Red blood cells in hemorrhagic shock: a critical role for glutaminolysis in fueling alanine transamination in rats

Abstract Red blood cells (RBCs) are the most abundant host cell in the human body and play a critical role in oxygen transport and systemic metabolic homeostasis. Hypoxic metabolic reprogramming of RBCs in response to high-altitude hypoxia or anaerobic storage in the blood bank has been extensively described. However, little is known about the RBC metabolism following hemorrhagic shock (HS), the most common preventable cause of death in trauma, the global leading cause of total life-years lost. Metabolomics analyses were performed through ultra-high pressure liquid chromatography–mass spectrometry on RBCs from Sprague-Dawley rats undergoing HS (mean arterial pressure [MAP], <30 mm Hg) in comparison with sham rats (MAP, >80 mm Hg). Steady-state measurements were accompanied by metabolic flux analysis upon tracing of in vivo–injected 13C15N-glutamine or inhibition of glutaminolysis using the anticancer drug CB-839. RBC metabolic phenotypes recapitulated the systemic metabolic reprogramming observed in plasma from the same rodent model. Results indicate that shock RBCs rely on glutamine to fuel glutathione (GSH) synthesis and pyruvate transamination, whereas abrogation of glutaminolysis conferred early mortality and exacerbated lactic acidosis and systemic accumulation of succinate, a predictor of mortality in the military and civilian critically ill populations. Glutamine is here identified as an essential amine group donor in HS RBCs, plasma, liver, and lungs, providing additional rationale for the central role glutaminolysis plays in metabolic reprogramming and survival following severe hemorrhage.

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Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells

Haematologica ◽

10.3324/haematol.2020.266676 ◽

2020 ◽

pp. 0-0

Author(s):

Angelo D’Alessandro ◽

Ariel Hay ◽

Monika Dzieciatkowska ◽

Benjamin C. Brown ◽

Evan J Morrison ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Oxidative Damage ◽

Red Blood Cells ◽

Blood Cells ◽

Reactive Oxygen ◽

Metabolic Reprogramming ◽

Oxygen Species ◽

Common Amino Acid

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

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Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic

Nature Communications ◽

10.1038/s41467-021-22898-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xuqing Zhang ◽

Mengyao Luo ◽

Shamael R. Dastagir ◽

Mellissa Nixon ◽

Annie Khamhoung ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Red Blood Cells ◽

Blood Cells ◽

Checkpoint Inhibitors ◽

Critical Role ◽

Cellular Immunotherapy ◽

Tumor Control

AbstractCheckpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential ‘off-the-shelf’ in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

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Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00542.2014 ◽

2014 ◽

Vol 307 (12) ◽

pp. H1737-H1744 ◽

Cited By ~ 9

Author(s):

Brett S. Kirby ◽

Gabi Hanna ◽

Hansford C. Hendargo ◽

Timothy J. McMahon

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Metabolic Flux ◽

Vascular Wall ◽

Extracellular Atp ◽

Glycolytic Flux ◽

Glycolytic Intermediate ◽

Atp Production ◽

Intracellular Atp

Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ∼42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., “PIPA”) restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ∼120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo.

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Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162132 ◽

1990 ◽

Vol 48 (3) ◽

pp. 914-915

Author(s):

D.J.P. Ferguson ◽

A.R. Berendt ◽

J. Tansey ◽

K. Marsh ◽

C.I. Newbold

Keyword(s):

Plasmodium Falciparum ◽

Red Blood Cells ◽

Blood Cells ◽

Umbilical Vein ◽

Cell Types ◽

Amelanotic Melanoma ◽

Cytoplasmic Process ◽

Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

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The Ullrastructure of Platelet Participation in Hemostasis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656288 ◽

1965 ◽

Vol 13 (01) ◽

pp. 065-083 ◽

Cited By ~ 7

Author(s):

Shirley A. Johnson ◽

Ronaldo S. Balboa ◽

Harlan J. Pederson ◽

Monica Buckley

Keyword(s):

Platelet Aggregation ◽

Red Blood Cells ◽

Blood Cells ◽

Guinea Pigs ◽

Platelet Factor ◽

Mesenteric Vessels ◽

Platelet Aggregates ◽

Electron Lucent

SummaryThe ultrastructure of platelet aggregation in vivo in response to bleeding brought about by transection of small mesenteric vessels in rats and guinea pigs has been studied. Platelets aggregate, degranulate and separating membranes disappear in parallel with fibrin appearance which is first seen at several loci after 30 seconds of bleeding. About 40 per cent of the electron opaque granules, some of which contain platelet factor 3 have disappeared after one minute of bleeding while the electron lucent granules increase by 70 per cent suggesting that some of them may be empty vesicles. Most of the platelet aggregates of the random type disappear leaving clumped red blood cells entrapped by a network of fibrin fibers which emanate from the remains of platelet aggregates of the rosette type to maintain hemostasis.

