Hydroxyurea improves nitric oxide bioavailability in humanized sickle cell mice

2021 ◽  
Vol 320 (5) ◽  
pp. R630-R640
Author(s):  
Crystal M. Taylor ◽  
Malgorzata Kasztan ◽  
Randee Sedaka ◽  
Patrick A. Molina ◽  
Luke S. Dunaway ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Sickle Cell ◽  
Renal Injury ◽  
Tap Water ◽  
Elevated Plasma ◽  
Mortality And Morbidity ◽  
Hydroxyurea Treatment ◽  
No Bioavailability ◽  
Plasma Nitrite ◽  
Renal Vascular

Despite advancements in disease management, sickle cell nephropathy, a major contributor to mortality and morbidity in patients, has limited therapeutic options. Previous studies indicate hydroxyurea, a commonly prescribed therapy for sickle cell disease (SCD), can reduce renal injury in SCD but the mechanisms are uncertain. Because SCD is associated with reduced nitric oxide (NO) bioavailability, we hypothesized that hydroxyurea treatment would improve NO bioavailability in the humanized sickle cell mouse. Humanized male 12-wk-old sickle (HbSS) and genetic control (HbAA) mice were treated with hydroxyurea or regular tap water for 2 wk before renal and systemic NO bioavailability as well as renal injury were assessed. Untreated HbSS mice exhibited increased proteinuria, elevated plasma endothelin-1 (ET-1), and reduced urine concentrating ability compared with HbAA mice. Hydroxyurea reduced proteinuria and plasma ET-1 levels in HbSS mice. Untreated HbSS mice had reduced plasma nitrite and elevated plasma arginase concentrations compared with HbAA mice. Hydroxyurea treatment augmented plasma nitrite and attenuated plasma arginase in HbSS mice. Renal vessels isolated from HbSS mice also had elevated nitric oxide synthase 3 (NOS3) and arginase 2 expression compared with untreated HbAA mice. Hydroxyurea treatment did not alter renal vascular NOS3, however, renal vascular arginase 2 expression was significantly reduced. These data support the hypothesis that hydroxyurea treatment augments renal and systemic NO bioavailability by reducing arginase activity as a potential mechanism for the improvement on renal injury seen in SCD mice.

Spontaneous Pulmonary Hypertension in Transgenic Sickle Cell Mice - Hemodynamics and Histology Suggest Abnormal Nitric Oxide Production and Scavenging.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 208-208
Author(s):  
Lewis L. Hsu ◽  
Hunter C. Champion ◽  
Elizabeth Manci ◽  
Bhalchandra Diwan ◽  
Daniel Schimel ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Cardiac Catheterization ◽  
Sickle Cell ◽  
No Synthase ◽  
No Donor ◽  
Vascular Congestion ◽  
No Consumption ◽  
No Bioavailability

Abstract Pulmonary hypertension is increasingly recognized in sickle cell disease (SCD) as a strong risk factor for early mortality. The finding of pulmonary hypertension in other hemolytic anemias suggests that the mechanism is linked to hemolysis and/or thrombosis. Pathophysiologic roles of nitric oxide (NO) consumption and recurrent lung injury have been considered. Transgenic mice expressing exclusively human sickle hemoglobin (sickle mice)(Pastzy 1997) are well established models of severe hemolytic anemia and ischemic organ damage in SCD, and provide the opportunity to examine mechanisms of pulmonary hypertension with invasive studies. Hypotheses: Pulmonary hypertension will spontaneously occur in sickle mice but not age-matched colony controls, and severity will increase as the mice grow older. Methods: Male sickle mice were compared with age-matched hemizygotes from the same colony. Mice had cardiac catheterization for baseline hemodynamics, then challenges to assess pulmonary vascular responsiveness. A pathologist made blinded assessments of the pulmonary histology. Results: Cardiac catheterization showed pulmonary hypertension in all sickle mice, and blunted pulmonary vasodilation to all NO donor compounds as well as authentic NO gas. Computed tomography in vivo detected pulmonary vascular congestion. Older sickle mice had modestly increased vessel wall thickness and vascular congestion but no thrombi by histology. Older mice also appear to be in right heart failure. Sickle mouse lungs had decreased eNOS activity (measured by L-arginine to citrulline turnover) and loss of active eNOS dimer (measured by western blotting). Sickle mouse plasma had high NO consumption, consistent with increased NO scavenging by free hemoglobin released by steady state hemolysis. mean & SD hemizygote control (5 mo & 13 mo) 5 mo sickle 13 mo sickle Pulmonary Arterial Pressure (torr) 9.4 (0.7) 18.2 (0.5) 14.8 (0.3) Pulmonary Vascular Resistance 0.37 (0.6) 0.80 (0.07) 0.75 (0.04) Cardiac Output (ml/min) 14.2 (2) 17.1 (2) 12.2 (2) Vasodilation to NO & NO donors, or bradykinin (endothelium-dependent) normal blunted none Vasodilation to CGRP (NO-independent and endothelium-independent) normal normal blunted Hypoxic vasoconstriction (10%O2) normal enhanced enhanced Discussion: This is one of the few descriptions of spontaneous pulmonary hypertension in an animal, and implicates low NO bioavailability mediated by NO resistance/scavenging. Interestingly, pulmonary thromboembolism was not observed. Combined effects of NO scavenging and the loss of active eNOS dimer may explain paradoxical blunted responses to NO donor agents, blunted responses to NO synthase inhibition, and arginine supplementation observed in patients with SCD, despite increased NO synthase protein expression. It is also likely that aberrant superoxide formation from uncoupled monomeric NO synthase contributes to vascular NO scavenging. In conclusion, pulmonary hypertension, associated with a vasoconstrictor phenotype and low NO bioavailability, develops early in the sickle cell transgenic mouse.

