Eryptosis Of Human Erythrocytes Research Articles

Вплив амфіфільних сполук на форму та ериптоз еритроцитів людини в умовах постгіпертонічного шоку

The search for protective substances that can be used during red blood cell thawing and the study of their effects on red blood cells contribute to increasing the number and quality of viable cells after the cryopreservation cycle. We studied the effect of posthypertonic shock and amphiphilic compounds on the shape and eryptosis of human erythrocytes. The method of flow cytometry was used, this allows analyzing two parameters simultaneously, which increases the efficiency of research. The shape was assessed by the sphericity index (SphI), and eryptosis by the redistribution of phosphatidylserine to the membrane outer surface. It has been shown that sodium decylsulfate and chlorpromazine reduce erythrocyte damage in posthypertonic shock by 3.6 and 4.2 times, respectively. Sodium decylsulfate helps to preserve the shape of cells (SphI coefficient remains the same), while when chlorpromazine is used, the shape changes towards spherical (SphI coefficient changes 2 times). The study of the level of Annexin V FITC binding to phosphatidylserine in outer layer of membrane revealed a concentration-dependent increase in fluorescence when sodium decylsulfate was used, indicating a disorder of the bilayer asymmetry. In contrast, chlorpromazine did not change the distribution of phosphatidylserine. Comparison of two parameters of cell viability - the sphericity coefficient and anexin binding - allowed us to choose the conditions that are optimal for the use of the studied protective substances. Namely, it is advisable to use the lowest effective concentration of sodium decylsulfate (200 mcmol/l) for protective purposes. This ensures the preservation of the cell shape and minimal impact on the membrane asymmetry.

Desipramine induces eryptosis in human erythrocytes, an effect blunted by nitric oxide donor sodium nitroprusside and N-acetyl-L-cysteine but enhanced by Calcium depletion

ABSTRACT Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET. Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms. Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography. Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC. Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.

Antiproliferative Wnt inhibitor wogonin prevents eryptosis following ionophoric challenge, hyperosmotic shock, oxidative stress, and metabolic deprivation.

Anemia is a common complication of chemotherapy and may arise due to premature or suicidal death of red blood cells (RBCs). Prevention of RBC death thus lends itself as a promising strategy to counteract anemia. Wogonin (WGN; 5,7-dihydroxy-8-methoxyflavone) is a Wnt inhibitor derived from Scutellaria baicalensis plant with potent cytotoxic and antitumor potential. However, the nature of interaction of WGN with human RBCs is unknown. RBCs from healthy participants were exposed to different hemolytic and eryptotic stimuli for 24 or 48hr at 37°C in the presence and absence of 100μM WGN. Calcium overload was induced by 2μM ionomycin, hyperosmotic shock with excessive sucrose, oxidative stress by 2-phenethyl isothiocyanate (PEITC), and metabolic deprivation by exclusion of glucose. Hemolysis was estimated from extracellular hemoglobin, phosphatidylserine (PS) exposure by Annexin V-FITC, intracellular calcium by Fluo4/AM, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA). While WGN did not rescue the cells from the hemolytic activity of ionomycin, it reduced PS externalization by interfering with calcium influx under both ionomycin treatment and metabolic exhaustion. WGN also significantly inhibited PS exposure upon hyperosmotic shock, but the effect was independent of calcium entry. Moreover, WGN exhibited antihemolytic and anti-eryptotic activities against PEITC without appreciable reduction in ROS levels. Altogether, WGN prevents PEITC-induced hemolysis and suppresses eryptosis due to calcium accumulation, hyperosmotic shock, oxidative stress, and metabolic exhaustion. These novel insights may open new avenues into the therapeutic application of WGN for conditions in which anemia is commonly encountered, most notably cancer. PRACTICAL APPLICATIONS: The herbal supplement Sho-Saiko-To (Xiaochaihu-tang) is commonly prescribed to relieve symptoms of liver disease. Flavonoids from the herbal constituents of Sho-Saiko-To have demonstrated considerable anti-inflammatory, antioxidant, antimicrobial, antitumor, and immunomodulatory properties. In this work, we identify WGN, a major flavonoid in Sho-Saiko-To, as a novel inhibitor of hemolysis and eryptosis of human erythrocytes. Since inordinate erythrocyte death is a major obstacle in therapeutic drug development, our findings argue for the use of WGN as a natural alternative, either as a primary or an adjuvant drug, for a wide assortment of pathological conditions including cancer.

Inhibition of suicidal erythrocyte death by pyrogallol.

Pyrogallol, a polyphenolic component of Acacia nilotica has previously been reported to induce apoptosis of diverse cell types. Pyrogallol is in part effective by influencing gene expression and by interference with mitochondrial function. Despite lack of nuclei and mitochondria, erythrocytes may undergo eryptosis, a suicidal death apparent from phosphatidylserine translocation to the erythrocyte surface and cell shrinkage. Eryptosis is triggered by glucose depletion, by oxidation, by hyperosmotic cell shrinkage and by excessive Ca2+ entry. As enhanced eryptosis is a common cause of anemia, uncovering inhibitors and stimulators of eryptosis may, both, be of clinical interest. Here we tested, whether eryptosis of human erythrocytes is modified by pyrogallol. Utilizing flow cytometry, phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding and cell volume from forward scatter. Prior to determinations erythrocytes were incubated with or without glucose, without or with added oxidant tert-butyl-hydroperoxide (t-BOOH, 0.5mM), without or with added hyperosmotic sucrose (550mM) or without or with added Ca2+ ionophore ionomycin (1µM). Treatment of erythrocytes with pyrogallol (2-8µM) was without significant effect on annexin-V-binding and forward scatter. Glucose deprivation, t-BOOH, sucrose and ionomycin, each, triggered annexin-V-binding and decreased forward scatter. Pyrogallol significantly blunted the effects on annexin-V-binding but not on forward scatter. Pyrogallol thus blunts phosphatidylserine translocation in erythrocytes exposed to glucose depletion, oxidative stress, hyperosmotic shock and excessive Ca2+ entry.

Proeryptotic Activity of 4-Hydroxynonenal: A New Potential Physiopathological Role for Lipid Peroxidation Products.

Background: Eryptosis is a physiological, apoptosis-like death of injured erythrocytes crucial to prevent premature haemolysis and the pathological sequalae generated by cell-free haemoglobin. When dysregulated, the process is associated to several inflammatory-based pathologies. 4-Hydroxy-trans-2-nonenal (HNE) is an endogenous signalling molecule at physiological levels and, at higher concentrations, is involved in the pathogenesis of several inflammatory-based diseases. This work evaluated whether HNE could induce eryptosis in human erythrocytes. Methods: Measurements of phosphatidylserine, cell volume, intracellular oxidants, Ca++, glutathione, ICAM-1, and ceramide were assessed by flow cytometry. Scanning electron microscopy evaluated morphological alterations of erythrocytes. Western blotting assessed caspases. PGE2 was measured by ELISA. Adhesion of erythrocytes on endothelial cells was evaluated by gravity adherence assay. Results: HNE in the concentration range between 10–100 µM induces eryptosis, morphological alterations correlated to caspase-3 activation, and increased Ca++ levels. The process is not mediated by redox-dependent mechanisms; rather, it strongly depends on PGE2 and ceramide. Interestingly, HNE induces significant increase of erythrocytes adhesion to endothelial cells (ECs) that are in turn dysfunctionated as evident by overexpression of ICAM-1. Conclusions: Our results unveil a new physiopathological role for HNE, provide mechanistic details of the HNE-induced eryptosis, and suggest a novel mechanism through which HNE could exert pro-inflammatory effects.

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore.

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

The second generation tyrosine kinase inhibitor dasatinib induced eryptosis in human erythrocytes—An in vitro study

Dasatinib, a new tyrosine kinase inhibitor, is used clinically to kill chronic myelogenous leukemia and acute lymphoblastic leukemia through apoptosis. Obviously, anemia is developed in many patients receiving dasatinib for treatment. Until now, the mechanism for the cytotoxic effects of dasatinib in human erythrocytes is not fully understood. As many tyrosine kinases are found in human erythrocytes, it is therefore logical to hypothesize that dasatinib is able to induce apoptosis (or eryptosis) in human erythrocytes. True to our expectation, dasatinib inhibited tyrosine kinase and induced eryptosis in human erythrocytes with early denature of esterase, cell shrinkage, loss of membrane integrity with inside-out phosphatidylserine, increase in the cytosolic Ca2+ ion concentration ([Ca2+]i), caspase-3 activation and change in cellular redox state. Mechanistically, the rise of [Ca2+]i seems to be a key mediator in the dasatinib-mediated eryptosis because depletion of external Ca2+ could suppress the eryptotic effects. Also, dasatinib was able to reduce membrane fluidity in human RBCs. For the direct action on membrane, dasatinib permeabilized RBC ghosts in a way similar to digitonin. Taken together, we report here for the first time that dasatinib inhibited tyrosine kinase and induced eryptosis in human erythrocytes through Ca2+ loading and membrane permeabilization.

