Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.     

Effect of phosphatidylserine on free radical susceptibility in human diploid fibroblasts

Summary We studied the effect of phosphatidylserine (PdtSER) on oxygen metabolite toxicity in skin fibroblast cell lines from apparently normal subjects. Fibroblast damage was produced by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by xanthineoxidase (Xo). In order to quantify cell damage, we measured lactate dehydrogenase (LDH) activity in culture medium and cell viability in fibroblast cultures, with and without preincubation for 4 days with PdtSER 13 M, after Xo incubation. We found a significant increase of LDH activity in culture medium of cells without preincubation with PdtSER. No significant increase of LDH activity was observed in the same cell lines after preincubation with PdtSER.     

Red cell and platelet‐derived microparticles are increased in G6PD‐deficient subjects

In response to oxidative stress and during apoptosis, cells often shed microparticles (MPs), submicron elements carrying phosphatidylserine and protein antigens. Glucose‐6‐phosphate dehydrogenase (G6PD)‐deficient cells are extremely sensitive to oxidative damage that may lead to the formation of MPs. To determine whether G6PD deficiency alters membrane phospholipid asymmetry and increases MPs production, we determined the concentrations and cellular origins of MPs in G6PD‐deficient individuals using flow cytometry. G6PD‐deficient individuals showed an increase in circulating MPs concentrations as compared with G6PD‐normal individuals [1051/μL (865–2532/μL) vs. 258/μL (235–575/μL), P < 0.01]. MPs concentrations were significantly increased with the severity of G6PD deficiency. Median MPs concentrations from individuals with severe G6PD deficiency, and individuals with moderate G6PD deficiency were 2567/μL (1216–2532/μL) and 984/μL (685–2107/μL), respectively (P < 0.01). Importantly, G6PD enzymatic activity was significantly correlated with MPs concentrations with r² = 0.731. MPs found in G6PD deficiency individuals were largely derived from red blood cells (RBCs) (45%) and platelets (30%). Additionally, Atomic Force Microscopy was used to study the morphology and measures the diameter of MPs found in G6PD‐deficient individuals. The mean (SD) width and height of RMPs were 0. 41 (0.18) and 2.04 (0.14) μm, respectively. Together, these results indicate that MP concentration is significantly correlated with G6PD enzymatic activity and is increased in G6PD‐deficient as compared with G6PD‐normal individuals. Our data also provide an evidence for an alteration in cell membrane associated with a decreased in G6PD activity. However, the significance of MPs in G6PD deficiency needs further clarification.     

Suppression of atopic dermatitis in mice model by reducing inflammation utilizing phosphatidylserine-coated biodegradable microparticles

Controlling inflammatory response is important to avoid chronic inflammation in many diseases including atopic dermatitis (AD). In this research, we tried using a phosphatidylserine (PS)-coated microparticles in the AD mouse model for achieving the modulation of the macrophage phenotype to an anti-inflammatory state. Here, we prepared poly (D,L-lactic acid) microparticle coated with PS on the outside shell. We confirmed the cellular uptake of the PS-coated microparticle, which leads to the significant downregulation of the inflammatory cytokine production. In the mouse model of AD, the PS-coated microparticle was injected subcutaneously for a period of 12 days. The mice showed significant reduction in the development of AD symptoms comparing with the mice treated with the PC-coated microparticle.     

Preparation and Characterization of Cellulose Ether Liposomes for the Inhibition of Prion Formation in Prion-Infected Cells

Prion accumulation in the brain and lymphoreticular system causes fatal neurodegenerative diseases. Our previous study revealed that cellulose ethers (CE) have anti-prion activities in vivo and in prion-infected cells when administered at high doses. This study aims to improve the bioavailability of a representative CE using a liposomal formulation and characterized CE-loaded liposomes in cultured cells. The liposomal formulation reduced the EC₅₀ dose of CE by <1/200-fold in prion-infected cells. Compared to empty liposomes, CE-loaded liposomes were taken up much more highly by prion-infected cells and less by macrophage-like cells. Phosphatidylserine modification reduced the uptake of CE-loaded liposomes in prion-infected cells and did not change the anti-prion activity, whereas increased the uptake in macrophage-like cells. Polyethylene glycol modification reduced the uptake of CE-loaded liposomes in both types of cells and reduced the anti-prion activity in prion-infected cells. These results suggest that a liposomal formulation of CE is more practical than unformulated CE and showed that the CE-loaded liposome uptake levels in prion-infected cells were not associated with anti-prion activity. Although further improvement of the stealth function against phagocytic cells is needed, the liposomal formulation is useful to improve CE efficacy and elucidate the mechanism of CE action.     

