Downloaded from British Journal of Nutrition (2015), 114, 368–375 doi:10.1017/S0007114515002111 q The Authors 2015 https://www.cambridge.org/core Dietary indicaxanthin from cactus pear (Opuntia ficus-indica L. Mill) fruit prevents eryptosis induced by oxysterols in a hypercholesterolaemia- relevant proportion and adhesion of human erythrocytes to endothelial cell layers . IP address: Luisa Tesoriere, Alessandro Attanzio, Mario Allegra and Maria A. Livrea* 170.106.33.19 Department of Biological Chemical and Pharmaceutical Science and Technologies (STEBICEF), Universita` di Palermo, Via M. Cipolla 74, 90123 Palermo, Italy , on (Submitted 22 January 2015 – Final revision received 6 May 2015 – Accepted 18 May 2015 – First published online 14 July 2015) 26 Sep 2021 at 20:54:52 Abstract Toxic oxysterols in a hypercholesterolaemia-relevant proportion cause suicidal death of human erythrocytes or eryptosis. This process proceeds through early production of reactive oxygen species (ROS), release of prostaglandin (PGE2) and opening of PGE2-dependent Ca channels, membrane phosphatidylserine (PS) externalisation, and cell shrinkage. The present study was the first to reveal that a , subject to the Cambridge Core terms of use, available at bioavailable phytochemical, indicaxanthin (Ind) from cactus pear fruit, in a concentration range (1·0–5·0 mM) consistent with its plasma level after a fruit meal, prevents PS externalisation and cell shrinkage in a dose-dependent manner when incubated with isolated healthy human erythrocytes exposed to an oxysterol mixture for 48 h. Dietary Ind inhibited ROS production, glutathione (GSH) depletion, PGE2 release and Ca2þ entry. Ind alone did not modify the erythrocyte redox environment or affect other parameters. Ex vivo spiking of normal human blood with the oxysterol mixture for 48 h induced eryptosis, resulting in the production of ROS and decreased levels of GSH, which was prevented by concurrent exposure to 5 mM-Ind. The adherence of eryptotic erythrocytes to the endothelium causes vascular tissue injury. Erythrocytes isolated from blood incubated with the oxysterol mixture plus 5 mM-Ind did not adhere to endothelial cell monolayers. Eryptotic erythrocytes may contribute to thrombotic complications in hypercholesterolaemia. Our findings suggest the positive effects of diets containing Ind on erythrocytes in hypercholesterolaemic subjects. Key words: Betalains: Dietary indicaxanthin: Eryptosis: Human erythrocytes: Hypercholesterolaemia: Oxysterols British Journal of Nutrition (1) (17) A physiological rate of programmed death, or eryptosis ,of various studies . Polyphenol phytochemicals have long https://www.cambridge.org/core/terms erythrocytes allows clearance of aged cells; however, an increased been considered as protective agents; however, recent atten- number of circulating eryptotic erythrocytes, as observed in var- tion has been focused on betalains(18), a group of pigments ious diseases(2–5), is dangerous and leads to complications. sharing the common structure of betalamic acid. Indicaxanthin Because of phosphatidylserine (PS) exposed at the erythrocyte (Ind; Fig. 1), the characteristic yellow pigment of cactus pear surface, eryptotic cells may activate coagulant enzymes(6),causing (Opuntia ficus-indica) fruit, is the immonium derivative of thrombosis and thrombo-occlusive disease(3,6–9),andadhereto proline with betalamic acid(19). Ind is a reducing and amphi- endothelial cells(8,10), promoting vascular damage. Our recent pathic molecule, interacts with and partitions in membranes, studies have for the first time demonstrated that a mixture of enters various cells, including erythrocytes, and counteracts oxysterols, known to be elevated in the serum of hypercholester- oxidative damage induced by various agents(20 – 26). Moreover, (11 – 13) . olaemic subjects , exerts a remarkable eryptotic activity it has been shown to modulate specific redox-driven signal- https://doi.org/10.1017/S0007114515002111 compared with healthy erythrocytes(14), an event that enhances ling pathways involved in the inflammatory response in the pathogenic potential of these cholesterol derivatives in the cultured endothelial and intestinal cells, and interfere with initiation and promotion of atherogenic processes(15,16). molecular mechanisms involved in the apoptotic activity of Evidence on the impact of dietary vegetable intake on the 7-ketocholesterol (7-KC) in a human monocyte/macrophage prevention of chronic diseases, including cancer, neurode- cell line(27 – 29). Dietary Ind that may be absorbed by generative disorders and atherosclerosis, has emerged from passive diffusion through the intestinal epithelium has been Abbreviations: 7-KC, 7-ketocholesterol; Ind, indicaxanthin; DCFDA, dichloro-dihydro-fluorescein