cells, all three extracts caused a concentration-dependent increase of ROS production, GSH, and ATP lowering, and appearance of phosphatidylserine on the external surface of cellular membranes where it was bound to annexin V-FITC; furthermore, apoptosis without activation of caspase-3 took place. The most effective was 60% ethanolic extract, which had the greatest total content of phenolic compounds and the greatest content of pentagalloyloglucose (PGG).

Mobilization of Lysosomal Calcium Regulates the Externalization of Phosphatidylserine during Apoptosis

A hallmark of apoptotic cells is the Ca2+-dependent appearance of phosphatidylserine (PS) at the cell surface as a result of its redistribution from the inner-to-outer plasma membrane leaflet. Although endoplasmic reticulum and mitochondrial Ca2+ are known to participate in apoptosis, their role in PS externalization has not been established. In this study, several organelle-specific fluorescent markers and Ca2+-sensitive probes were used to identify the source of Ca2+ critical to PS externalization. By employing Rhod-2AM, fluorescein-labeled high molecular weight dextran, and Calcium Green 1, we provide evidence that lysosomes respond to apoptotic stimuli by releasing their luminal Ca2+ to the cytosol. Cells treated with the cytosolic phospholipase A2 inhibitor, cPLA2alpha, had no effect on caspase activation but exhibited a significant decrease in lysosomal Ca2+ release and externalization of PS in response to apoptotic stimuli. Similarly, cells depleted of lysosomal Ca2+ underwent programmed cell death yet failed to externalize PS. These data indicate that although Ca2+ release from other intracellular organelles to the cytosol is adequate for apoptosis, the release of Ca2+ from lysosomes is critical for PS externalization.

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

BackgroundRNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.Methodology/Principal FindingsIn search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC50) ranging from 0.2 to 13 µM and to leukemia cell lines with IC50 values ranging from 2.4 to 82 µM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC50 = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.Conclusions/SignificanceThese results demonstrate that barnase is a potent toxic agent for targeting to cancer cells.

MAPK inhibitors and pifithrin-alpha block cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. The aim of this study was to investigate the effect of pifithrin-alpha (PFTα; a specific p53 inhibitor) and mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on apoptotic signaling transduction mechanism induced by Cin in human hepatoma PLC/PRF/5 (CD95-negative) cells. Using XTT assay, Cin exhibited a powerful cytotoxic effect and apoptotic induction in PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 μM Cin as characterized by morphological changes and the appearance of phosphatidylserine on the outer surface of the plasma membrane. Cin down-regulated the expression of Bcl- XL, up-regulated mutant p53 and Bax proteins and promoted caspase-3 to active forms, as well as cleaving poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. This could be supported by the activation and phosphorylation of MAPKs, including JNK, ERK and p38 kinases. Pre-incubation with PFTα and specific MAPK inhibitors significantly diminished the effect of Cin-induced apoptosis. The activities of anti-apoptotic (Bcl- XL) and pro-apoptotic (Bax) proteins were remarkably affected by PFTα and PD98059 pre-treatment. PFTα effectively blocked PARP cleavage in cells treated with Cin, and also markedly prevented the phosphorylation of JNK, p38 and ERK proteins. These results suggest that p53 induction and MAPK signaling pathways are required for Cin-mediated apoptosis in PLC/PRF/5 cells.

Fluorescent Detection of Apoptotic Cells by Using Zinc Coordination Complexes with a Selective Affinity for Membrane Surfaces Enriched with Phosphatidylserine

The appearance of phosphatidylserine on the membrane surface of apoptotic cells (Jurkat, CHO, HeLa) is monitored by using a family of bis(Zn2+-2,2'-dipicolylamine) coordination compounds with appended fluorescein or biotin groups as reporter elements. The phosphatidylserine affinity group is also conjugated directly to a CdSe/CdS quantum dot to produce a probe suitable for prolonged observation without photobleaching. Apoptosis can be detected under a wide variety of conditions, including variations in temperature, incubation time, and binding media. Binding of each probe appears to be restricted to the cell membrane exterior, because no staining of organelles or internal membranes is observed.