Nitric Oxide Effects in Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-48-sci-48
Author(s):  
Lori Styles
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Oxidant Stress ◽  
Cell Disease ◽  
No Donors ◽  
Skin Ulcers ◽  
No Bioavailability ◽  
Inhaled No

Sickle cell disease (SCD) is a complex hemoglobinopathy characterized by microvascular occlusion and hemolytic anemia. Patients suffer from a myriad of both acute and chronic problems affecting virtually every organ system. Historically, microvascular occlusion has been the focus of scientific investigations into these manifestations and the chronic hemolysis of SCD was overlooked. More recently, however, the importance of the pathophysiology of hemolysis has been appreciated and related to a subset of the clinical manifestations of SCD, including pulmonary hypertension, priapism, skin ulcers, and possibly stroke. This subphenotype of SCD has been convincingly related to impaired nitric oxide (NO) homeostasis due to hemolysis. NO has pleiotropic effects including vaso-dilatory, antioxidative, anti-adhesion, and anti-thrombotic properties, which are all potentially important in the pathophysiology of SCD. Perturbation of NO homeostasis, therefore, could profoundly impact patients with SCD. Animal and human data support a state of “NO resistance” in SCD patients. Human studies have shown that SCD patients have a decreased response to exogenous NO donors and that is likely due to the scavenging of NO by free plasma hemoglobin that results from ongoing hemolysis. “NO resistance” is further augmented by the increased levels of reactive oxygen species (ROS) known to occur in SCD patients. High levels of ROS favor additional hemolysis through increased oxidant stress on the sickle red blood cell and reduce NO bioavailability by inactivation of circulating NO. With the substantial human and animal data to support a role for “NO resistance” in the pathophysiology of SCD, investigation with NO-based therapy have begun. Several approaches to overcoming “NO resistance” can be devised including increasing the precursors to NO, decreasing hemolysis, direct NO donors, and decreasing oxidant stress. To date, studies evaluating arginine (NO precursor), inhaled NO, and sildenafil (NO donor) have been reported. Oral arginine showed no benefit in a large clinical trial, and a preliminary trial of inhaled NO had only minimal benefit. Sildenafil may be more promising and is under further study. Lastly, although impaired NO bioavailability has been related to a subset of patients with pulmonary hypertension, skin ulcers and priapism, it will be important to determine what impact NO has on other manifestations, such as vaso-occlusive pain episodes and whether NO modulation can also be used therapeutically in this setting.

