Enhanced Suicidal Erythrocyte Death Contributing to Anemia in the Elderly

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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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“…Eryptosis is stimulated by a wide variety of small molecules [2, 9-63]. Moreover, eryptosis is enhanced in elderly individuals [64]. It is sensitive to erythrocyte age [65] and enhanced eryptosis is observed following erythrocyte storage [66, 67].…”

Section: Introductionmentioning

confidence: 99%

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

Abed

Thiel

Towhid

et al. 2017

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Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca²⁺-activity ([Ca²⁺]_i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca²⁺]_i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca²⁺]_i, increase of ceramide abundance and caspase activation.

“…Eryptosis is stimulated by hyperosmotic shock [50], oxidative stress [50], energy depletion [50], or a myriad of xenobiotics [50, 57-115]. Augmented eryptosis is observed in several clinical conditions including iron deficiency [50], dehydration [116], hyperphosphatemia [117], chronic kidney disease (CKD) [118-121], hemolytic-uremic syndrome [122], diabetes [123], hepatic failure [57], malignancy [124, 125], arteriitis [126], sepsis [127], sickle-cell disease [50], beta-thalassemia [50], Hb-C and G6PD-deficiency [50], Wilsons disease [127], as well as advanced age [128]. Eryptosis further increases during storage of blood for transfusion [129].…”

Section: Introductionmentioning

confidence: 99%

Triggering of Suicidal Erythrocyte Death by Gefitinib

Bhuyan

Wagner

Cao

et al. 2017

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Background/Aims: The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca²⁺ entry and/or oxidative stress. Methods: Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺. Gefitinib further significantly increased DCFDA fluorescence. Conclusions: Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca²⁺ and paralleled by oxidative stress.