Phosphatidylserine Exposition Research Articles

Assessing mitochondria-targeted acyl hydroquinones on the mitochondrial platelet function and cytotoxic activity: Role of the linker length

IntroductionThe use of triphenylphosphonium cation (TPP+) linked to phenolic compounds by alkyl chains has a significant relevance as a mitochondrial delivery strategy in biomedicine because it affects mitochondrial bioenergetics in models of noncommunicable diseases such as cancer and cardiovascular-related conditions. Studies indicate that a long alkyl chain (10–12 carbon) increases the mitochondrial accumulation of TPP+-linked drugs. In contrast, other studies show that these compounds are consistently toxic to micromolar concentrations (as observed in platelets). In the present study, we evaluated the in vitro effect of three series of triphenylphosphonium-linked acyl hydroquinones derivates on the metabolism and function of human platelets using 3–9 carbons for the alkyl linker. Those were assessed to determine the role of the length of the alkyl chain linker on platelet toxicity. MethodsHuman platelets were exposed in vitro to different concentrations (2–40 μM) of every compound; cellular viability, phosphatidylserine exposition, mitochondrial membrane potential (ΔΨm), intracellular calcium release, and intracellular ROS generation were assessed by flow cytometry. An in silico energetic profile was generated with Umbrella sampling molecular dynamics (MD). Results and conclusionsThere was an increase in cytotoxic activity directly related to the length of the acyl chain and lipophilicity, as seen by three techniques, and this was consistent with a decrease in ΔΨm. The in silico energetic profiles point out that the permeability of the mitochondrial membrane may be involved in the cytotoxicity of phosphonium salts. This information may be relevant for the design of new TPP+ -based drugs with a safe cardiovascular profile.

Anticancer Activity and Molecular Targets of Piper cernuum Substances in Oral Squamous Cell Carcinoma Models.

Oral squamous cell carcinoma (OSCC) is a worldwide public health problem, with high morbidity and mortality rates. The development of new drugs to treat OSCC is paramount. Piper plant species have shown many biological activities. In the present study, we show that dichloromethane partition of Piper cernuum (PCLd) is nontoxic in chronic treatment in mice, reduces the amount of atypia in tongues of chemically induced OSCC, and significantly increases animal survival. To identify the main active compounds, chromatographic purification of PCLd was performed, where fractions 09.07 and 14.05 were the most active and selective. These fractions promoted cell death by apoptosis characterized by phosphatidyl serine exposition, DNA fragmentation, and activation of effector caspase-3/7 and were nonhemolytic. LC-DAD-MS/MS analysis did not propose matching spectra for the 09.07 fraction, suggesting compounds not yet known. However, aporphine alkaloids were annotated in fraction 14.05, which are being described for the first time in P. cernuum and corroborate the observed cytotoxic activity. Putative molecular targets were determined for these alkaloids, in silico, where the androgen receptor (AR), CHK1, CK2, DYRK1A, EHMT2, LXRβ, and VEGFR2 were the most relevant. The results obtained from P. cernuum fractions point to promising compounds as new preclinical anticancer candidates.

Exposition of Phosphatidylserine in Lewis Carcinoma Cells under the Action of Anphen Sodium and Hydrogen Peroxide

Exposition of Phosphatidylserine in Lewis Carcinoma Cells under the Action of Anphen Sodium and Hydrogen Peroxide

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells

BackgroundRickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector–pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26.MethodsThe proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined.ResultsTick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth.ConclusionsTaken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.Graphical

N-3 Polyunsaturated fatty acids induce acute myeloid leukemia cell death associated with mitochondrial glycolytic switch and Nrf2 pathway activation.