Erythrocytic phosphatidylserine exposure and hemostatic alterations in β-thalassemia intermediate patients

Introduction

Hypercoagulable state is one of the common findings in beta-thalassemia intermedia (β-TI), particularly in splenectomized patients, with infrequent blood transfusion. Abnormality of the red blood cells (RBC) membrane due to oxidative damage is suggestive of possible etiologies. Membrane lipid peroxidation increases the exposure of phosphatidylserine (PS) that plays a role in the activation of coagulation factors V and X, subsequently initiating thrombosis. Our aim of this study was to find the probable correlation of the alteration of the PS on the RBC outer membrane with the hypercoagulable state in the β-TI patients.

Materials and methods

Our cross-sectional study was conducted on 39 splenectomized β-TI patients and 38 age-matched healthy controls. The mean age was 37 years. Analysis of the PS exposure on the RBCs was performed by fluorescein isothiocyanate (FITC) conjugated AV protein .Measurement of the coagulation factors X, V and antithrombin III (AT-III) was performed. We also checked the D-dimer levels .Analysis was performed by SPSS16.

Results

Fluorescence of FITC-Annexin V labeling on patients RBCs were higher than healthy controls; (2.8 ± 2.2%) of the patients versus (0.4 ± 0.18%) in the control group and was statistically significant (P < 0.05). Mean levels of factor X and AT-III of the patients as compared with the control group decreased and showed significant difference (P < 0.05).

Conclusions

Circulation of thalassemic RBCs, which abnormally possess PS on RBC membrane outer surface, suggests the possibility of the gradual consumption of the coagulation factors in the presence of a chronic coagulability state.     

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane‐dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel‐filtered platelets, intact or loaded with calcium‐sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine‐positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine‐negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha‐granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine‐positive platelets formation several‐fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate‐limiting, for subpopulation segregation. Inhibition of mitogen‐activated protein kinase kinase (U0126) and glycoprotein IIb‐IIIa (Monafram^®) was without effect, whereas inhibitors of phosphatidylinositol 3‐kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.     

Platelet apoptosis in paediatric immune thrombocytopenia is ameliorated by intravenous immunoglobulin

To evaluate the role of intravenous immunoglobulin (IVIg) in platelet apoptosis in paediatric immune thrombocytopenia, we investigated the platelets of 20 paediatric patients with acute immune thrombocytopenia (ITP), before and after IVIg treatment. Healthy children with platelet counts in the normal range and children with thrombocytopenia due to chemotherapy were enrolled as controls. All ITP patients presented with platelet counts <20 × 10⁹/l and bleeding symptoms. Markers of apoptosis, including activated caspase‐3, ‐8 and ‐9, phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), as well as platelet‐derived microparticle formation, were analysed by flow cytometry. After IVIg treatment, platelet counts increased to >20 × 10⁹/l in all patients. ITP patients had significantly increased proportions of platelets with activated caspase‐3, ‐8 and ‐9, with PS exposure, and with decreased ΔΨm, and demonstrated increased microparticle formation. Except for ΔΨm, these markers for apoptosis were reduced by IVIg treatment. Platelets of children with thrombocytopenia after chemotherapy also demonstrated increased microparticle formation and decreased ΔΨm, but no activation of caspases 3, 8 and 9 or PS exposure. In conclusion, in acute paediatric ITP, enhanced platelet apoptosis is seen at diagnosis that normalizes after IVIg treatment.     

Increased oxidative stress is associated with balanced increases in hepatocyte apoptosis and proliferation in glycerol-3-phosphate acyltransferase-1 deficient mice

The absence of mouse mitochondrial glycerol-3-phosphate acyltransferase-1 (Gpat1-/-) increases the amount of arachidonate in liver phospholipids and increases beta-hydroxybutyrate and acyl-carnitines, suggesting an elevated rate of liver fatty acid oxidation. We asked whether these alterations might increase reactive oxygen species (ROS), apoptosis, or hepatocyte proliferation. Compared to wildtype controls, liver mitochondria from Gpat1-/- mice showed a 20% increase in the rate of ROS production and a markedly increased sensitivity to the induction of the mitochondrial permeability transition. Mitochondrial phosphatidylethanolamine and phosphatidylcholine from Gpat1-/- liver contained 21% and 67% more arachidonate, respectively, than wildtype controls, and higher amounts of 4-hydroxynonenal, a product of arachidonate peroxidation. Oxidative stress was associated with an increase in apoptosis, and with 3-fold and 15-fold higher TUNEL positive cells in liver from young and old Gpat1-/- mice, respectively, compared to age-matched controls. Compared to controls, bromodeoxyuridine labeling was 50% and 7-fold higher in livers from young and old Gpat1-/- mice, respectively, but fewer glutathione-S-transferase positive cells were present. Thus, Gpat1-/- liver exhibits increased oxidative stress and sensitivity of the mitochondrial permeability transition pore, and a balanced increase in apoptosis and proliferation.     

Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation

Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze‐thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m  ml^?1. The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short‐term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART.