Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death

Alfhili, Mohammad A.; Lee, Myon‐Hee

doi:10.1007/978-1-0716-1514-0_11

Cited by 12 publications

(6 citation statements)

References 23 publications

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“…E-20-4544) and conducted according to the Declaration of Helsinki. Heparinized blood samples were collected from 12 healthy volunteers and RBCs were isolated by centrifugation at 3000 rpm for 20 min and repeated washing in phosphate-buffered saline (PBS) [ 37 ]. For complete blood count analysis, K 2 -EDTA-anticoagulated whole blood (WB) was used [ 38 ].…”

Section: Methodsmentioning

confidence: 99%

“…LA and PA were made as 100 mM stock solutions in dimethylsulfoxide (DMSO) and stored at −80 °C. Ringer solutions were prepared essentially as described elsewhere [ 37 ], containing 1 mM of CaCl 2 when present.…”

Section: Methodsmentioning

confidence: 99%

“…Forward scatter (FSC) and side scatter (SSC) of light were measured by flow cytometry as surrogates for cell size and surface granularity, respectively [ 37 ].…”

Section: Methodsmentioning

confidence: 99%

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Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis

Alfhili

Aljuraiban

2021

Cells

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Cardiovascular diseases (CVD) are a leading cause of mortality worldwide, and dietary habits represent a major risk factor for dyslipidemia; a hallmark of CVD. Saturated fatty acids contribute to CVD by aggravating dyslipidemia, and, in particular, lauric acid (LA) raises circulating cholesterol levels. The role of red blood cells (RBCs) in CVD is increasingly being appreciated, and eryptosis has recently been identified as a novel mechanism in CVD. However, the effect of LA on RBC physiology has not been thoroughly investigated. RBCs were isolated from heparin-anticoagulated whole blood (WB) and exposed to 50–250 μM of LA for 24 h at 37 °C. Hemoglobin was photometrically examined as an indicator of hemolysis, whereas eryptosis was assessed by Annexin V-FITC for phosphatidylserine (PS) exposure, Fluo4/AM for Ca2+, light scatter for cellular morphology, H2DCFDA for oxidative stress, and BODIPY 581/591 C11 for lipid peroxidation. WB was also examined for RBC, leukocyte, and platelet viability and indices. LA caused dose-responsive hemolysis, and Ca2+-dependent PS exposure, elevated erythrocyte sedimentation rate (ESR), cytosolic Ca2+ overload, cell shrinkage and granularity, oxidative stress, accumulation of lipid peroxides, and stimulation of casein kinase 1α (CK1α). In WB, LA disrupted leukocyte distribution with elevated neutrophil-lymphocyte ratio (NLR) due to selective toxicity to lymphocytes. In conclusion, this report provides the first evidence of the pro-eryptotic potential of LA and associated mechanisms, which informs dietary interventions aimed at CVD prevention and management.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis

Alfhili

Aljuraiban

2021

Cells

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“…The characteristics of eryptosis involves dysfunctional ion exchange, cell shrinkage, ceramide accumulation, cell membrane vesiculation due to cytosolic calcium overload, and membrane phospholipid scrambling with alteration of the cell membrane asymmetry and phosphatidylserine (PS) exposure at the cell surface [ 9 , 10 ]. Ca 2+ influx opens Ca 2+ -sensitive K + channels, or Gardos channels, leading to subsequent loss of KCl, hyperpolarization of the membrane, loss of water, and cell shrinkage [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanisms and Pathophysiological Significance of Eryptosis

Alghareeb

Alfhili

Fatima

2023

IJMS

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Despite lacking the central apoptotic machinery, senescent or damaged RBCs can undergo an unusual apoptosis-like cell death, termed eryptosis. This premature death can be caused by, or a symptom of, a wide range of diseases. However, various adverse conditions, xenobiotics, and endogenous mediators have also been recognized as triggers and inhibitors of eryptosis. Eukaryotic RBCs are unique among their cell membrane distribution of phospholipids. The change in the RBC membrane composition of the outer leaflet occurs in a variety of diseases, including sickle cell disease, renal diseases, leukemia, Parkinson’s disease, and diabetes. Eryptotic erythrocytes exhibit various morphological alterations such as shrinkage, swelling, and increased granulation. Biochemical changes include cytosolic Ca2+ increase, oxidative stress, stimulation of caspases, metabolic exhaustion, and ceramide accumulation. Eryptosis is an effective mechanism for the elimination of dysfunctional erythrocytes due to senescence, infection, or injury to prevent hemolysis. Nevertheless, excessive eryptosis is associated with multiple pathologies, most notably anemia, abnormal microcirculation, and prothrombotic risk; all of which contribute to the pathogenesis of several diseases. In this review, we provide an overview of the molecular mechanisms, physiological and pathophysiological relevance of eryptosis, as well as the potential role of natural and synthetic compounds in modulating RBC survival and death.

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“…Heparinized blood samples were collected to isolate RBCs (2500 RPM, 20 min, RT), and, following washing two times in PBS, cells were stored in PBS or Ca 2+ -free Ringer solution at 4 • C for a maximum of 48 h. Exposure to 10-800 µM of RA was carried out at 5% hematocrit (11.0 × 10 5 cells/µL) in PBS or Ringer solutions for 24 h at 37 • C. In some experiments, cells were cotreated with 400 µM of RA in the presence or absence of 1 µM of PKC inhibitor staurosporin (STSP), 100 µM of p38 inhibitor SB203580, 20 µM of CK1a inhibitor D4476, or 500 µM of ATP. Additionally, whole blood in EDTA was diluted 1:2 with PBS and exposed to 800 µM RA for 24 h at 37 • C. Control and experimental cells from the same subject were used in distinct experiments to account for potential individual variation [16].…”

Section: Blood Collection and Experimental Designmentioning

confidence: 99%

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC

Alghareeb,

Alfhili,

Alsughayyir

2023

Molecules

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Background: Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. Methods: RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10–800 μM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. Results: RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. Conclusions: RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.

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Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death

Cited by 12 publications

References 23 publications

Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis

Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis

Molecular Mechanisms and Pathophysiological Significance of Eryptosis

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC

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