Genomic variants in the ASS1 gene, involved in the nitric oxide biosynthesis and signaling pathway, predict hydroxyurea treatment efficacy in compound sickle cell disease/β-thalassemia patients

2016 ◽  
Vol 17 (4) ◽  
pp. 393-403 ◽  
Author(s):  
Constantina Chalikiopoulou ◽  
Anastasia-Gerasimoula Tavianatou ◽  
Argyro Sgourou ◽  
Alexandra Kourakli ◽  
Dimitra Kelepouri ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Sickle Cell Disease ◽  
Signaling Pathway ◽  
Sickle Cell ◽  
Treatment Efficacy ◽  
Cell Disease ◽  
Genomic Variants ◽  
Hydroxyurea Treatment

scholarly journals Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

2008 ◽  
Vol 295 (1) ◽  
pp. H39-H47 ◽  
Author(s):  
Dhananjay K. Kaul ◽  
Xiaoqin Zhang ◽  
Trisha Dasgupta ◽  
Mary E. Fabry
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Reactivity ◽  
Microvascular Function ◽  
Cell Disease ◽  
No Bioavailability ◽  
Cox 2 ◽  
And Oxidative Stress

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human α- and βS-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E2(PGE2) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.

scholarly journals Transfusion of Stored Autologous Blood Does Not Alter Reactive Hyperemia Index in Healthy Volunteers

2012 ◽  
Vol 117 (1) ◽  
pp. 56-63 ◽  
Author(s):  
Lorenzo Berra ◽  
Andrea Coppadoro ◽  
Binglan Yu ◽  
Chong Lei ◽  
Ester Spagnolli ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Autologous Blood ◽  
Reactive Hyperemia ◽  
Plasma Concentrations ◽  
Mortality And Morbidity ◽  
Plasma Hemoglobin ◽  
Reactive Hyperemia Index ◽  
Potassium Lactate ◽  
Randomization Scheme ◽  
Plasma Nitrite

Background Transfusion of human blood stored for more than 2 weeks is associated with increased mortality and morbidity. During storage, packed erythrocytes progressively release hemoglobin, which avidly binds nitric oxide. We hypothesized that the nitric oxide mediated hyperemic response after ischemia would be reduced after transfusion of packed erythrocytes stored for 40 days. Methods and Results We conducted a crossover randomized interventional study, enrolling 10 healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced packed erythrocytes, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index measurements were performed before and 10 min, 1 h, 2 h, and 4 h after transfusion. The reactive hyperemia index during the first 4 h after transfusion of 40-day compared with 3-day stored packed erythrocytes was unchanged. Plasma hemoglobin and bilirubin concentrations were higher after transfusion of 40-day than after 3-day stored packed erythrocytes (P = 0.02, [95% CI difference 10-114 mg/l] and 0.001, [95% CI difference 0.6-1.5 mg/dl], respectively). Plasma concentrations of potassium, lactate dehydrogenase, haptoglobin, and cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored packed erythrocytes, but not after transfusion of 3-day stored packed erythrocytes (P = 0.01, [95% CI difference 0.446-0.66 μM]). Conclusions Transfusion of autologous packed erythrocytes stored for 40 days is associated with increased hemolysis, an unchanged reactive hyperemia index, and increased concentrations of plasma nitrite.

Sexual Dimorphism in Whole Blood Nitrite and Susceptibility to Hypoxic Sickle Cell Vaso-Occlusive Injury in Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2327-2327
Author(s):  
Lewis L. Hsu ◽  
Andre Dejam ◽  
Mildred Pelletier ◽  
Audrey E. Cochard ◽  
Bhalchandra Diwan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Sexual Dimorphism ◽  
Liver Injury ◽  
Sickle Cell ◽  
Whole Blood ◽  
Ischemia Reperfusion ◽  
Ischemic Injury ◽  
Hypoxic Exposure ◽  
No Bioavailability ◽  
Hypoxia Reoxygenation

