Oxysterol Mixture in Hypercholesterolemia-Relevant Proportion Causes Oxidative Stress-Dependent Eryptosis

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

“…Nevertheless, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell shrinkage [33] and cell membrane scrambling with exposure of phosphatidylserine at the cell surface [34]. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ) [34], formation of ceramide [35], oxidative stress [36], caspase activation [37,38,39,40,41], activation of casein kinase 1α [42,43], Janus-activated kinase JAK3 [44], protein kinase C [45], or p38 kinase [46], as well as inhibition or knockout of AMP activated kinase AMPK [47], cGMP-dependent protein kinase [38], PAK2 kinase [48], sorafenib sensitive kinases [49] and sunitinib sensitive kinases [50]. …”

Section: Introductionmentioning

confidence: 99%

Stimulation of Suicidal Erythrocyte Death by PRIMA-1

Faggio

Alzoubi

Calabrò

et al. 2015

Background: The anticarcinogenic drug PRIMA-1 (p53 reactivation and induction of massive apoptosis 1) induces suicidal death of tumor cells, an effect in large part attributed to the up-regulation of the proapoptotic transcription factor p53. Erythrocytes are lacking gene transcription but are nevertheless able to enter eryptosis, a suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i) and ceramide formation. The present study tested whether PRIMA-1 stimulates eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and ROS formation from DCFDA fluorescence. Results: A 48 h exposure of human erythrocytes to PRIMA-1 (25 µM) significantly increased the percentage of annexin-V-binding cells without significantly influencing [Ca²⁺]_i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca²⁺ or by the caspase-3 inhibitor zVAD. PRIMA-1 (100 µM) further increased the ceramide abundance at the cell surface and ROS formation. Conclusions: PRIMA-1 stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance and ROS formation.

“…Moreover, eryptosis is triggered by activated casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, p38 kinase, and PAK2 kinase [25], as well as by inhibited or lacking AMP activated kinase AMPK, cGMP-dependent protein kinase, sorafenib sensitive kinases and sunitinib sensitive kinases [25]. Stimulators of eryptosis further include diverse xenobiotics [25,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57]. …”

Section: Introductionmentioning

confidence: 99%

Stimulation of Suicidal Erythrocyte Death by the Antimalarial Drug Mefloquine

Bissinger

Barking

Alzoubi

et al. 2015

Background: The antimalarial drug mefloquine has previously been shown to stimulate apoptosis of nucleated cells. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), and ceramide. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide abundance from specific antibody binding. Results: A 48 h treatment of human erythrocytes with mefloquine significantly increased the percentage of annexin-V-binding cells (≥5 µg/ml), significantly decreased forward scatter (≥5 µg/ml), significantly increased ROS abundance (5 µg/ml), significantly increased [Ca²⁺]_i (7.5 µg/ml) and significantly increased ceramide abundance (10 µg/ml). The up-regulation of annexin-V-binding following mefloquine treatment was significantly blunted but not abolished by removal of extracellular Ca²⁺. Even in the absence of extracellular Ca²⁺, mefloquine significantly increased annexin-V-binding. Conclusions: Mefloquine treatment leads to erythrocyte shrinkage and erythrocyte membrane scrambling, effects at least partially due to induction of oxidative stress, increase of [Ca²⁺]_i and up-regulation of ceramide abundance.