Acute Myeloid Leukemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. n-3 polyunsaturated fatty acids (PUFAs), present in fish oil (FO) at high concentrations, have antitumoral properties in various cancer models. We investigated the effects of two n-3 PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), in AML cell lines and primary AML blasts. EPA and DHA induced a dose-dependent decrease in cell viability in five AML cell lines, which was also observed with FO, but not SO (devoid of n-3 PUFAs) in cell lines and primary leucoblasts. Mitochondrial energy metabolism shifted from oxidative respiration to glycolytic metabolism in the U937, MOLM-13, and HL-60 cell lines. This phenomenon was associated with major disorganization of the mitochondrial network and mitochondrial swelling. Transcriptomic analysis after 6 h and 24 h of exposure to FO revealed a Nrf2 activation signature, which was confirmed by evidence of Nrf2 nuclear translocation in response to oxidative stress, but insufficient to prevent cell death following prolonged exposure. Apoptosis studies showed consistent phosphatidylserine exposition among the AML cell lines tested and a reduced mitochondrial membrane potential. The cell-killing effect of FO was additive with that of cytarabine (AraC), by the Chou and Talalay method, and this combination effect could be reproduced in primary AML blasts. Altogether, our results show deleterious effects of n-3 PUFAs on mitochondrial metabolism of AML cells, associated with oxidative stress and Nrf2 response, leading to cell death. These observations support further investigation of n-3 PUFA addition to standard chemotherapy in AML.

Isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives induce regulated necrosis-like cell death on Leishmania (Leishmania) mexicana.

Leishmaniasis is a neglected tropical disease caused by the parasite of the genus Leishmania. About 13 million people are infected worldwide, and it is estimated that 350 million are at risk of infection. Clinical manifestations depend on the parasite species and factors related to the host such as the immune system, nutrition, housing, and financial resources. Available treatments have severe side effects; therefore, research currently focuses on finding more active and less toxic compounds. Quinoxalines have been described as promising alternatives. In this context, 17 isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives were evaluated as potential leishmanicidal agents. Their effect on the cell metabolism of Leishmania mexicana promastigotes and their cytotoxic effects on the J774.A1 cell line and on erythrocytes were evaluated, and their selectivity index was calculated. Compounds T-069 (IC50=1.49μg/mL), T-070 (IC50=1.71μg/mL), T-072 (IC50=6.62μg/mL), T-073 (IC50=1.25μg/mL), T-085 (IC50=0.74μg/mL), and T-116 (IC50=0.88μg/mL) were the most active against L. mexicana promastigotes and their mechanism of action was characterized by flow cytometry and microscopy. Compound T-073, the most selective quinoxaline derivative, induced cell membrane damage, phosphatidylserine exposition, reactive oxygen species production, disruption of the mitochondrion membrane potential, and DNA fragmentation, all in a dose-dependent manner, indicating the induction of regulated necrosis. Light and transmission electron microscopy showed the drastic morphological changes induced and the mitochondrion as the most sensitive organelle in response to T-073. This study describes the mechanism by which active isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide quinoxalines affect the parasite.

Synthesis and evaluation of a radiolabeled bis-zinc(II)-cyclen complex as a potential probe for in vivo imaging of cell death.

The exposition of phosphatidylserine (PS) from the cell membrane is associated with most cell death programs (apoptosis, necrosis, autophagy, mitotic catastrophe, etc.), which makes PS an attractive target for overall cell death imaging. To this end, zinc(II) macrocycle coordination complexes with cyclic polyamine units as low-molecular-weight annexin mimics have a selective affinity for biomembrane surfaces enriched with PS, and are therefore useful for detection of cell death. In the present study, a 11C-labeled zinc(II)-bis(cyclen) complex (11C-CyclenZn2) was prepared and evaluated as a new positron emission tomography (PET) probe for cell death imaging. 11C-CyclenZn2 was synthesized by methylation of its precursor, 4-methoxy-2,5-di-[10-methyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester] phenol (Boc-Cyclen2) with 11C-methyl triflate as a prosthetic group in acetone, deprotection by hydrolysis in aqueous HCl solution, and chelation with zinc nitrate. The cell death imaging capability of 11C-CyclenZn2 was evaluated using in vitro cell uptake assays with camptothecin-treated PC-3 cells, biodistribution studies, and in vivo PET imaging in Kunming mice bearing S-180 fibrosarcoma. Starting from 11C-methyl triflate, the total preparation time for 11C-CyclenZn2 was ~40min, with an uncorrected radiochemical yield of 12 ± 3% (based on 11C-CH3OTf, n = 10), a radiochemical purity of greater than 95%, and the specific activity of 0.75-1.01GBq/μmol. The cell death binding specificity of 11C-CyclenZn2 was demonstrated by significantly different uptake rates in camptothecin-treated and control PC-3 cells in vitro. Inhibition experiments for 18F-radiofluorinated Annexin V binding to apoptotic/necrotic cells illustrated the necessity of zinc ions for zinc(II)-bis(cyclen) complexation in binding cell death, and zinc(II)-bis(cyclen) complexe and Annexin V had not identical binding pattern with apoptosis/necrosis cells. Biodistribution studies of 11C-CyclenZn2 revealed a fast clearance from blood, low uptake rates in brain and muscle tissue, and high uptake rates in liver and kidney, which provide the main metabolic route. PET imaging using 11C-CyclenZn2 revealed that cyclophosphamide-treated mice (CP-treated group) exhibited a significant increase of uptake rate in the tumor at 60min postinjection, compared with control mice (Control group). The results indicate that the ability of 11C-CyclenZn2 to detect cell death is comparable to Annexin V, and it has potential as a PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy.

