scholarly journals Effects of Quercetin on Neutrophil Extracellular Trap Formation in Sickle Cell Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2024-2024
Author(s):  
Bindu Parachalil Gopalan ◽  
Brenda Merriweather ◽  
Anna Conrey ◽  
Ankit Saxena ◽  
Evi X. Stavrou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Neutrophil Activation ◽  
Neutrophil Extracellular Trap ◽  
Lower Percentage ◽  
Cell Disease ◽  
Sytox Green ◽  
Extracellular Trap ◽  
Anti Inflammatory

Abstract Introduction: Sickle Cell Disease (SCD) is characterized by chronic inflammation with innate immune cell activation, especially observed in neutrophils. Emerging evidence implicates the imbalance between neutrophil extracellular trap (NET) formation and degradation as having a central role in the pathophysiology of thromboinflammation and venous thrombosis. Although NETosis and NET formation influences venous thromboembolism (VTE) pathophysiology, little is known about baseline and agonist-induced NETosis in SCD. We hypothesized that systemic neutrophil activation would lead to higher baseline and agonist induced NETosis in SCD and would influence phenotypic variability. To test this hypothesis, we assessed baseline and agonist induced NETosis in patients with SCD and ethnic matched controls. We also investigated the anti-inflammatory effects of flavonoid Quercetin on neutrophil activation. Methods: Neutrophils negatively selected from citrate anticoagulated blood using an immunomagnetic bead based kit (MACSxpress® Miltenyi Biotec) were either fixed immediately to assess baseline NETosis or stimulated with fMLP (1 µM) for 1 hour to assess agonist-induced NETosis. To study flavonoid anti-inflammatory effects, neutrophils were pretreated with Quercetin (100 µM) for 30 min prior to fixation and fMLP stimulation. NETosis was assessed by flow cytometry. Extracellular DNA extrusion on neutrophils was detected by gating the neutrophil population staining with Sytox green. Sytox green positive neutrophils that were positive for both myeloperoxidase (MPO) and tri-Citrullinated Histones (H3Cit) were defined as undergoing NETosis. In some experiments, NET formation was independently confirmed by image flow cytometry (AMNIS). Results: Subjects included SCD patients (genotype SS n=11) and ethnic matched controls (genotype AA, n=11) with a median age of 49 years (p=0.58) and a predominance of males (70%). All SCD patients were at least 60 days remote from an acute painful vaso-occlusive crisis or blood transfusion and were receiving hydroxyurea. The white cell and absolute neutrophil counts were higher in SCD patients (mean ± SD 8.77 ± 1.52 and 5.07 ± 1.78 x 10 9/L) when compared with controls (mean ± SD 5.33 ± 1.05 and 2.8 ± 0.95 x 10 9/L). Subsequent data are presented as median percentages with interquartile ranges (IQR). A subgroup of the study population demonstrated spontaneous NETosis (27%; SS = 4; AA = 4) and were therefore excluded from our analysis. Contrary to expectations, SCD patients exhibited a lower percentage of NETosis at baseline compared to controls (20 % (11, 36) vs. 33 % (15, 58); p=0.22). Similarly, neutrophils from SCD patients exhibited lower agonist-induced NETosis compared to controls (42% (19, 47) vs. 51% (37, 70); p=0.15) (Fig 1 A and B) Pretreatment of neutrophils from SCD patients with Quercetin appeared to inhibit basal levels of NETosis (6%, (2, 26) vs. 20% (11, 36) p=0.08) although this effect was not appreciable in controls (33% (11, 58) vs. 33% (15, 58) p=0.41) (Fig 1 C and D). Neutrophils from SCD patients that were pretreated with Quercetin and then stimulated with fMLP demonstrated significantly reduced NETosis compared to untreated neutrophils (17.1% (10, 38) vs 41.7% (19, 47) p=0.007) although this effect was not significant in controls (35% (17, 72) vs 50.7% (37, 70) p=0.11) Fig 1 E and F. Our ongoing experiments will demonstrate the effects of more specific inhibitors of neutrophil activation (e.g. R406) in human and mouse models of SCD. Conclusion: These preliminary data suggest lowered NETosis in SCD patients despite neutrophil activation in the systemic inflammatory environment that are partially explained by hydroxyurea treatment. The results also support further evaluation of anti-inflammatory therapies to reduce neutrophil activation in SCD and ameliorate thrombo-inflammatory disease pathology. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Use of anti-inflammatory analgesics in sickle-cell disease

