Peg-Cohb Mediated Carbon Monoxide and Oxygen Transfer to Hypoxic Red Blood Cells Prevent, Slow, and/or Reverse Sickling in Vitro.

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 967-967
Author(s):  
Peter Buontempo ◽  
Rick Yglesias ◽  
Yuli Chen ◽  
Catherine Buontempo ◽  
Ronald G. Jubin ◽  
...  
Keyword(s):  
Blood Flow ◽  
Sickle Cell ◽  
Cell Morphology ◽  
Oxygen Transfer ◽  
Blood Cells ◽  
Normal Cell ◽  
Cell Disease ◽  
Normal Blood Flow ◽  
O2 Transfer

Abstract Background: In Sickle Cell disease (SCD), a single amino acid substitution in the beta globin chain converts HbA to sickle genotype HbS. This genetic change promotes HbS polymerization upon deoxygenation that can promote occlusion of small blood vessels that is often associated with increased blood viscosity, and circulatory inflammation. PEGylated-carboxyhemoglobin (PEG-COHb; SANGUINATE) was designed as a novel therapeutic agent to initially release carbon monoxide (CO) and then transfer oxygen (O2) to hypoxic tissue and cells. Delivery of either CO and/or O2 to hypoxic, sickled red blood cells (RBCs) should return cells to a more normal cell morphology and help re-establish normal blood flow and rheology. PEG-COHb was shown to mediate transfer of either a CO or O2/CO mixture and restore normal morphology to hypoxic, sickled RBCs in vitro. Studies are now focused on the potential therapeutic implications of delaying or slowing sickling, which should maintain normal blood flow through hypoxic microvasculature. Unsickling is expected to be expedited by O2 transfer by PEG-COHb. To examine these potential therapeutic effects, current in vitro studies examined the effects of time and dose of PEG-COHb to not only reverse, but also prevent or delay sickling by transferring CO as well as expedite atmospheric O2 transfer to the sickled RBCs. Methods: Reversal of sickling studies were conducted by deoxygenating RBCs from healthy (control) and SCD volunteers in followed by treatment with either PEG-COHb, fully oxygenated PEG-Hb (PEG-O2Hb) or PEG-BSA for 2 hours. For prevention of sickling studies, fully oxygenated RBC suspensions were treated with increasing amounts of PEG-COHb and then subjected to hypoxia for 3 hours. Time-dose effects were quantified by area under the curve (AUC) analysis. O2 transfer studies were conducted by treating hypoxic, sickled RBCs to increasing concentrations of PEG-COHb and raising the pO2 from 3.8mm to 40mm. In all studies, the fractions of CO-Hb, O2-Hb and reduced Hb were determined by co-oximetry and sickled RBCs were quantified by imaging flow cytometry of fixed RBC specimens. Results: PEG-COHb-mediated delivery of either CO or O2 can unsickle hypoxic SCD RBCs. Controls exhibited gas exchange similar to SCD RBCs. Interestingly, sickle reversion time-course studies showed differential kinetics between the CO and O2 capacity to cause unsickling. AUC analysis at 20 minutes demonstrated that both CO and O2 reversed sickling by 41% and 42%, respectively. PEG-O2Hb was able to exert substantial unsickling by 5 minutes, where PEG-COHb showed a delayed, more pronounced effect, peaking approximately 20 to 40 minutes post-treatment. When fully oxygenated SCD RBCs were pretreated with PEG-COHb prior to oxygenation, sickling was inhibited with an IC50 of 2.5±0.6 mg per mL in deoxygenated saline (PBS). In addition, treatment concentrations below IC50 values had increased time-dose AUC values indicating that although, not completly inhibited, sickling was delayed. Oxygen transfer facilitation studies indicated that PEG-COHb increased the rate of unsickling as measured by AUC by 50% and 15% at 4 and 2 mg per mL, respectively. These levels are within the expected therapeutic dosage of SANGUINATE. Summary: RBCs from patients with SCD undergo a conformational shift upon deoxygenation resulting in HbS polymerization and morphological changes of the RBCs. The occlusive and fragile properties of sickled RBCs are responsible for the development of the numerous comorbidities associated with SCD. It is only when the fraction of oxygenated or carboxylated HbS reaches a sufficient level that reversion to normal cell morphology occurs which promotes vascular perfusion. These experiments showed a concentration and time-dependent effect of PEG-COHb ability to deliver both O2 and CO to sickled RBC. These data suggested that PEG-COHb is a promising gas transfer agent that has the potential to improve sickle cell morphology by reversing sickling; the underlying pathology of sickle cell disease co-morbidities. Disclosures Buontempo: Prolong Pharmaceuticals: Employment. Yglesias:Prolong Pharmaceuticals: Employment. Chen:Prolong Pharmaceuticals: Employment. Buontempo:Prolong Pharmaceuticals: Employment. Jubin:Prolong Pharmaceuticals: Employment. Abuchowski:Prolong Pharmaceuticals: Employment.