Haemostasis Alterations in Sickle Cell Disease.

Sickle cell disease (SCD) is considered by many workers to be a hypercoagulable state. The vaso-occlusion in sickle cell disease is complex, involving adhesive processes between sickle red blood cells, leukocytes, and the activated endothelium. Loss of normal membrane phospholipids asymetry with the appearance of phosphatidylserine on the erythrocyte surface has been documented as promoting an exacerbation of the anaemia and an activation of coagulation. The aim of this study was to monitor changes in coagulation and platelet activation (monitored by measuring the levels of procoagulant phospholipids microparticles - PPM) in SCD (both in steady state and crisis). Coagulation screening tests (PT, APPT), Protein S (PS), D-Dimer (D-Di) and PPM were measured in plasma samples obtained from 18 patients with SCD (8 in crisis state and 10 in steady state) and in twenty one normal volunteers as control. All blood samples were collected and the plasma prepared in the same hospital using the same conditions. All plasma samples were stored at -80°C until assayed. PT, aPTT, PS and D-dimers were measured using reagents from Diagnostica Stago, Asniéres, France. PPM was monitored with a novel factor Xa-based coagulation assay. In this assay shortened clotting times are associated with increased levels of PPM and thus platelet activation.APTT Sec.PT %PPM SecPS %D-Di μg/lControls34.6 (29.4–39.6)93.1 (79–109)57.5 (51.1–74.9)88.8 (62–112)0.22 (0.22–0.45)Sickle cell – in crisis37.3 (33.4–47.4)71.1 (56.5–86.5)44.6 (34.0–60.0)48.9 (25.0–89.5)1.6 (0.6–2.6)Sickle cell – steady state.38.0 (33.0–47.5)75.0 (61.0–93.0)52.2 (42.5–62.8)84.7 (76.5–103.0)0.7 (0.2–1.0)In sickle cell crisis a significant increase in both coagulation and platelet activation, as monitored by PPM, was found (all at p < 0.001). The decrease in % PT and PS levels confirms the findings of others. In steady state only DDi levels are significantly higher than controls. Further studies are needed to explain these findings. Although this was a small pilot study and further studies are needed it is interesting to speculate on the possible usefulness of the PPM assay in monitoring SCD crisis.

Biophysical studies of a synthetic mimic of the apoptosis‐detecting protein annexin v

AbstractA Zn2+‐dipicolylamine coordination compound is shown to sense the presence of anionic phospholipids in a membrane bilayer. The sensor contains two dipicolylamine subunits attached to an anthracene scaffold, which exhibits a maximum absorbance at 380 nm, and undergoes an enhancement in fluorescence intensity when exposed to membranes enriched in phosphatidylserine. For these reasons, the compound is referred to as PSS‐380 (Phosphatidylserine Sensor, 380 nm). The fluorescence emission of PSS‐380 is enhanced up to tenfold by the presence of vesicles containing the anionic phospholipids phosphatidylserine, phosphatidylglycerol, or phosphatidic acid. No enhancement in fluorescence is observed upon exposure to vesicles containing only zwitterionic phosphatidylcholine, or exposure to monodispersed (non‐aggregated) anionic phospholipids. The sensing effect is cooperative; not only does association to the vesicles increase if the vesicles have raised levels of anionic phospholipid, but the maximum fluorescence at sensor saturation is also enhanced. It appears that sensing is triggered by the three‐component self‐assembly of sensor, Zn2+, and the anionic membrane surface, which leads to diminished photo‐induced electron transfer (PET) quenching. The utility of PSS‐380 in flow cytometry and fluorescence microscopy is demonstrated by using the molecule to detect the appearance of phosphatidylserine on the plasma membrane surface of various cell lines. Thus, PSS‐380 can identify apoptotic cells in the same way as the commonly used protein reagent annexin V.