2017 ◽  
Vol 42 (5) ◽  
pp. 656-660 ◽  
Author(s):  
J. Han ◽  
S. L. Saraf ◽  
J. P. Lash ◽  
V. R. Gordeuk
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Disease ◽  
Anti Inflammatory

Nitrated Lipids in a Murine Model of Sickle Cell Disease at Baseline.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3758-3758
Author(s):  
Jeffrey Schwartz ◽  
Paul R.S. Baker ◽  
Francisco J. Schopfer ◽  
Bruce A. Freeman
Keyword(s):  
Linoleic Acid ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ion Trap ◽  
Internal Standard ◽  
Wild Type ◽  
Cell Disease ◽  
Inflammatory Signaling ◽  
No Bioavailability ◽  
Anti Inflammatory

Abstract Introduction Sickle cell disease (SCD) is increasingly recognized as a disorder of inflammatory homeostasis. One major focus in recent years has been understanding the role of Nitric Oxide (NO) in the pathophysiology of SCD. NO is a critical mediator of inflammatory pathways and current evidence supports the precept that NO bioavailability is decreased in SCD, resulting in normal concentrations of NO at baseline but an inability to increase NO during stress. Nitrated fatty acids, such as Nitrolinoleate (LNO2), have recently been reported as potent and abundant anti-inflammatory signaling mediators with the ability to cause vasorelaxation and inhibition of platelet and neutrophil activation. Evidence supports their anti-inflammatory signaling is mediated through the release of NO and NO-related products. LNO2 has not previously been described in patients with SCD and our objective was to quantify LNO2 in a murine model of SCD at baseline. Methods Whole blood was obtained from transgenic sickle cell and wild type mice (n = 5 and 6, respectively). Blood was centrifuged and separated into plasma and packed red blood cells (RBCs). These biological samples were prepared for lipid analysis by the method of Bligh and Dyer; care was taken so that the pH of the extraction milieu was consistently maintained at 7 so as to avoid artifactual nitration. Samples were analyzed for free LNO2 content by electrospray ionization tandem mass spectrometry. Using a hybrid triple quadrupole ion trap mass spectrometer, MRM transitions were monitored that specifically identified nitrated linoleic acid species; these species were concomitantly confirmed by the qualitative analytical abilities of the ion trap. The presence of nitrated linoleic acid was confirmed by HPLC chromatographic retention times, MS/MS “fingerprints” and was quantitated by the inclusion of a known quantity of 13C-labeled LNO2. Results LNO2 concentration was calculated as a function of the ratio of analyte to internal standard peak areas by using an internal standard curve linear over five orders of magnitude. Free LNO2 in the RBCs and plasma of 5 transgenic sickle cell mice were 3.97 ± 2.56 nM and 12.37 ± 9.83 nM, respectively. Free LNO2 in the RBCs and plasma of 6 wild type mice were 9.49 ± 8.32 nM and 14.91 ± 10.08 nM, respectively. There were no significant differences in LNO2 concentration between any of the groups. Conclusions LNO2 is present in both transgenic sickle cell mice and wild type mice in comparable concentrations at baseline. As a mediator of NO anti-inflammatory signaling, this is consistent with human studies showing comparable concentrations of NO metabolites at baseline between sickle cell patients and healthy controls. Further study of LNO2 in sickle cell disease is warranted to better understand its role in the inflammatory process associated with acute stress, such as vaso-occlusive pain crisis and acute chest syndrome, when NO bioavailability is decreased.

Heme Induces Endothelial Tissue Factor Expression: Potential Role in Hemostatic Activation in Patients with Intravascular Hemolysis Including Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1902-1902
Author(s):  
Yamaja Setty ◽  
Suhita Gayen Betal ◽  
Jie Zhang ◽  
Nigel S Key ◽  
Marie Stuart
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Dependent Manner ◽  
Cell Disease ◽  
Intravascular Hemolysis ◽  
Tnf Α ◽  
Endothelial Tissue