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Cytotoxicity of chitosan ultrafine nanoshuttles on MCF-7 cell line as surrogate model for breast cancer

Current Drug Delivery ◽

10.2174/1567201817666200719005440 ◽

2020 ◽

Vol 17 ◽

Author(s):

Tarek Faris ◽

Gamaleldin I. Harisa ◽

Fars K. Alanazi ◽

Mohamed M. Badran ◽

Afraa Mohammad Alotaibi ◽

...

Keyword(s):

Red Blood Cells ◽

Factorial Design ◽

Cell Line ◽

Cell Lines ◽

Blood Cells ◽

Sodium Tripolyphosphate ◽

Physicochemical Characterization ◽

Spherical Shape ◽

Mcf 7

Aim: This study aimed to explore an affordable technique for the fabrication of Chitosan Nanoshuttles (CSNS) at the ultrafine nanoscale less than 100 nm with improved physicochemical properties, and cytotoxicity on the MCF-7 cell line. Background: Despite several studies reported that the antitumor effect of CS and CSNS could achieve intracellular compartment target ability, no enough available about this issue and further studies are required to address this assumption. Objectives: The objective of the current study was to investigate the potential processing variables for the production of ultrafine CSNS (> 100 nm) using Box-Benhken Design factorial design (BBD). This was achieved through a study of the effects of processing factors, such as CS concentration, CS/TPP ratio, and pH of the CS solution, on PS, PDI, and ZP. Moreover, the obtained CSNS was evaluated for physicochemical characteristics, morphology Also, hemocompatibility, and cytotoxicity using Red Blood Cells (RBCs) and MCF-7 cell lines were investigated. Methods: Box-Benhken Design factorial design (BBD) was used in the analysis of different selected variables. The effects of CS concentration, sodium tripolyphosphate (TPP) ratio, and pH on particle size, Polydispersity Index (PDI), and Zeta Potential (ZP) were measured. Subsequently, the prepared CS nanoshuttles were exposed to stability studies, physicochemical characterization, hemocompatibility, and cytotoxicity using red blood cells and MCF-7 cell lines as surrogate models for in vivo study. Result: The present results revealed that the optimized CSNS have ultrafine nanosize, (78.3±0.22 nm), homogenous with PDI (0.131±0.11), and ZP (31.9±0.25 mV). Moreover, CSNS have a spherical shape, amorphous in structure, and physically stable. Also, CSNS has biological safety as indicated by a gentle effect on red blood cell hemolysis, besides, the obtained nanoshuttles decrease MCF-7 viability. Conclusion: The present findings concluded that the developed ultrafine CSNS has unique properties with enhanced cytotoxicity. thus promising for use in intracellular organelles drug delivery.

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Multimodal Diagnostics of Microrheologic Alterations in Blood of Coronary Heart Disease and Diabetic Patients

Diagnostics ◽

10.3390/diagnostics11010076 ◽

2021 ◽

Vol 11 (1) ◽

pp. 76

Author(s):

Anastasia Maslianitsyna ◽

Petr Ermolinskiy ◽

Andrei Lugovtsov ◽

Alexandra Pigurenko ◽

Maria Sasonko ◽

...

Keyword(s):

Coronary Heart Disease ◽

Heart Disease ◽

Red Blood Cells ◽

Blood Cells ◽

Optical Methods ◽

Diabetic Patients ◽

Aggregation Index ◽

Significant Difference

Coronary heart disease (CHD) has serious implications for human health and needs to be diagnosed as early as possible. In this article in vivo and in vitro optical methods are used to study blood properties related to the aggregation of red blood cells in patients with CHD and comorbidities such as type 2 diabetes mellitus (T2DM). The results show not only a significant difference of the aggregation in patients compared to healthy people, but also a correspondence between in vivo and in vitro parameters. Red blood cells aggregate in CHD patients faster and more numerously; in particular the aggregation index increases by 20 ± 7%. The presence of T2DM also significantly elevates aggregation in CHD patients. This work demonstrates multimodal diagnostics and monitoring of patients with socially significant pathologies.

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Rhythmic glucose metabolism regulates the redox circadian clockwork in human red blood cells

Nature Communications ◽

10.1038/s41467-020-20479-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ratnasekhar Ch ◽

Guillaume Rey ◽

Sandipan Ray ◽

Pawan K. Jha ◽

Paul C. Driscoll ◽

...