Di-n-butyl phthalate, butylbenzyl phthalate and their metabolites induce haemolysis and eryptosis in human erythrocytes

Phthalates have been extensively used as plasticizers in various fields, including food, cosmetic, and pharmaceutical industry. Those compounds do not form covalent bonds to substances they are being added to, and thus they may migrate easily and penetrate various products used every day. They may reach organisms with air, food, or by a direct skin contact. Significant levels of phthalates and their metabolites are found in urine, breast milk, blood serum, venous blood, and cord blood.The purpose of this study was to assess the simple toxicity (haemolysis) and programmed death (eryptosis) caused by following phthalates: di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP) and their metabolites: mono-n-butyl phthalate (MBP) and mono-benzyl phthalate (MBzP) in vitro in human RBCs. RBCs were incubated with the above mentioned compounds at concentrations ranging between 0.5 and 500 μg/mL for 24 h.Obtained results demonstrated that DBP and BBP possess higher haemolytic properties compared to their metabolites. The lethal concentration (LC50) was determined. The value was 126.37 ± 5.94 μg/mL for DBP, and 103.65 ± 4.03 μg/mL for BBP, and for metabolites the LC50 value was over 500 μg/mL.All compounds induced eryptosis causing translocation of phosphatidylserine, increased cytosolic calcium ions level, increased caspase-3 and calpain activation in human erythrocytes. BBP caused translocation of phosphatidylserine at a lower concentration compared to DBP. In case of other parameters, more pronounced changes were evoked by DBP at lower concentrations. Metabolites showed a significantly lower toxicity compared to parent compounds.

Quantitative detection of eryptosis in human erythrocytes using tunable resistive pulse sensing and annexin-V-beads.

Toxicological assessments of human red blood cells (RBCs) are important in human health because RBCs are the most abundant cell type in our body. Erythrotoxicology testing guidelines using hemolysis have been established as a standard (e.g. by the ASTM International). However, many xenobiotics promote eryptosis (apoptosis in human RBCs) without causing hemolysis. Based on the major features of eryptosis, i.e. cell shrinkage and translocation of phosphatidylserine (PS) to the outer lipid bilayer of the plasma membrane, we report here a novel approach utilizing the quantitative tunable resistive pulse sensing (TRPS) technology, a widely adopted technique for characterizing nanoparticles in the field of nanotechnology, to measure the degree of eryptosis in a non-optical manner. With the TRPS system, we were able to determine PS externalization with microbeads functionalized with annexin-V for PS binding, cell swelling and shrinkage in physiological buffers (cell volume: 86 ± 12 fL) and solutions of different osmolarities with or without apoptotic trigger. After setting these standards, we then evaluated the toxicity of Polyphyllin D (PD), a potential anti-cancer drug that kills more liver cancer cells with multi-drug resistance, in erythrocytes to prove our concept. Data revealed that PD induced PS externalization and shrinkage in RBCs in a dose-dependent manner. Moreover, another feature of eryptosis, as small as 5 fL, was detected thus showing the PD-induced erythrotoxicity in human cells. Taken together, our results indicate that our approach using annexin-V-beads and TRPS is simple, safe and convenient, using only a small volume (35 μL) to evaluate the erythrotoxicity of xenobiotics.

Inhibitory Effect of Furosemide on Non-Selective Voltage-Independent Cation Channels in Human Erythrocytes

Background: Furosemide, a loop diuretic inhibiting the renal tubular Na⁺,K⁺,2Cl^- cotransporter, has been shown to decrease cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in platelets and erythrocytes. [Ca²⁺]_i in erythrocytes is a function of Ca²⁺ permeable cation channels. Activation of those channels e.g. by energy depletion or oxidative stress leads to increase of [Ca²⁺]_i, which in turn triggers eryptosis, a suicidal erythrocyte death characterized by cell membrane scrambling. The present study was performed to explore whether furosemide influences the cation channels and thus influences eryptosis. Methods: Cation channel activity was determined by whole-cell patch clamp, [Ca²⁺]_i utilizing Fluo3 fluorescence and annexin V binding to estimate cell membrane scrambling with phosphatidylserine exposure. Results: A 45 min exposure to furosemide (10 and 100 µM) slightly, but significantly decreased cation channel activity and [Ca²⁺]_i in human erythrocytes drawn from healthy individuals. ATP-depletion (> 3 hours, +37°C, 6 mM ionosine and 6 mM iodoacetic acid) enhanced the non-selective cation channel activity, increased [Ca²⁺]_i and triggered cell membrane scrambling, effects significantly blunted by furosemide (10 – 100 µM). Oxidative stress by exposure to tert-butylhydroperoxide (0.1 –1 mM) similarly enhanced the non-selective cation channels activity, increased [Ca²⁺]_i and triggered cell membrane scrambling, effects again significantly blunted by furosemide (10 – 100 µM). Conclusions: The present study shows for the first time that the loop diuretic furosemide applied at micromolar concentrations (10 – 100 µM) inhibits non-selective cation channel activity in and eryptosis of human erythrocytes.