Abstract Murine vasoregulation and ischemia-reperfusion injury studies indicate greater nitric oxide synthase (NOS) activity in females than in males, linked to estrogen upregulation of endothelial NOS-3 (eNOS). Plasma levels of nitrite ions have been used as an index of NOS activity in vivo. Our research on blood nitrite in humans indicates that eNOS-derived NO is stabilized as nitrite, transported by erythrocytes, and bioactivated by a nitrite reductase activity of deoxyhemoglobin (Dejam 2005 Blood 106:734–739). Transgenic mice expressing exclusively human sickle hemoglobin (sickle mice) originally from Berkeley (Pastzy, 1997) are susceptible to vaso-occlusive ischemic injury from hypoxia, and increased NO availability may protect against this ischemic injury. Hypothesis: Females will have higher whole blood nitrite levels, which will be a marker of NO bioavailability and protection against ischemic challenges such as sickle cell vaso-occlusive liver injury after hypoxia-reoxygenation. Methods: Whole blood samples were stabilized with a ferricyanide solution as previously described (Dejam 2005), then analyzed for nitrite levels using reductive chemiluminescence. C57BL6 mice were studied at baseline, during normobaric hypoxia (10% O2), or 1 hour after injection of the NOS inhibitor L-N5-(1-iminoethyl)-ornithine (L-NIO, 30 mg/kg) vs saline. Sickle mice had hypoxia-reoxygenation challenge to induce sickle vaso-occlusive injury: 2 hours of hypoxia followed by blood sample 18 hours later for serum alanine aminotransferase (ALT) as a marker of ischemic injury. Results: Average whole blood nitrite was 16% (SEM 7%) lower in males than in females, with much individual variation. Hypoxic exposure as short as 15 minutes lowered nitrite by 40% (SEM 7%). The magnitude of L-NIO effect was similar, lowering nitrite by 30% (SEM 9%). Female sickle mice and C57BL6 mice of both sexes showed no rise in ALT after hypoxia-reoxygenation, but male sickle mice showed 3-fold rise in ALT (p < 0.05). Ovariectomized female sickle mice had elevated ALT that was no different from males. Discussion: Murine whole blood nitrite levels show sexual dimorphism consistent with estrogen upregulation of eNOS. L-NIO inhibition of NOS was associated with a fall in nitrite level, consistent with inhibition of nitric oxide production. The remaining nitrite after NOS inhibition is likely to be controlled by the several eNOS-independent influences on nitrite, such as dietary nitrite and nitrate, and systemic inflammation. Although hypoxia was expected to upregulate eNOS production of NO, nitrite levels fell significantly with moderate hypoxia, consistent with reduction of nitrite to NO by deoxyhemoglobin. Male sickle mice and ovariectomized females are susceptible to liver injury with hypoxic-reoxygenation challenge, consistent with a role for female hormones in upregulating protective NO bioavailability. Thus, modest changes in NO bioavailability are potentially enough to protect against sickle cell vaso-occlusive injury. Future studies to correlate whole blood nitrite levels with vasoregulation and ischemic protection are feasible with this new method for experimental measurement of whole blood nitrite.

Helicobacter Pylori Infection and Migraine

Cephalalgia ◽  
2002 ◽  
Vol 22 (3) ◽  
pp. 222-225 ◽  
Author(s):  
I Ciancarelli ◽  
C Di Massimo ◽  
MG Tozzi-Ciancarelli ◽  
G De Matteis ◽  
C Marini ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Helicobacter Pylori ◽  
Migraine Without Aura ◽  
Oxidative Status ◽  
Helicobacter Pylori Infection ◽  
Igg Antibodies ◽  
No Bioavailability ◽  
Nitrite Nitrate ◽  
Plasma Nitrite

The study is aimed to ascertain whether the Helicobacter pylori ( Hp) infection is responsible for the vulnerability to oxidative stress observed in migraineurs. Hp sero-logical positivity was assessed by ELISA evaluation of specific IgA and IgG antibodies in 30 subjects (11 males and 19 females) suffering from migraine without aura during the headache-free period. The Hp infection was detected in 16.7% of migraineurs. Plasma accumulation of peroxidative substances (TBA-RS), an index of systemic oxidative status, was increased in migraineurs without Hp infection with respect to controls ( P< 0.001), while no significant differences of TBA-RS were found in migraineurs with or without Hp infection. Unmodified values of plasma nitrite/nitrate concentrations, expression of systemic nitric oxide (NO), were obtained in migraineurs in comparison to controls indicating that Hp infection does not modify the plasma oxidative status and the systemic NO bioavailability of migraineurs. In conclusion, our results do not support any specific correlation between Hp infection and migraine.

scholarly journals Vitamin E Alleviates Renal Injury, but Not Hypertension, during Chronic Nitric Oxide Synthase Inhibition in Rats

2001 ◽  
Vol 12 (12) ◽  
pp. 2585-2593
Author(s):  
Diana M. Attia ◽  
A. Marjan G. Verhagen ◽  
Erik S. G. Stroes ◽  
Ernst E. van Faassen ◽  
Hermann-Josef Gröne ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Vitamin E ◽  
Endothelial Function ◽  
Vascular Injury ◽  
Renal Injury ◽  
Specific Inhibitor ◽  
No Synthase ◽  
Resonance Spectroscopy ◽  
Tubulointerstitial Injury ◽  
Renal Vascular