Abstract Plasma levels of heme in the 20 to 600 μM range are found in clinical conditions associated with intravascular hemolysis including paroxysmal nocturnal hemoglobinuria and sickle cell disease, conditions also associated with a thrombotic tendency. Objectives: To investigate whether heme, an inflammatory mediator and a product of intravascular hemolysis in patients with hemolytic anemia including sickle cell disease (SCD), could modulate hemostasis by an effect on endothelial tissue factor (TF) expression. Additionally, in SCD patient-related studies, we assessed whether any association existed between whole blood TF activity (WBTF) and levels of surrogate markers of intra-vascular hemolysis including lactate dehydrogenase (LDH) and reticulocyte counts. Methods: Following incubation of human endothelial cells (from umbilical vein and/or lung microvasculature) with heme (1 to 100 μM) for various times (30 minutes to 8 hours), levels of TF protein were assessed using ELISA, flow cytometry and/or Western blotting; and TF mRNA by a semi-quantitative RT-PCR. An assay for TF functional activity was performed using a chromogenic tenase activity kit where specificity of TF activity was tested in antibody-blocking experiments. Three TF-specific antibodies including a rabbit polyclonal and two mouse monoclonal (clones hTF-1 and TF9-10H10) antibodies were used in assays involving TF protein analysis. All experiments were performed in media containing polymyxin B to neutralize any potential endotoxin contamination. In patient-related studies, 81 subjects with SCD (1 to 21 years) were evaluated for levels of WBTF, LDH, and reticulocyte counts and data analyzed for potential relationships. Results: Heme induced TF protein expression on the surface of both macro- and micro-vascular endothelial cells in a concentration-dependent manner with 12- to 50-fold induction noted (ELISA assays) between 1 and 100 μM heme (P<0.05, n=3 to 6). Complementary flow cytometry studies showed that the heme-mediated endothelial TF expression was quantitatively similar to that induced by the cytokine TNF-α. Heme also up-regulated endothelial expression of TF mRNA (8- to 26-fold, peak expression at 2 hours postagonist treatment), protein (20- to 39-fold, peak expression at 4 hours) and procoagulant activity (5- to 13-fold, peak activity at 4 hours post-agonist treatment) in a time-dependent manner. Time-course of heme-mediated TF antigen expression paralleled induction of procoagulant activity with antibody blocking studies demonstrating specificity for TF protein. Potential involvement of endogenously released cytokines including IL-1α and TNF-α in mediating the heme effect was next explored. We found that the latter cytokines are not involved, since antibodies against IL-1α and TNF-α, and an IL-1- receptor antagonist failed to block heme-induced endothelial TF expression. Inhibition of heme-induced TF mRNA expression by sulfasalazine and curcumin suggested that the transcription factor NFκB was involved in mediating heme-induced effect. In patient-related studies, whole blood TF levels in SCD correlated positively with both LDH (r=0.72, p<0.000001), and reticulocyte count (r=0.60, p<0.000001). Conclusions: Our findings demonstrate that heme induces TF expression in endothelial cells, and that the observed effects occurred at patho-physiologically relevant heme concentrations. Our results suggest that heme-induced endothelial TF expression may provide a pathophysiologic link between the intravascular hemolytic milieu and the hemostatic perturbations previously noted in patients with hemolytic anemia including sickle cell disease.

A Novel Connection Between Stress Erythropoiesis and Coagulation Activation in Sickle Cell Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 905-905
Author(s):  
Julia E. Brittain ◽  
David Manly ◽  
Leslie V. Parise ◽  
Nigel Mackman ◽  
Kenneth I. Ataga
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Tissue Factor ◽  
Sickle Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Cell Disease ◽  
Coagulation Activation ◽  
Monocytic Cells ◽  
Stress Erythropoiesis