Clomipramine kills Trypanosoma brucei by apoptosis

Drug repositioning, i.e. use of existing medicals to treat a different illness, is especially rewarding for neglected tropical diseases (NTD), since in this field the pharmaceutical industry is rather reluctant to spend vast investments for drug development. NTDs afflict primarily poor populations in under-developed countries, which minimizes financial profit. Here we investigated the trypanocidal effect of clomipramine, a commercial antipsychotic drug, on Trypanosoma brucei. The data showed that this drug killed the parasite with an IC50 of about 5μM. Analysis of the involved cell death mechanism revealed furthermore an initial autophagic stress response and finally the induction of apoptosis. The latter was substantiated by a set of respective markers such as phosphatidylserine exposition, DNA degradation, loss of the inner mitochondrial membrane potential and characteristic morphological changes. Clomipramine was described as a trypanothione inhibitor, but as judged from our results it also showed DNA binding capacities and induced substantial morphological changes. We thus consider it likely that the drug induces a multifold adverse interaction with the parasite’s physiology and induces stress in a way that trypanosomes cannot cope with.

Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes

Leishmaniasis is an overlooked tropical disease affecting approximately 1 million people in several countries. Clinical manifestation depends on the interaction between Leishmania and the host's immune response. Currently available treatment options for leishmaniasis are limited and induce severe side effects. In this research, we tested nitro-heterocyclic compounds (BSF series) as a new alternative against Leishmania. Its activity was measured in Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes using MTT colorimetric assay. Additionally, we assessed the phosphatidylserine exposure by promastigotes, measured by flow cytometry, as well as nitric oxide production, measured by Griess' method. The nitro-heterocyclic compounds (BSF series) showed activity against L. (L.) infantum promastigotes, inducting the phosphatidylserine exposition by promastigotes, decreasing intracellular amastigotes and increasing oxide nitric production. The selectivity index was more prominent to Leishmania than to macrophages. Compared to amphotericin b, our compounds presented higher IC50, however the selectivity index was more specific to parasite than to amphotericin b. In conclusion, these nitro-heterocyclic compounds showed to be promising as an anti-Leishmania drug, in in vitro studies.

Abstract 209: A quantitative analysis of paclitaxel-induced mitotic catastrophe of MDA-MB-231 cells by high frequency ultrasound