scholarly journals The Effect of Rigid Red Blood Cells on Platelet Adhesion in Blood Flow and Implications in Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 986-986
Author(s):  
Alison Leigh Banka ◽  
Mark Shamoun ◽  
Mario Gutierrez ◽  
Tyler Tanski ◽  
Lola Eniola-Adefeso
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Platelet Adhesion ◽  
Model System ◽  
Cell Disease ◽  
The Impact

Introduction: Sickle cell disease (SCD) occurs due to a mutation in the β-subunit of hemoglobin, causing stiffening of red blood cells (RBCs) and leading to RBC sickling and vaso-occlusive crises (VOC) in SCD patients. While sickled RBCs remain a hallmark of SCD, they are prone to lysis and represent a small fraction of the total RBCs present in patients at a given time. The remaining RBCs maintain a normal, discoid shape and are either healthy or stiff due to polymerization of the hemoglobin β-globin subunit. In healthy blood flow, RBCs form a core in the center of the vessel and the remaining cells, platelets and white blood cells (WBCs), marginate towards the endothelium. However, the increased stiffness of RBCs in SCD disrupts this neat segregation of blood cells to different areas of the blood vessel and can contribute to VOC, the root cause of many acute and chronic complications for SCD patients. Despite the presence of normally shaped, stiffened RBCs in SCD patients, the impact of these RBCs on other cell types in blood flow is currently not well understood. Our laboratory previously demonstrated that the presence of artificially rigidified RBCs leads to an expansion of the RBC core and significantly decreases WBC adhesion to an inflamed endothelium in vitro. Here, we examine the impact of stiffened RBCs on platelet adhesion to a damaged endothelium in vitro by first using a model system with artificially rigidified RBCs and second, utilizing SCD patient blood to further support our model and understand platelet-RBC interactions in SCD patients. Methods: In our model system, we artificially rigidified RBCs taken from healthy donors and reconstituted them into whole blood before perfusing the mixture over an activated, damaged endothelium using a parallel plate flow chamber. We quantified platelet adhesion to the endothelium in comparison to healthy, non-rigidified controls using fluorescent microscopy. To determine if our model findings translated to SCD, we recruited a cohort of hemoglobin SS and SC patients during routine visits and similarly perfused their whole blood over the same damaged endothelium and quantified platelet adhesion. Results and conclusions: The inclusion of artificially rigidified RBCs in otherwise healthy subject blood flow significantly increased platelet adhesion to a damaged endothelium with a maximum increase in platelet adhesion of six-fold over a healthy, non-rigid control in our model system. Both RBC rigidity and the percentage of RBCs that were artificially rigidified had a large impact on the increase in platelet adhesion. SCD platelet adhesion to the damaged endothelium model varied from donor to donor based on variables such as treatment method and disease severity. Overall, this work experimentally elucidates the biophysical impact of stiffened RBCs on platelet adhesion using both an artificial model utilizing healthy blood as well as SCD blood, which can help determine the mechanism of action causing VOC in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rheological Effects of L-Glutamine in Patients with Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3567-3567
Author(s):  
Celeste K. Kanne ◽  
Varun Reddy ◽  
Vivien A. Sheehan
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Viscosity ◽  
Whole Blood ◽  
Blood Cells ◽  
Cell Disease ◽  
Phase 3 ◽  
Whole Blood Viscosity