Abstract Abstract 905 Introduction: Sickle cell disease (SCD) is associated with a hypercoagulable state. Multiple studies show that plasma from these patients exhibit: 1) increased thrombin generation; 2) decreased levels of natural anticoagulant proteins; and 3) a defect in the activation of fibrinolysis. The mechanism of coagulation activation in SCD is presumed to be multi-factorial, with contributions from abnormal erythrocyte phospholipid asymmetry and induction of tissue factor (TF) following hemolysis. In addition, hemolysis in SCD leads to elevated levels of erythropoietin (EPO) in patients, increased reticulocyte counts and the presence of stress (or shift) reticulocytes in circulating blood. These stress reticulocytes retain expression of the α4b1 integrin and are demonstrably adhesive to vascular factors in SCD. We have previously reported that these stress reticulocytes bind to blood monocytes in SCD patients via the α4b1 integrin, but the effect of this interaction on either cell remained unknown in SCD. Objective: With the increasing evidence that hemolysis and subsequent stress erythropoiesis associates with coagulation activation, we sought to evaluate the role of erythropoietin and the effect of stress reticulocyte adhesion to monocytes on coagulation activation in SCD patients. Methods: Coagulation activation in plasma samples was examined by evaluating TF activity on microparticles derived from patients with SCD. Stress reticulocytes were visualized and enumerated from these same patients using Wright Giemsa stained blood smears counter stained with new methylene blue to detect reticulocytes. Reticulocytes were scored as a stress reticulocytes based on the amount of punctuate reticular material, cell size, and presence of nuclear material. Stress reticulocyte induction of monocyte tissue factor expression was measured by flow cytometry after incubation of THP-1 monocytic cells with purified SS RBCs or control RBCs. To determine if induced THP-1 TF expression was due stress reticulocyte binding, THP-1 TF expression was examined in the presence or absence of known inhibitors of the monocyte/stress reticulocyte interaction. TF expression on CD14+ monocytes was examined in whole blood from SCD patients using flow cytometry. Plasma erythropoietin levels were quantified by ELISA. Results: We found that direct binding of the stress reticulocyte increased THP-1 TF expression 2.5 fold. This increase in TF expression was completely ablated by function blocking antibodies against the α4 integrin, but not by an isotype-matched control IgG. In whole blood samples, we also found increased TF expression on CD14+ monocytes with stress reticulocytes directly bound, compared to those monocytes in the same patient without stress reticulocytes bound (p = 0.002, n =3).We noted a strong correlation between stress reticulocyte count and TF activity on plasma microparticles in SCD (rspearman = 0.8656, CI = 0.5382 – 0.9660, p = 0.0006, n=11). Furthermore, we found that EPO induced α4b1 activation on the stress reticulocyte. This activation may promote both adhesion to the monocyte and an increase in TF expression. Consequently, we noted a strong trend towards an association of EPO with microparticle TF activity in SCD (rspearman = 0.5740, CI=-0.06 – 0.8780, p=0.068, n= 11) suggesting that EPO, by promoting the interaction between the stress reticulocyte and the monocyte, may contribute to TF activity in SCD. Conclusion: Taken together, we find that stress reticulocyte adhesion to monocytes and monocytic cells induces TF expression and may promote TF activity in patients. These data suggest a novel connection between stress erythropoiesis and coagulation activation in SCD. Disclosures: No relevant conflicts of interest to declare.

Mitophagy Induction Is a Potential Therapeutic Approach for Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 267-267
Author(s):  
Ramasamy Jagadeeswaran ◽  
Benjamin Alejandro Vazquez ◽  
Vinzon Ibanez ◽  
Maria A Ruiz ◽  
Robert E Molokie ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Therapeutic Approach ◽  
Research Funding ◽  
Single Point Mutation ◽  
Cell Disease ◽  
Blood Disorder ◽  
In Vivo Treatment