Abstract In recent years, high-frequency ultrasound (HFUS) has shown promise of non-invasively detecting tumor death in response to chemotherapy. However, those studies have been limited to ultrasound B-mode and immunohistochemical image comparison over extended periods of time (≥24 hours). The purpose of this study was to evaluate the relationship of quantified HFUS spectral parameters to the dynamics of tumor cell death in MDA-MB-231 cell samples exposed to paclitaxel chemotherapy. Several ultrasound parameters were investigated, including the, attenuation, spectral slope, spectral y-intercept, midband fit, estimated scatterer diameter and estimated scatterer concentration. Changes in these spectral-based parameters were evaluated in their capacity to respond to various cell death events, including mitochondrial depolarization, caspase activation, phosphatidylserine exposition, and membrane selective permeability compromise, as determined by flow cytometry analysis. Standard histology and transmission electron microscopy (TEM) permitted further investigation of possible candidates responsible for the changes detected using ultrasound. Based on TEM, negative TUNEL staining, and cell cycle analysis it was found that paclitaxel at 1mM induces mitotic catastrophe as the main cell death modality. The spectral slope correlated strongly (r2 = 0.89) with the percentage of cells arrested at the G2/M checkpoint and exhibited an opposite trend to that of systems undergoing classical apoptosis, suggesting that it may be a prime biophysical marker for differentiating modalities of tumor cell death. Midband fit increases through time suggested an increase in the concentration of acoustic scatterers. The formation of condensed chromatin condensates of a 1 micron or greater diameter in the course of paclitaxel-induced mitotic catastrophe, as revealed by TEM, were hypothesized to act as large intracellular scatterers and correlated with the increase in ultrasound midband fit. Midband fit showed the strongest correlation of all parameters (r2 = 0.99) when plotted against the percentage of the cell population expected to possess these scattering structures, establishing the first quantitative correlation of ultrasound parameters to a cell death index. These findings support that HFUS spectral parameters may be used to quantify the degree of cell death and predict the efficacy of chemotherapeutic treatment within a short post-treatment timeframe. Citation Format: Maurice M. Pasternak, Gregory J. Czarnota. A quantitative analysis of paclitaxel-induced mitotic catastrophe of MDA-MB-231 cells by high frequency ultrasound. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 209. doi:10.1158/1538-7445.AM2015-209

Ostreopsis cf. ovata induces cytoskeletal disorganization, apoptosis, and gene expression disregulation on HeLa cells

The dinoflagellates of the genus Ostreopsis Schmidt are toxic species involved in the occurrence of massive blooms. These dinoflagellates are known to produce palytoxin and ovatoxins, which are considered amongst the most poisonous phycotoxins in the world. In this work, aqueous and methanolic extracts of one Ostreopsis cf. ovata Ionian strain were tested on the human-derived HeLa cell line, and cytotoxicity, cytoskeletal rearrangement, and apoptosis induction were recorded by morphological and molecular analysis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test revealed high toxicity for both extracts, which activity was inhibited by ouabain, which suggests the main involvement of palytoxin-like molecules in the observed toxic effects of this strain. Overall, the effects induced by O. cf. ovata on HeLa cells were dependent on the type of adopted extracts, being both toxic, but with the methanolic extract more toxic than the aqueous one and able to induce cell death by apoptosis. This process was evidenced by both phosphatidylserine exposition and upregulation of caspase-3 gene expression. The different effect of the two extracts on cell death is indicative of their different composition and/or activity. Our findings represent the starting step for the characterization of novel bioactive molecules.

Biological Functions of Plasma Membrane-Derived Extracellular Vesicles and Their Role in Diseases

Plasma Membrane-derived Extracellular Vesicles (PMEVs) are membrane-coated vesicles of diameter 0.1 to 1.5µm, carrying various proteins inherent in their parental cells. PMEVs are released when cells undergo activation/apoptosis via blebbing and shedding and have a function in intercellular communication. Exposition of phosphatidylserine (PS) on the outer membrane leaflet that mark them as a biologically distinct entity could also explain a role for PMEVs in phagocytosis and thrombosis. The purpose of this review is to outline and discuss some of the functions of PMEVs in detail to throw more light on its biological effects as more research delve into emerging therapies targeting the microvesicle communication system. The role of PMEVs as a differentiation agent and therefore its possible use in differentiation therapy is discussed. In some experiments, the myeloid differentiating agents all trans retinoic acid (ATRA), phorbol 12-myristate 13-acetate (PMA) and histamine, which inhibit promonocyte proliferation, induced an intracellular Ca2+-mediated PMEV release from HL-60 promonocytes. These PMEVs caused HL-60 cells to enter G0/G1 cell cycle arrest and induce terminal monocyte-to-macrophage differentiation through TGF-β1 mediation. The review also discusses the relationship between PMEVs and diseases where it is known that patients with certain inflammatory diseases show increased PMEV levels in the plasma. The review conclude on the fact that PMEVs have a lot of biological functions that are beneficial to the physiological functions in humans and therefore more work is required to elucidate their composition and the mechanisms involved in exertion of their effects.

Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

Label-free real-time acoustic sensing of microvesicle release from prostate cancer (PC3) cells using a Quartz Crystal Microbalance

Using a Quartz Crystal Microbalance with dissipation monitoring, QCM-D (label-free system) measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, we showed the attachment, over a 60min period, of a monolayer of PC3 cells to the gold electrodes of the quartz crystal sensor, which had been rendered hydrophilic. That MVs were released upon BzATP stimulation of cells was confirmed by NTA analysis (average 250nm diameter), flow cytometry, showing high phosphatidylserine exposition and by fluorescent (Annexin V Alexa Fluor® 488-positive) and electron microscopy. Over a period of 1000s (16.7min) during which early apoptosis increased from 4% plateauing at 10% and late apoptosis rose to 2%, the Δf increased 20Hz, thereupon remaining constant for the last 1000s of the experiment. Using the Sauerbrey equation, the loss in mass, which corresponded to the release of 2.36×106MVs, was calculated to be 23ng. We therefore estimated the mass of an MV to be 0.24pg. With the deposition on the QCM-D of 3.5×107MVs over 200s, the decrease in Δf (Hz) gave an estimate of 0.235pg per MV.

Neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells.

BackgroundUpon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. In this study we investigated the potential role of myeloperoxidase in the endothelial cell injury caused by neutrophil-derived microparticles.ResultsMicroparticles were produced by activating human neutrophils with a calcium ionophore and characterized by flow cytometry and transmission and scanning electron microscopy. Myeloperoxidase activity was measured by luminol-dependent chemiluminescence. Neutrophil microparticles-induced injuries and morphological alterations in human umbilical vein endothelial cells (HUVECs) were evaluated by microscopy and flow cytometry. Neutrophil microparticles were characterized as structures bounded by lipid bilayers and were less than 1 μm in diameter. The microparticles also expressed CD66b, CD62L and myeloperoxidase, which are all commonly expressed on the surface of neutrophils, as well as exposition of phosphatidylserine. The activity of the myeloperoxidase present on the microparticles was confirmed by hypochlorous acid detection. This compound is only catalyzed by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. The addition of sodium azide or taurine inhibited and reduced enzymatic activity, respectively. Exposure of HUVEC to neutrophil microparticles induced a loss of cell membrane integrity and morphological changes. The addition of sodium azide or myeloperoxidase-specific inhibitor-I consistently reduced the injury to the endothelial cells. Taurine addition reduced HUVEC morphological changes.ConclusionsWe have demonstrated the presence of active myeloperoxidase in neutrophil microparticles and that the microparticle-associated myeloperoxidase cause injury to endothelial cells. Hence, the microparticle-associated myeloperoxidase-hydrogen peroxide-chloride system may contribute to widespread endothelial cell damage in conditions of neutrophil activation as observed in vasculitis and sepsis.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Pro-apoptotic effects of Amblyomin-X in murine renal cell carcinoma “in vitro”

Renal cell carcinoma (RCC) is one of the most lethal urologic cancers and is highly resistant to both radiotherapy and chemotherapy. The recombinant protein Amblyomin-X, characterized as a Kunitz-type protease inhibitor, was obtained from a cDNA library from the salivary glands of the Amblyomma cajennense tick. This paper reports the biological effect of Amblyomin-X on inducing cell death by apoptotic process in vitro. For this purpose, the changes in morphological aspects of cells, the phosphatidylserine exposition and DNA degradation were evaluated after treatment with Amblyomin-X. We found that Amblyomin-X was able to induce apoptosis in Renca cells in a dose-dependent manner. So, the results presented here open perspectives for new researches and developing for Amblyomin-X in the treatment of RCC.