Detached endothelial cells and microparticles as sources of tissue factor activity

Introduction Cytokine activation of endothelial cell monolayers is associated with cell detachment, microparticle shedding from plasma membranes, and phosphatidylserine appearance in the plasma membrane outer leaflets. While tissue factor expression on activated endothelial cells and microparticles is well documented, the contribution of detached endothelial cells to tissue factor activity is less clear. We studied tissue factor expression and the role of tissue factor pathway inhibitor on adherent and detached endothelial cells and on microparticles following endothelial cell activation with TNF-α. Materials and methods Detached endothelial cells and microparticles were obtained from cultures of human umbilical vein endothelial cells by differential centrifugation of cell culture supernatant. For microparticle capture, an antibody directed against CD146 was used. Functional tissue factor activity was measured by chromogenic assay and tissue factor antigen by ELISA. Endothelial cell and microparticle morphology was examined by light and transmission electron microscopy. Results After cell activation for 22 h, functional tissue factor activity was distributed as follows: 60%, adherent endothelial cells; 35%, detached cells; and 5%, microparticles. Tissue factor protein followed a similar distribution. Cell detachment was 47%. Electron microscopy demonstrated shedding of microparticles with a diameter of 0.1–0.6 μm. Cy3–annexin V revealed increased phosphatidylserine on activated adherent endothelial cells and microparticles. Pre-incubation of adherent and detached endothelial cells and microparticles with anti-tissue factor antibody blocked factor Xa production. Pre-incubation with anti-tissue factor pathway inhibitor antibody increased tissue factor activity of adherent endothelial cells 2.8-fold, detached cells 1.4-fold, and microparticles 45-fold. Conclusions Detached endothelial cells as well as microparticles from activated endothelial cell monolayers express tissue factor activity, and this activity on microparticles is markedly inhibited by microparticle-associated tissue factor pathway inhibitor.

Protection against amyloid beta-peptide (1–42)-induced loss of phospholipid asymmetry in synaptosomal membranes by tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester: Implications for Alzheimer's disease

Amyloid-beta (1-42) [Abeta (1-42)] deposition in the brain is a hallmark of Alzheimer's disease (AD) and has been shown to induce apoptosis and disrupt cellular ion homeostasis. Abeta (1-42) induces membrane lipid peroxidation, and 4-hydroxynonenal (HNE) and 2-propenal (acrolein) are the two reactive products of lipid peroxidation, which structurally modify proteins by covalent interaction and inhibit enzyme function. Phosphatidylserine (PS), an aminophospholipid, is sequestered in the inner leaflet of the plasma membrane in nonstimulated cells. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine in the outer leaflet of the membrane. The ATP-requiring enzyme, flippase, maintains phospholipid asymmetry of PS. Here, we have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by Abeta (1-42). Flippase activity depends on a critical cysteine residue, a putative site of covalent modification by the Abeta (1-42)-induced lipid peroxidation products, HNE or acrolein. The present study is aimed to investigate the protective effects of tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester (FAEE) on Abeta (1-42) induced modulation in phospholipid asymmetry in the synaptosomal membranes. Pretreatment of synaptosomes with D609 and FAEE significantly protected Abeta (1-42)-induced loss of phospholipid asymmetry in synaptosomal membranes. Our results suggest that D609 and FAEE exert protective effects against Abeta (1-42) induced apoptosis. The increase in intracellular Ca(2+) might not be the sole cause for the loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in the modulation of phospholipid asymmetry. The results of this study are discussed with relevance to neuronal loss in the AD brain.

Decreased Total Body Irradiation (TBI)-Induced Apoptosis in Murine Hematopoietic Side Population Cells Compared to Non-Side Population Cells.