Abstract Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide. A single point mutation of the sixth amino acid of β-globin causes glutamic acid to be replaced by valine, rendering the hemoglobin susceptible to polymerization when deoxygenated. SCD patients suffer from the wide variety of disease manifestations including chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. In addition to the HbS polymerization-mediated rigid and fragile sickle-shaped red blood cell (RBC) formation, an excessive formation of intracellular reactive oxygen species (ROS) occurs in SCD red blood cells, which accelerates their hemolysis. This causes the release of ROS, free extracellular hemoglobin, hemin, and inflammatory cytokines that trigger disease progression. We analyzed levels of ROS in SCD patient RBCs and observed a higher fraction of intracellular ROS positive RBC in SCD (HbSS) compared to control (HbAA) RBC of adults [Control (HbAA): 7.1%± 1.4 %, n=11; SCD (HbSS): 25.3 % ± 4.3%, n=9; p<0.0004]. We also made the novel observation that mature RBCs from SCD patients abnormally contain mitochondria as evidenced by flow cytometry analysis of blood samples of 36 SCD patients and 14 normal human control subjects.[Control (HbAA):0.4 % ± 0.04%, n=14; SCD (HbSS): 7.8%± 0.9%, n=30; p<0.0001]. Further subset analysis from SCD patients with HbSC showed mitochondrial retention in their mature RBCs [HbSC: 2.2%± 0.6%,n=6 p<0.01], however to a lesser degree than patients with HbSS. Transmission electron microscopy confirmed the presence of mitochondria in mature RBC of patients with SCD. ROS analysis between mitochondria positive vs. negative fractions showed that mitochondria-positive (TMRM+) RBC fractions have higher levels of ROS compared to mitochondria-negative (TMRM-) RBC fractions. This data strongly suggests that retained mitochondria significantly contribute to the production of ROS in SCD RBCs. Similar to humans, a higher fraction of RBCs of SCD mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/J) retain mitochondria compared to control mice RBC [Control (HbAA): 0.29% ± 0.18%; SCD (HbSS): 16.68%± 1.9%, p<0.0001]. While investigating RN-1, a lysine specific demethylase-1 (LSD-1) inhibitor, as a HbF inducing agent, we observed that SCD mice treated with RN-1 showed a reduction in the fraction of RBCs which retain mitochondria. Therefore, we investigated mitophagy-inducing drugs as a possible useful therapeutic approach for SCD by administering mitophagy-inducing agent Sirolimus. SCD mice treated with RN-1 (5mg), or Sirolimus (5mg) had a significant decrease in the fraction of mitochondria containing RBCs (RN1: 4.96± 1.0%, p<0.0005; Sirolimus: 6.4% ± 1.8%, p<0.002). We observed a reduction of ROS in mature RBCs coupled with decreased mitochondrial retention in RBCs after in vivo treatment with RN1 or Sirolimus as measured by co-staining of TMRM, APC-conjugated CD71antibody, and CM-H2DCFDA. We also observed a significant improvement in RBC survival after the in vivo treatment with Sirolimus or RN-1. RBC survival was measured by flow cytometry and calculated biotin positive circulating RBCs after 2 days of in vivo labeling [SCD treated with vehicle control: 40 %± 2.6%; SCD treated with RN1 (2.5mg): 69.9 ± 2.6%, p<0.004; Sirolimus (5mg): 67.5% ± 6.1%, p<0.04]. Based on this data, mitophagy-inducing drugs have the potential to be a novel therapeutic approach for the treatment of SCD patients. Disclosures Jagadeeswaran: Acetylon: Research Funding. DeSimone:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine. Lavelle:Acetylon: Research Funding. Rivers:Acetylon: Research Funding.

Simvastatin Reduces the in Vitro Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2489-2489 ◽  
Author(s):  
Andreia A Canalli ◽  
Renata P. Ferreira ◽  
Sara T.O. Saad ◽  
Nicola Conran ◽  
Fernando F. Costa
Keyword(s):  
Cell Adhesion ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Leukocyte Adhesion ◽  
Cell Disease ◽  
Tnf Α ◽  
Cell Layers ◽  
Anti Inflammatory ◽  
Vitro Effect

Abstract Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation, leukocyte and red cell adhesion. Markers of neutrophil activation are also increased in SCD, in association with increased levels of circulating cytokines and increased leukocyte adhesion. In animal models, vaso-occlusion causes hypoxia/reperfusion, leading to vascular endothelium damage and an inflammatory response. We postulate that anti-inflammatory agents may reduce the participation of activated endothelium in the vaso-occlusive process. Statins are commonly used to treat arteriosclerosis and have anti-inflammatory effects that include a regulatory action on endothelial function, reduced oxidative stress and inflammation. The objective of this study was to investigate the in vitro effect of simvastatin on the adhesion of sickle neutrophils to activated endothelial cell layers (HUVEC). Neutrophils (Neu) were isolated from the peripheral blood of healthy controls (ConNeu) and SCD (SCDNeu) individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106 cell/ml in Ham’s F12 K) to cultured human umbilical vein endothelial cells (HUVEC) grown to confluence was assessed using static adhesion assays. HUVEC cells were treated with or without 1 μg/ml simvastatin for 6 hours in the absence or presence of a 10nM TNF-α activating stimulus (3 hours) before allowing adhesion of Neu to the cell layers (30 min, 37°C, 5%CO2). Neu from SCD patients demonstrated a significantly greater adhesion to HUVEC than ConNeu (20.5 ± 1.9% compared to 13.8 ± 1.7 %; n=15; p&lt;0.02; Mann Whitney test). Subsequently, Neu from patients and controls were allowed to adhere to endothelial layers previously treated with simvastatin; adhesion was not significantly different to the adhesion of Neu to nonsimvastatin treated HUVEC (16.7 ± 3.2% for ConNeu; n=8, p&gt;0.05 and 19.8 ±2.7% for SCDNeu; n=11, p&gt;0.05, paired t test). Pre-treatment of HUVEC with the cytokine TNF-α increased the adhesion of SCD and Con Neu to HUVEC (40.9 ± 5.4%; 28.9 ± 5.0%, respect, N&gt;8, P&lt;0.01 compared to adhesion to non-activated HUVEC). Interestingly, when the endothelium layer was protected with simvastatin and then stimulated with TNF-α, SCDNeu adhesion was significantly diminished (reduced to 31.3% ± 3.6%; n=11, p&lt;0.005 comp. to adhesion to non-simvastatin-treated HUVEC); in contrast, no difference in the adhesion of ConNeu to HUVEC treated with TNF-α and simvastatin was observed (31.9 ± 5.8%, n=8, p&gt;0.05 for ConNeu). In conclusion, data indicate that under in vitro inflammatory conditions, simvastatin appears to protect endothelium layers and reduces SCD leukocyte adhesion. We speculate that statins may have anti-inflammatory properties and, as such, may be useful for diminishing endothelial activation and, in turn, preventing the adhesion of leukocytes adhesion to the vascular wall in SCD, a mechanism that is essential to the vaso-occlusive process.