Atorvastatin does not Affect Ischaemia-Induced Phosphatidylserine Exposition in Humans in-vivo

Statins can induce pharmacologic preconditioning and thereby reduce infarct size. Cellular phosphatidylserine (PS) exposition occurs in the course of ischaemia and reperfusion and has been associated with injury. In this experiment we studied the effect of atorvastatin on PS exposition after a standardised ischaemia and reperfusion challenge. In a double-blind randomised cross-over trial 30 healthy volunteers were allocated to 3 day treatment with atorvastatin (80 mg/day) and placebo (n = 24), or placebo treatment twice (n = 6). At the end of each treatment period, volunteers underwent 10 minutes of forearm ischaemic exercise. At reperfusion radiolabeled annexin A5 was administered intravenously and Gamma camera imaging of both hands was performed 1 and 4 hours after reperfusion. Annexin A5 targeting was not different between atorvastatin treatment (26.1 ± 9.8% and 24.0 ± 9.5% respectively at 1 and 4 hours after reperfusion) and placebo treatment (25.6 ± 11.0% and 24.5 ± 10.7%) (p = 0.99). Our time control experiment did not reveal a carry-over effect. Our results show that treatment with atorvastatin 80 mg does not reduce forearm PS exposition after ischaemic exercise. This suggests that the role of PS exposure in the prevention of ischemia and reperfusion injury by short term treatment with atorvastatin is limited.

Angiotensin II Type 1 Receptor Blockade Does Not Enhance Apoptotic Cell Death During Ischemia and Reperfusion in Humans In Vivo

Despite the theoretical benefits, angiotensin II type 1 receptor antagonists seem to enhance rather than reduce morbidity and mortality after myocardial infarction compared with angiotensin-converting enzyme inhibitors. This may result from unopposed angiotensin II type 2 receptor stimulation, which is associated with enhanced apoptotic cell death and increased infarct size. We studied whether the clinical effectiveness of irbesartan is hampered by enhanced apoptotic activity, detected by exposition of phosphatidylserines, during ischemia and reperfusion in humans in vivo. Twenty healthy male volunteers were randomized to a 1-week treatment with irbesartan (300 mg/d) or placebo in a double-blind fashion. After treatment, all participants underwent 10 minutes of ischemic exercise of the nondominant forearm. Upon reperfusion, Tc-99m-labeled Annexin A5 was administered, and 1 and 4 hours afterward, both hands were scanned using a gamma camera. Targeting of annexin A5, expressed as the percentage difference in radioactivity in the area of interest (thenar muscle) between experimental and control hand, did not differ between participants treated with irbesartan or placebo. Therefore, irbesartan does not enhance phosphatidylserine exposition in humans in vivo. The results of this study do not support enhanced apoptotic activity after treatment with irbesartan in a setting of ischemia and reperfusion.

Photopheresis with UV-A light and 8-methoxypsoralen leads to cell death and to release of blebs with anti-inflammatory phenotype in activated and non-activated lymphocytes

Background: Extracorporeal photopheresis is a therapy for treatment of autoimmune diseases, cutaneous T-cell lymphoma, organ graft rejection as well as graft-versus-host diseases. The exact mechanism how the combination of 8-methoxypsoralen plus UV-A irradiation (PUVA) acts is still unclear. We investigated the cell death of activated and non-activated lymphocytes after PUVA treatment as well as the rate of released blebs and their antigen composition. Results: In presence of 8-MOP, UV-A light highly significantly increased the cell death of activated lymphocytes. The same was observed to a lesser extent in non-activated cells. Blebs derived from activated lymphocytes after PUVA treatment showed the highest surface exposition of phosphatidylserine. These blebs also displayed a high exposure of the antigens CD5 and CD8 as well as a low exposure of CD28 and CD86. Conclusion: PUVA treatment exerts anti-inflammatory effects by inducing apoptosis and apoptotic cell-derived blebs with immune suppressive surface composition.