Vitamin D-Rich Diet in Mice Modulates Erythrocyte Survival

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Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca²⁺ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca²⁺ activity ([Ca²⁺]_i) with Fluo3, oxidative stress with 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

Section: Introductionmentioning

confidence: 99%

Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death

Jèmaà¹,

Fezai²,

Bissinger³

et al. 2017

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“…Enhanced eryptosis is observed in diverse clinical conditions including iron deficiency [33], dehydration [92], hyperphosphatemia [93], vitamin D excess [94], chronic kidney disease (CKD) [95-100], hemolytic-uremic syndrome [101], diabetes [102], hepatic failure [103, 104], malignancy [33, 105, 106], sepsis [107], sickle-cell disease [33], beta-thalassemia [33], Hb-C and G6PD-deficiency [33], as well as Wilson´s disease [107]. The enhanced eryptosis leads to rapid clearance of the phosphatidylserine exposing erythrocytes from circulating blood [33].…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Suicidal Erythrocyte Death by Reversine

Jèmaà

Fezai

Läng³

2017

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Background/Aims: The A3 adenosine receptor antagonist reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine) influences cellular differentiation, inhibits cell proliferation, induces cell-cycle arrest, triggers apoptosis, causes cell swelling with polyploidy and stimulates autophagy. The effect on apoptosis involves mitochondria and caspases. Erythrocytes are lacking mitochondria but express caspases and are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), energy depletion and oxidative stress. The present study explored, whether reversine influences eryptosis. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), Ca²⁺ loading (30 minutes treatment with 1 µM Ca²⁺ ionophore ionomycin), or oxidative stress (15 min exposure to 0.3 mM tert-butylhydroperoxide). Results: A 48 hours exposure of human erythrocytes to reversine (1-10 µM) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, Ca²⁺ loading, and oxidative stress were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of each, Ca²⁺ loading, energy depletion and oxidative stress on annexin-V-binding were significantly blunted in the presence of reversine (1-10 µM). The effect of ionomycin, but not the effects of energy depletion and oxidative stress on forward scatter were again significantly blunted in the presence of reversine (≥1 µM]. Conclusions: Reversine is a powerful inhibitor of cell membrane scrambling following energy depletion, Ca²⁺ loading and oxidative stress.

“…Several substances inhibit eryptosis [113][114][115][116]. Enhanced eryptosis is observed in diverse clinical conditions including iron deficiency [58], vitamin D excess [117], chronic kidney disease (CKD) [118][119][120][121][122][123], hemolytic-uremic syndrome [124], autoimmune hemolytic anemia [125], diabetes [126], hypertension and dyslipidemia [127], hepatic failure [128], malignancy [129][130][131], arteritis [132], systemic lupus erythematosus [133], sepsis [134,135], malaria [58,136,137], sicklecell disease [58], beta-thalassemia [58], Hb-C and G6PD-deficiency [58], Wilsons disease [134], as well as advanced age [138]. Eryptosis further increases following storage for transfusion [67,68,83,139] and is enhanced in erythrocytes from newborns exposed to oxidative stress [58,140].…”

Section: Introductionmentioning

confidence: 99%

Stimulation of Eryptosis by Afatinib

Bhuyan

Läng

2018

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Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca 2+ entry, oxidative stress, and/or ceramide. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca 2+ ] i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca 2+ . Conclusions: Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca 2+ entry, oxidative stress, and ceramide.