Keyword(s):

Glucose Metabolism ◽

Red Blood Cells ◽

Blood Cells ◽

Mammalian Cells ◽

Metabolic Flux ◽

Pentose Phosphate ◽

Human Red Blood Cells ◽

Integral Process ◽

Metabolic Oscillations ◽

Human Rbcs

AbstractCircadian clocks coordinate mammalian behavior and physiology enabling organisms to anticipate 24-hour cycles. Transcription-translation feedback loops are thought to drive these clocks in most of mammalian cells. However, red blood cells (RBCs), which do not contain a nucleus, and cannot perform transcription or translation, nonetheless exhibit circadian redox rhythms. Here we show human RBCs display circadian regulation of glucose metabolism, which is required to sustain daily redox oscillations. We found daily rhythms of metabolite levels and flux through glycolysis and the pentose phosphate pathway (PPP). We show that inhibition of critical enzymes in either pathway abolished 24-hour rhythms in metabolic flux and redox oscillations, and determined that metabolic oscillations are necessary for redox rhythmicity. Furthermore, metabolic flux rhythms also occur in nucleated cells, and persist when the core transcriptional circadian clockwork is absent in Bmal1 knockouts. Thus, we propose that rhythmic glucose metabolism is an integral process in circadian rhythms.

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Lactate secreted by PKM2 upregulation promotes Galectin-9-mediated immunosuppression via inhibiting NF-κB pathway in HNSCC

Cell Death and Disease ◽

10.1038/s41419-021-03990-4 ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Hanyue Chang ◽

Qiaoshi Xu ◽

Jiayi Li ◽

Mingyu Li ◽

Zhiyuan Zhang ◽

...

Keyword(s):

Critical Role ◽

Lactate Production ◽

Metabolic Reprogramming ◽

Migration And Invasion ◽

Histone Deacetylase 3 ◽

Proliferation And Metastasis ◽

Immunosuppressive Microenvironment ◽

Transcriptional Complex

AbstractPyruvate kinase M2 as a key rate-limiting enzyme in glycolysis, it plays a critical role in metabolic reprogramming and carcinogenesis. However, whether PKM2 can promote immunosuppressive microenvironment formation remains unknown in head and neck squamous cell carcinoma (HNSCC). PKM2 expression was detected using immunohistochemical staining. The biological functions of PKM2 were investigated in vitro and in vivo. Lactate production and the expression of Galectin-9, a critical immunosuppression molecule, were detected after PKM2 knockdown and overexpression in HNSCC cells. The mechanism of lactate regulating Galectin-9 expression through NF-κB signaling was explored in vitro. Overexpression of PKM2 correlates with poor prognosis in HNSCC patients. Silencing PKM2 markedly inhibits proliferation and metastasis capacity in vivo and in vitro, and vice versa. The glycolysis and glycolytic capacity are significantly decreased after PKM2 silencing. Lactate secretion induced by PKM2 significantly promotes migration and invasion capacity. Furthermore, a positive correlation between PKM2 and Galectin-9 expression is observed in HNSCC tissues. The induction of Galectin-9 expression by PKM2 can be affected by a lactate transporter inhibitor. Mechanically, lactate impeded the suppressive transcriptional complex formation of NF-κB and histone deacetylase 3 (HDAC3), which released the transcription of Galectin-9 mediated by NF-κB signaling. Our findings demonstrate that lactate produced by PKM2 upregulation promotes tumor progression and Galectin-9-mediated immunosuppression via NF-κB signaling inhibition in HNSCC, which bridges metabolism and immunosuppression. The novel PKM2-lactate-Galectin-9 axis might be a potential therapeutic target in HNSCC.

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Role of Calcium in Phosphatidylserine Externalisation in Red Blood Cells from Sickle Cell Patients

Anemia ◽

10.1155/2011/379894 ◽

2011 ◽

Vol 2011 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

Erwin Weiss ◽

David Charles Rees ◽

John Stanley Gibson

Keyword(s):

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Phosphatidylserine Exposure ◽

Channel Inhibition ◽

Cation Conductance ◽

Gardos Channel ◽

Cation Permeability

Phosphatidylserine exposure occurs in red blood cells (RBCs) from sickle cell disease (SCD) patients and is increased by deoxygenation. The mechanisms responsible remain unclear. RBCs from SCD patients also have elevated cation permeability, and, in particular, a deoxygenation-induced cation conductance which mediates entry, providing an obvious link with phosphatidylserine exposure. The role of was investigated using FITC-labelled annexin. Results confirmed high phosphatidylserine exposure in RBCs from SCD patients increasing upon deoxygenation. When deoxygenated, phosphatidylserine exposure was further elevated as extracellular [] was increased. This effect was inhibited by dipyridamole, intracellular chelation, and Gardos channel inhibition. Phosphatidylserine exposure was reduced in high saline. levels required to elicit phosphatidylserine exposure were in the low micromolar range. Findings are consistent with entry through the deoxygenation-induced pathway (), activating the Gardos channel. [] required for phosphatidylserine scrambling are in the range achievablein vivo.

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