The side population (SP) cells of the murine bone marrow (BM) contain primitive hematopoietic stem cells (HSCs). To understand the effect of irradiation on these cells, C57BL/6NHsd mice were irradiated to 10 Gy total body dose and sacrificed 24 hours later. The bone marrow was removed and stained with Hoechst dye, and Annexin V as an early marker of apoptosis. The SP cells were analyzed by flow cytometry. Following irradiation, there was no change in the frequency of SP cells in total nucleated BM cells between non-irradiated (0.081 ± 0.033%) and irradiated mice (0.093 ± 0.027%). In contrast, there was a decrease in total nucleated BM and SP cells. SP cells decreased by 60.1% from 44873 ± 22099 SP cells per harvest to 17550 ± 4805 SP cells per harvest 24 hours after 10 Gy. The number of lymphocytes in the irradiated BM decreased from 25.7% to 4.5% after irradiation. Since the majority of the SP cells are located in the lymphocyte sorting region, there was a 6-fold decrease in the percent of SP cells found in the live lymphocyte region following irradiation (0.469 ± 0.185% compared to 2.923 ± 1.913%).Annexin V was used to stain for the appearance of phosphatidylserine (PS) on the cell surface as an early marker for apoptosis. The appearance of Annexin V positive cells did not increase significantly with irradiation in SP compared to non-irradiated SP cells (26 ± 8% and 25 ± 12%, respectively). In contrast, the percent of apoptotic cells in the non-SP fraction was significantly increased following irradiation compared to the non-irradiated cells (35 ± 8% and 25 ± 6%, respectively) (p=0.02). Our data indicate that irradiation was not inducing apoptosis in SP cells but in non-SP cells. It might suggest that SP cells could be more resistant to radiation damage. Future investigations will be performed to understand the functional activity of SP cells from lethally irradiated mouse BM and the molecular mechanism of those SP cells in resistance to irradiation damage.

EFFECTS OF VITAMIN E ON THE CINNAMALDEHYDE‐INDUCED APOPTOTIC MECHANISM IN HUMAN PLC/PRF/5 CELLS

1. Cinnamaldehyde has been shown to be effective in inducing cell apoptosis in a number of human cancer cells. The aim of the present study was to investigate the effect of vitamin E on the apoptotic signalling mechanism induced by cinnamaldehyde in human hepatoma PLC/PRF/5 cells. 2. Using the XTT assay, cinnamaldehyde exhibited a powerful antiproliferative effect on PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 micromol/L cinnamaldehyde, as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. 3. The apoptotic effect induced by cinnamaldehyde could be further supported by the release of cytochrome c, Smac/Diablo and Omi/HtrA2 from mitochondria to the cytosol and activation of caspase 3. Cinnamaldehyde also upregulated the expression of pro-apoptotic protein (Bax) and down-regulated the levels of anti-apoptotic proteins, such as Bcl-2 and the inhibitor of apoptosis protein family (X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein (cIAP)-1 and cIAP-2). 4. Cinnamaldehyde induces the generation of reactive oxygen species (ROS) in cells. Following the pre-incubation of PLC/PRF/5 cells with anti-oxidants, it was found that 100 micromol/L vitamin E significantly diminished the effect of cinnamaldehyde-induced apoptosis, whereas a lesser effect was seen with on 100 micromol/L N-acetyl-L-cysteine. Vitamin E effectively blocked the release of cytochrome c, Smac/Diablo and Omi/HtrA2 from mitochondria to the cytosol in cells treated with cinnamaldehyde. Vitamin E also markedly suppressed caspase 3 activation. The expression of apoptotic inhibitors (XIAP, cIAP-1, cIAP-2) and anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins was affected by vitamin E pretreatment. 5. Taken together, the results suggest that cinnamaldehyde triggers apoptosis possibly through the mitochondrial pathway. Pretreatment with vitamin E markedly prevented cinnamaldehyde-mediated apoptosis, which was associated with the modulation of XIAP, cIAP-1, cIAP-2, Bcl-2 and Bax protein activity.