ABSTRACT. Chronic nitric oxide (NO) synthase inhibition in rats causes hypertension, renal vascular injury, and proteinuria. NO deficiency increases superoxide (O2−) activity, but the effects of antioxidant treatment on renal injury have not been studied in this model. Exposure of rats toNω-nitro-l-arginine (l-NNA) for 4 d markedly decreased NO-dependent relaxation in aortic rings and increased glomerular and renal interstitial monocyte influx, but renal O2−activity was not increased. After 7 d, BP and proteinuria were significantly increased. After 21 d ofl-NNA treatment, rats displayed severe hypertension, decreased GFR, marked proteinuria, glomerular ischemia, renal vascular and tubulointerstitial injury, and complete loss of NO-dependent relaxation. Renal O2−activity was markedly increased [lucigenin-enhanced chemiluminescence (LEC), 279 ± 71versus50 ± 7 counts/10 mg,P< 0.01; electron paramagnetic resonance spectroscopy, 0.57 ± 0.05versus0.34 ± 0.04 U/10 mg,P< 0.05]. Apocynin, a specific inhibitor of NADPH oxidase, and diphenyleneiodonium, an inhibitor of flavin-containing enzymes, completely inhibited LEC signalsin vitro, whereas allopurinol had no effect, indicating that NAD(P)H oxidase plays a major role in superoxide production in the kidney. Endothelial function remained impaired during cotreatment with α-tocopherol and there was no effect on hypertension or tubulointerstitial injury, but glomerular ischemia, decreases in GFR, and renal vascular injury were prevented and proteinuria was ameliorated. Renal LEC signals were intermediate between control andl-NNA-alone values (181 ± 84 counts/10 mg). Chronic NO synthase inhibition in rats results in marked increases in renal cortical O2−activity, mediated by flavin-dependent oxidases. The absence of early increases in renal O2−activity, in the presence of endothelial dysfunction and macrophage influx, indicates that increased renal O2−activity is neither attributable to NO deficiencyper senor solely related to macrophage influx. The improvement of glomerular function and amelioration of renal vasculitis and proteinuria with vitamin E cotreatment indicate that oxidants are involved in the pathogenesis of renal injury in this model. However, markedly impaired endothelial function and unabated hypertension persist with vitamin E treatment and seem to be directly attributable to NO deficiency.

A perinatal nitric oxide donor increases renal vascular resistance and ameliorates hypertension and glomerular injury in adult fawn-hooded hypertensive rats

2008 ◽  
Vol 294 (6) ◽  
pp. R1847-R1855 ◽  
Author(s):  
Maarten P. Koeners ◽  
Branko Braam ◽  
Dionne M. van der Giezen ◽  
Roel Goldschmeding ◽  
Jaap A. Joles
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Blood Flow ◽  
Vascular Resistance ◽  
Renal Injury ◽  
Renal Vascular Resistance ◽  
Perinatal Exposure ◽  
Hypertensive Rats ◽  
No Donor ◽  
Renal Vascular

Enhancing perinatal nitric oxide (NO) availability persistently reduces blood pressure in spontaneously hypertensive rats. We hypothesize that this approach can be generalized to other models of genetic hypertension, for instance those associated with renal injury. Perinatal exposure to the NO donor molsidomine was studied in fawn-hooded hypertensive (FHH) rats, a model of mild hypertension, impaired preglomerular resistance, and progressive renal injury. Perinatal molsidomine increased urinary NO metabolite excretion at 8 wk of age, i.e., 4 wk after treatment was stopped ( P < 0.05). Systolic blood pressure was persistently reduced after molsidomine (42-wk females: 118 ± 3 vs. 141 ± 5 and 36-wk males: 139 ± 4 vs. 158 ± 4 mmHg; both P < 0.001). Perinatal treatment decreased glomerular filtration rate ( P < 0.05) and renal blood flow ( P < 0.01) and increased renal vascular resistance ( P < 0.05), without affecting filtration fraction, suggesting persistently increased preglomerular resistance. At 4 wk of age natriuresis was transiently increased by molsidomine ( P < 0.05). Molsidomine decreased glomerulosclerosis ( P < 0.05). Renal blood flow correlated positively with glomerulosclerosis in control ( P < 0.001) but not in perinatally treated FHH rats. NO dependency of renal vascular resistance was increased by perinatal molsidomine. Perinatal enhancement of NO availability can ameliorate development of hypertension and renal injury in FHH rats. Paradoxically, glomerular protection by perinatal exposure to the NO donor molsidomine may be due to persistently increased preglomerular resistance. The mechanisms by which increased perinatal NO availability can persistently reprogram kidney function and ameliorate hypertension deserve further study.