Hydroxyurea-Induced Changes of Components Involved In the Modulation of Adenosine Levels, In Blood Cells From Sickle Cell Disease Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2674-2674
Author(s):  
Rodrigo Alexandre Panepucci ◽  
Ana Cristina S Pinto ◽  
Carolina Dias-Carlos ◽  
Felipe Saldanha-Araujo ◽  
Patricia VB Palma ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Blood Cell ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Potential Effect ◽  
T Test ◽  
Lower Percentage ◽  
Cell Disease ◽  
Local Synthesis

Abstract Abstract 2674 Introduction. Recent studies have demonstrated the role of high adenosine levels in priapism episodes in a mouse model of sickle cell disease (SCD). Interestingly, adenosine signaling is related to several physiopathological processes that may relate to clinical features observed in patients with SCD. Adenosine (ADO) is a purine nucleoside that plays diverse roles in distinct physiological contexts. Extracellular ADO production occurs sequentially by the ectonucleotidases CD39 (which converts ATP and ADP to 5′-AMP) and CD73 (which convert 5′-AMP to ADO). Moreover, ADO levels are controlled by its conversion to inosine by the enzyme Adenosine Deaminase (ADA). ADA can be anchored in the cell membrane by CD26, leading to an increased localized action and consequently, to reduced local concentrations of adenosine. Hydroxyurea (HU) is the only drug approved by FDA to reduce vaso-occlusive episodes in patients with SCD, partly by the induction of fetal hemoglobin (HbF) and reduction of polymerization of HbS. However, the clinical improvement of patients is not always associated with increased HbS levels, indicating the potential effect of HU on other processes. Given the known (or proposed) contribution of distinct blood cell types in the physiopathology of SCD, in this study, we aimed to evaluate the possible modulation in the expression of CD39, CD73 and CD26 on lymphocytes and monocytes from SCD patients, in HU treated patients. Methods. The expression of CD39, CD73 and CD26 was evaluated by flow cytometry on total lymphocytes (CD3+) and monocytes (CD14+) in the peripheral blood (PB) of 12 patients treated with HU, 21 untreated and seven control healthy individuals. Results. On average, while less than 0.3% and 1.7% of monocytes of controls and untreated patients express CD26, respectively; in patients treated with HU, more than 10% of the monocytes express CD26 (p=0.0171, unpaired T-test). Additionally, in treated patients, a significantly lower percentage of lymphocytes express CD39, as compared to untreated (p=0.0431, unpaired T-test). The CD73 protein was not expressed by monocytes, and there was no modulation of its levels in lymphocytes. Conclusions. During inflammation (a processes associated with the physiopathology of SCD), the extracellular concentration of adenosine is increased and distinct blood cell types localize to the affected tissue. The results indicate a potential mechanism of action of HU in SCD patients, mediated by the increased expression of CD26 on monocytes (with subsequent co-localization of the enzyme ADA) and by the decreased expression of CD39 on lymphocytes. As a result of the observed changes, a decrease in the local synthesis of adenosine, associated with its increased conversion to inosine, would be expected. Thus, HU may drive the reduction of adenosine levels, thereby reducing the aggravating effects of this molecule in different physiopathological processes affected in patients with SCD. Supported by: FAPESP, CNPQ, FINEP and INSERM. Disclosures: No relevant conflicts of interest to declare.

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