Background: ENDARITM (oral pharmaceutical L-glutamine powder) received FDA approval in 2017 as a treatment for sickle cell disease (SCD). A pivotal phase 3 clinical study conducted by Emmaus Medical, Inc. showed that L-glutamine resulted in a lower incidence of vaso-occlusive crises (VOC) as well as a lower rate of hospitalizations and shorter hospital stays. No changes in standard clinical laboratory values were noted. The clinical improvements associated with sickle cell complications are believed to be due to an increase in the proportion of the reduced form of nicotinamide adenine dinucleotides in the red blood cells (RBC) of patients with SCD, reducing the oxidative stress. While the endpoints in the phase 3 study are clinically important, it is essential that we identify biomarkers or measurable laboratory changes that can serve as endpoints for future clinical trials assessing dose optimization and the efficacy and safety of L-glutamine in SCD individuals, including those with hepatic and renal dysfunction. RBC rheology is markedly abnormal in SCD; blood is more viscous for a given hematocrit than normal individuals, dense red blood cells (DRBC) are packed with HbS, potentiating sickling, and RBCs are less deformable than those of HbAA or HbAS individuals. High whole blood viscosity, high DRBCs, and poor RBC deformability are associated with higher rates of VOC. Given the demonstrated reduction in pain events, we hypothesized that L-glutamine might improve RBC rheology and sought to test this in vitro and in vivo using a battery of rheological tests. Methods: For the in vitro study, 6 mL of whole blood was drawn into an EDTA vacutainer from ten pediatric patients with sickle cell anemia (HbSS or HbSβ0) during routine clinical checkups under an IRB approved protocol. The cohort included 3 female and 7 male patients, ages 2-19 years old. All patients were on a steady dose of hydroxyurea and did not receive a transfusion within the 3 months prior to sample collection. A 200 mM stock solution of L-glutamine and water was mixed and filtered under light-protected conditions. Aliquots were stored at -20°C to avoid multiple freeze/thaw cycles. L-glutamine was added to 3 mL of whole blood for a final concentration of 1 mM (average in vivo L-glutamine plasma concentration in patients with SCD treated with L-glutamine); 3 mL of the same patient sample with water added served as a control. After a 24-hour incubation period at 4°C, whole blood viscosity was measured using a cone and plate viscometer at 37°C (DV3T Rheometer, AMETEK Brookfield, USA), %DRBCs were measured on an ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Inc., USA), and deformability measured using a Laser Optical Rotational Red Cell Analyzer (Lorrca®) (RR Mechatronics, the Netherlands) with the Oxygenscan module. The Oxygenscan measures RBC deformability at normoxia (Elmax), deformability upon deoxygenation (EImin), and point of sickling (PoS), the oxygen tension at which deformability begins to decline, reflecting the patient-specific pO2 at which sickling begins. Paired samples (with and without added L-glutamine) were analyzed using Student's t-test. For the in vivo study, rheological tests were performed on peripheral blood from one patient (18-year-old male on hydroxyurea) at baseline and treated with L-glutamine as part of his routine clinical care. Results and conclusions: Addition of L-glutamine in vitro significantly reduced the PoS, meaning RBCs incubated with L-glutamine could tolerate a lower pO2 before sickling compared to the control. RBCs incubated with L-glutamine also had significantly higher EImin, meaning deoxygenated RBCs were more flexible and deformable. Whole blood viscosity at 45s-1 and 225s-1 did not change significantly following incubation with L-glutamine; %DRBCs also did not change significantly (Table 1). The in vivo patient sample tested exhibited a similar improvement in PoS and EImin (Figure 1). We therefore propose to further test the performance of the PoS and EImin as possible biomarkers of response to L-glutamine in vivo. If validated, these biomarkers may also help further elucidate the mechanisms of action of L-glutamine in SCD. Disclosures No relevant conflicts of interest to declare.

Preclinical Studies for Sickle Cell Disease Gene Therapy Using Bone Marrow CD34+ Cells Modified with a βAS3-Globin Lentiviral Vector

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3119-3119
Author(s):  
Fabrizia Urbinati ◽  
Zulema Romero Garcia ◽  
Sabine Geiger ◽  
Rafael Ruiz de Assin ◽  
Gabriela Kuftinec ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Blood Cells ◽  
Globin Gene ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

scholarly journals In vitro investigation of the antisickling properties of aqueous fruits extracts of Citrus paradisi, Musa acuminata and Malus domestica

2020 ◽  
Vol 13 (3) ◽  
pp. 203-209
Author(s):  
Augustine O. Odibo ◽  
Ifunanya R. Akaniro ◽  
Emmanuel M. Ubah
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Malus Domestica ◽  
Blood Cells ◽  
Musa Acuminata ◽  
Citrus Paradisi ◽  
Cell Disease ◽  
In Vitro Investigation

Sickle cell disease (SCD) is a genetic blood disorder that affects the shape and transportation of red blood cells (RBCs) in blood vessels, leading to various clinical complications. Many drugs that are available for treating the disease are insufficiently effective, toxic, or too expensive. Therefore, there is a pressing need for safe, effective, and inexpensive therapeutic agents from indigenous plants used in ethnomedicine. In the current study, the potentials of aqueous extracts of Citrus paradisi, Musa acuminata, Malusdomestica fruit in sickle cell disease management were investigated in vitro using P-hydroxybenzoic acid and normal saline as positive and negative control respectively. The method employed the inhibition of sodium metabisulphite induced sickling of HbSS red blood cells, collected from confirmed sickle cell patients. Results obtained showed that; 50 mg/ml aqueous extract of Musa acuminata showed the lowest sickling inhibition (80.3%) at 60 minutes while 1 mg/ml gave the highest inhibition of 97.3% at 90 minutes. For Citrus paradisi, lowest (83.3%) and highest (98%) sickling inhibitions were obtained with 25 mg/ml and 50 mg/ml fruit extracts at 30 minutes and 60 minutes respectively. Malus domestica had the highest antisickling activities of 99% and 99.33% respectively at 30 and 60 minutes. In all, the sickling inhibition was least with Musa acuminata but highest with Malus domestica. Also, highest inhibitions were observed at 60 minutes (optimum time) and 10 mg/ml (optimum concentration). This study has demonstrated that; Malus domestica, Citrus paradisi and Musa acuminata possess antisickling potentials useful in the management or therapy of sickle cell diseases.

scholarly journals Vaso-Occlusion in Sickle Cell Disease: Is Autonomic Dysregulation of the Microvasculature the Trigger?

2019 ◽  
Vol 8 (10) ◽  
pp. 1690 ◽  
Author(s):  
Saranya Veluswamy ◽  
Payal Shah ◽  
Christopher Denton ◽  
Patjanaporn Chalacheva ◽  
Michael Khoo ◽  
...  
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Critical Role ◽  
Microvascular Blood Flow ◽  
Cell Disease ◽  
Hemoglobin S

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by polymerization of hemoglobin S upon deoxygenation that results in the formation of rigid sickled-shaped red blood cells that can occlude the microvasculature, which leads to sudden onsets of pain. The severity of vaso-occlusive crises (VOC) is quite variable among patients, which is not fully explained by their genetic and biological profiles. The mechanism that initiates the transition from steady state to VOC remains unknown, as is the role of clinically reported triggers such as stress, cold and pain. The rate of hemoglobin S polymerization after deoxygenation is an important determinant of vaso-occlusion. Similarly, the microvascular blood flow rate plays a critical role as fast-moving red blood cells are better able to escape the microvasculature before polymerization of deoxy-hemoglobin S causes the red cells to become rigid and lodge in small vessels. The role of the autonomic nervous system (ANS) activity in VOC initiation and propagation has been underestimated considering that the ANS is the major regulator of microvascular blood flow and that most triggers of VOC can alter the autonomic balance. Here, we will briefly review the evidence supporting the presence of ANS dysfunction in SCD, its implications in the onset of VOC, and how differences in autonomic vasoreactivity might potentially contribute to variability in VOC severity.

scholarly journals Differential Efficacy of Anti-Sickling and Anti-Inflammatory Mechanisms in a Fluorescent Intravital Microscopy Dorsal Skinfold Vaso-Occlusion Model in Sickle Cell Disease Townes Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2264-2264
Author(s):  
Sarah Sturtevant ◽  
Alejandra Macias-Garcia ◽  
Sriram Krishnamoorthy ◽  
Arjan van der Flier ◽  
Alexandra Hicks ◽  
...  
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Blood Cells ◽  
Ex Vivo ◽  
Endothelial Activation ◽  
Cell Disease ◽  
Occlusion Model ◽  
Hypoxia Reoxygenation

Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

scholarly journals GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro

2018 ◽  
Vol 10 (2) ◽  
Author(s):  
Kobina Dufu ◽  
Donna Oksenberg
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Oxygen Affinity ◽  
Microvascular Blood Flow ◽  
Cell Disease ◽  
Hypoxic Conditions