Inhibition of Increased Sickle Neutrophil Adhesion to Fibronectin and ICAM-1 by a Nitric Oxide Donor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3776-3776 ◽  
Author(s):  
Andreia A. Canalli ◽  
Nicola Conran ◽  
Sara T.O. Saad ◽  
Fernando F. Costa
Keyword(s):  
Nitric Oxide ◽  
Cell Adhesion ◽  
Sickle Cell ◽  
Leukocyte Adhesion ◽  
Second Messenger ◽  
Nitric Oxide Donor ◽  
Neutrophil Adhesion ◽  
Adhesive Properties ◽  
No Bioavailability ◽  
Nitric Oxide Metabolites

Abstract Leukocytes play an important role in sickle cell disease (SCD); increased numbers of leukocytes are associated with increased SCD morbidity and mortality and leukocytes participate significantly in the vaso-occlusive process. Nitric oxide (NO), a signaling gas involved in vaso-relaxation and platelet aggregation regulation, has recently been implicated as important in SCD pathophysiology. NO plasma bioavailability is thought to be decreased in SCD, and NO therapy has been proposed for SCD and vaso-occlusive crises treatment. The possible consequences of decreased NO bioavailability in SCD are innumerous and have yet to be fully explored. As NO is an important inflammatory mediator and may be important for the inhibition of leukocyte adhesion and migration mechanisms, we compared nitric oxide metabolites and cGMP levels (second messenger for NO) in neutrophils from normal subjects and SCD patients (SCD neutrophils) and then compared to the adhesive properties of these cells and observed the effect of NO donating agents on this adhesion. Neutrophils were isolated from whole blood by separating on a Ficoll gradient. NO metabolites/cGMP level were measured in neutrophil extracts using specific assays. Levels of nitric oxide metabolites in SCD neutrophils did not differ signicantly from those in normal neutrophils (17.34 ± 7.02%, n=10; 16.30 ± 7.00%, n=8; respectively). Furthermore, levels of cGMP (0.11 ± 0.02 pMol/1x107 neutrophils, n=9) in SCD neutrophils were not significantly different to those observed in normal neutrophils (0.142 ± 0.036 pMol/1x107 neutrophils, n=7; p>0.05). Cell adhesion to fibronectin and ICAM-1 was compared using static adhesion assays. Neutrophils from SCD patients demonstrated a significantly greater adhesion to FN-coated plates (FN= 20mg/ml) than normal neutrophils (18.73 ± 6.24%, n=18; 13.52 ± 5.15%, respectively, n=15; p=0.016). Similarly, neutrophils from SCD patients demonstrated significantly higher adhesion to ICAM-1 (10mg/ml) when compared to normal neutrophils (19.51 ± 9.02%, n=13; 10.98 ± 3.95%, respectively n=9; p=0.025). Co-incubation of SCD neutrophils with a nitric oxide donor, sodium nitroprusside (SNP,10mM) abolished their increased adhesion to both FN and ICAM-1 to levels similar to those of normal neutrophil adhesion (16.53 ± 2.27 %, reduced to 12.61 ± 1.98%, n=8; p= 0.002 for fibronectin and 19.12 ± 3.00% reduced to 16.49 ± 2.60, n=8; p=0.009 for ICAM-1). In contrast, SNP did not affect normal neutrophil adhesion to either FN (11.59 ± 1.02% and 12.67 ± 1.35%, following incubation n=11; p>0.05) or ICAM-1 (12.33 ± 0.25% to 11.95 ± 0.62%, n= 5; p> 0.05) significantly. Although NO bioavailability may be decreased in the plasma of SCD patients due to NO sequestration by cell-free hemoglobin, our data indicate that NO dynamics are not significantly altered in the leukocytes of patients in SCD, as indicated by levels of NO metabolites and the major second messenger for NO, cGMP, are not significantly different in sickle cell neutrophils. We speculate that increased neutrophil adhesive properties in SCD may not be the consequence of decreased NO bioavailability, rather, other factors such as altered cytokine levels may mediate increased cell adhesion. However, whilst increased adhesion appears not to be mediated by NO-dependent mechanisms, an NO donor significantly reduced increased SCD neutrophil adhesion to both FN and ICAM-1, indicating that NO and NO donating drugs may also benefit SCD patients by reducing increased leukocyte adhesion, a mechanism important for the vaso-occlusive process.

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