diacetate; FACS, fluorescence-activated cell sorting; GSH, glutathione; HUVEC, human umbilical vein endothelial cells; PS, phosphatidylserine; ROS, reactive oxygen species. * Corresponding author: M. A. Livrea, email [email protected] Downloaded from Indicaxanthin prevents eryptosis by oxysterols 369 were pre-incubated for 1 h in the absence or presence of Ind https://www.cambridge.org/core + before adding a mixture of oxysterols, followed by incubation N COOH for 48 h. The mixture of oxysterols at final concentrations of 7 mM-7-KC, 2 mM-TRIOL, 4 mM-a-epox, 1 mM-7a-OH, 2 mM-7b- OH and 4 mM-b-epox (20 mMof total oxysterols) was added to the cells using tetrahydrofuran at a 0·1 % (v/v) final concen- tration. Preliminary experiments showed that under these conditions, tetrahydrofuran did not have any effect on erythro- cytes; therefore, erythrocytes incubated with the solvent were . IP address: HOOC N COOH H used as the control. Indicaxanthin 170.106.33.19 Fig. 1. Molecular structure of indicaxanthin. Measurement of phosphatidylserine externalisation and forward scatter Erythrocytes washed in Ringer solution were adjusted at , on 1·0 £ 106 cells/ml with the combining buffer according to 26 Sep 2021 at 20:54:52 found to be unmodified in human blood and exhibits high the manufacturer’s protocol (eBioscience, Inc.). Cell suspen- bioavailability, reaching plasma concentrations of several sion (100 ml) was added to a new tube, and incubated with micromolars after a cactus pear fruit meal(30 – 32). Therefore, 5 ml Annexin V-FITC at room temperature for 15 min in the the aim of the present study was to investigate whether dietary dark. Fluorescence-activated cell sorting (FACS) analysis was Ind affected oxysterol-induced eryptosis in human erythro- carried out using an Epics XLe flow cytometer with Expo32 cytes. For this purpose, a mixture of oxysterols in hyper- , subject to the Cambridge Core terms of use, available at software (Beckman Coulter). Cells were analysed by forward cholesterolaemia-relevant proportions(11 – 13) and Ind scatter, and annexin V fluorescence intensity was measured concentrations consistent with the plasma levels achieved in the fluorescence channel FL-1 with an excitation wave- after a dietary intake of cactus pear fruits(32) were considered. length of 488 nm and an emission wavelength of 530 nm. The study was performed with either isolated erythrocytes or At least 1 £ 104 cells were examined. after ex vivo spiking of whole blood with oxysterols in the presence or absence of Ind. Finally, the effect of dietary Ind on the adherence of oxysterol-treated erythrocytes to endo- thelial cell monolayers (human umbilical vein endothelial Measurement of cytosolic calcium cells; HUVEC) was explored. Intracellular Ca2þ concentration was measured by the cell- permeable probe fluo-3 AM, whose fluorescence directly represents the ion concentration, as reported previously(14). Materials and methods Fluorescent intensities were analysed by FACS analysis. Ca2þ- British Journal of7-KC, Nutrition cholestan-3b,5a,6b-triol (TRIOL), 5a,6a-epoxycholesterol dependent fluorescence intensity was measured by FACS (a-epox), 5b,6b-epoxy-cholesterol (b-epox), 7a-hydroxy- analysis in the fluorescence channel FL-1 with an excitation https://www.cambridge.org/core/terms cholesterol (7a-OH) and 7b-hydroxy-cholesterol (7b-OH) were wavelength of 488 nm and an emission wavelength of 530 nm. 4 obtained from Avanti Polar Lipids, Inc. All other reagents At least 1 £ 10 cells were examined. and chemicals were obtained from Sigma Chemical Co., unless otherwise indicated. Measurement of PGE2 Preparation of indicaxanthin After incubation, as indicated above, PGE2 released (in pg/ml) from erythrocytes (1 £ 109 cells/ml) was quantified using a Ind was isolated from cactus pear (Opuntia ficus-indica Prostaglandin E2 Enzyme Immunoassay Kit (Cayman Chemical L. Mill) fruit pulp (yellow cultivar) by methanol extraction, Corporation, Inc.) in accordance with the manufacturer’s . (29) https://doi.org/10.1017/S0007114515002111 purified and quantified as reported previously . protocol. Briefly, after incubation, the cells were pelleted by centrifugation at 48C, for 5 min, at 450 g. Samples of the super- natant were diluted at 1:2·5 with assay buffer. Then, a 100 ml Cells and incubation conditions sample, a 50 ml alkaline phosphatase PGE2 conjugate and a Erythrocytes collected from the blood of healthy volunteers 50 ml monoclonal anti-PGE2 EIA antibody were applied to (n 5; age 25–68 years; normal BMI range), with informed con- goat anti-mouse IgG microtitre plates, and incubated at room sent, were isolated by centrifugation (2000 g,48C, 20 min) on a temperature for 2 h. After washing, 200 mlofp-nitrophenyl Ficoll gradient (Biochrom KG). Erythrocytes (0·4