Sickle cell disease is characterized by hemolytic anemia, vasoocclusion and early mortality. Polymerization of hemoglobin S followed by red blood cell sickling and subsequent vascular injury are key events in the pathogenesis of sickle cell disease. Sickled red blood cells are major contributors to the abnormal blood rheology, poor microvascular blood flow and endothelial injury in sickle cell disease. Therefore, an agent that can prevent and or reverse sickling of red blood cells, may provide therapeutic benefit for the treatment of sickle cell disease. We report here that GBT440, an anti-polymerization agent being developed for the chronic treatment of sickle cell disease, increases hemoglobin oxygen affinity and reverses in vitro sickling of previously sickled red blood cells under hypoxic conditions. Our results suggest that besides preventing sickling of red blood cells, GBT440 may mitigate vasoocclusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo.

scholarly journals Optimized Beta-Globin Expression and Enucleation from Induced Red Blood Cells for In Vitro Modeling of Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2359-2359
Author(s):  
Tolulope O Rosanwo ◽  
Melissa Kinney ◽  
Martha A Clark ◽  
Linda T Vo ◽  
R. Grant Rowe ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Research Funding ◽  
Disease Modeling ◽  
Sequencing Analysis ◽  
Beta Globin ◽  
Cell Disease

Abstract Human induced pluripotent stem cells (hiPSCs) hold remarkable capacity for disease modeling and the development of novel therapeutic treatments for sickle cell disease (SCD). hiPSCs can theoretically produce all cell types including induced red blood cells (iRBCs). Sickle cell patients, in particular, could benefit from autologous, engineered red blood cells (RBCs). Many patients possess rare Rh phenotypes, are allo-sensitized to blood products and are at risk of iron overload from recurrent transfusions. Therefore, the generation of personalized iRBCs is attractive. Yet, in vitro iRBC production has been hampered by an inability of these cells to differentiate into terminally-mature, enucleated, beta globin-expressing RBCs. Here, we describe updated strategies to improve in vitro production of iRBCs. hiPSCs from sickle cell patients as well as those with normal hemoglobin were differentiated into hematopoietic stem progenitor cells (HSPCs) and immortalized via the overexpression of a previously characterized set of transcription factors promoting self-renewal and multipotency under the control of a doxycycline-regulated promoter. Utilizing an in vitro protocol incorporating increasing concentrations of human plasma, HSPCs differentiated from these lines proceed through terminal erythroid differentiation, including the formation of CD71-/GlyA+/α4 integrin-/Band 3+ cells. Plasma-stimulated iRBCs achieved robust enucleation (11-60.7%) and underwent fetal to adult globin-switching. Further, nearly 21% of the enucleated iRBCs were RNA negative erythrocytes 5-8 microns in diameter. RNA sequencing analysis reveals similar transcriptional profiles between iRBCs and peripheral blood CD34+- derived cultured RBCs (cRBCs) yet distinct differences between SCD and WT iRBCs. SCA iRBCs have increased extracellular matrix organization, cell-cell adhesive properties and up-regulation of hypoxia-response genes. Heme metabolism, DNA repair, fatty acid metabolism and oxidative phosphorylation are all impaired in SCD iRBCs. Assessment of cell physiology exposes membrane damage in SCD iRBCs with increased phalloidin permeability in comparison to wild type controls. Intriguingly, SCD iRBCs co-expressing gamma and beta-globin also demonstrate sickling under hypoxic conditions. With the development of expandable source of erythroid progenitors capable of producing mature red cells, we now aim to utilize this platform for robust disease modeling and autologous cell therapy. Figure. Figure. Disclosures Heeney: Pfizer: Research Funding; Sancilio Pharmaceuticals: Consultancy, Research Funding; Astra Zeneca: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee.

Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

1996 ◽  
Vol 76 (03) ◽  
pp. 322-327 ◽  
Author(s):  
Dominique Helley ◽  
Amiram Eldor ◽  
Robert Girot ◽  
Rolande Ducrocq ◽  
Marie-Claude Guillin ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Annexin V ◽  
Mean Value ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Cell Disease ◽  
Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

Microfluidic electrical impedance assessment of red blood cell mediated microvascular occlusion

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Yuncheng Man ◽  
Debnath Maji ◽  
Ran An ◽  
Sanjay Ahuja ◽  
Jane A Little ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Electrical Impedance ◽  
Cell Disease ◽  
Blood Disorders

Alterations in the deformability of red blood cells (RBCs), occurring in hemolytic blood disorders such as sickle cell disease (SCD), contributes to vaso-occlusion and disease pathophysiology. However, there are few...

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