Sickle Cell Disease Patients In Steady State Research Articles

Altered levels of cytokines and inflammatory mediators in plasma and leukocytes of sickle cell anemia patients and effects of hydroxyurea therapy

Inflammation, cell adhesion to vascular endothelium, and endothelial injury contribute to sickle cell anemia (SCA) vaso-occlusion. Although alterations in inflammatory cytokines and biomarkers have been related, reports have been conflicting, and a conclusive role for these molecules in the disease remains to be established. Furthermore, the effect of hydroxyurea therapy (HU) on the release of inflammatory mediators is not understood. This study aimed to determine plasma levels and leukocyte gene expressions of inflammatory mediators in healthy controls, steady-state SCA patients, and SCA patients on HU therapy. TNF-alpha, IL-8, and PGE(2) levels were significantly higher in the plasma of SCA individuals when compared with control individuals. HU therapy was associated with a significant reversal of augmented TNF-alpha and, interestingly, increased plasma anti-inflammatory IL-10. IFN-gamma, IL-10, cyclooxygenase 2 (COX-2), and inducible NO synthase (iNOS) gene expressions were unaltered in SCA mononuclear cells (MC); however, gene expressions of TNF-alpha, IL-8, and the protective enzyme heme oxygenase-1 (HO-1) were significantly higher. HU therapy was not associated with significantly altered SCA MC inflammatory gene expression, although COX-2 mRNA expression was decreased. In SCA neutrophils, gene expressions of IL-8, IFN-gamma, iNOS, and HO-1 were significantly higher than those of control subjects. Patients on HU demonstrated lower iNOS and higher IL-10 neutrophil gene expressions. Taken together, data suggest that alterations in the gene expressions and productions of a number of pro- and anti-inflammatory mediators are present in SCA and importantly, in those patients on HU therapy. Knowledge of these pathways may contribute to further the understanding of the pathophysiology of this disease.

High expression of the cGMP‐specific phosphodiesterase, PDE9A, in sickle cell disease (SCD) and the effects of its inhibition in erythroid cells and SCD neutrophils

Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP-dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti-inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue-specific elevation of cGMP, we studied the gene expressions of cGMP-specific PDEs (-1A, -5A and -9A) in the reticulocytes and neutrophils of healthy controls, steady-state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34(+)-derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73-6691, significantly increased production of the gamma-globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.

The moderating effect of haemoglobin A<sub>2</sub> on morbidity in sickle cell anaemia patients

The presence of high blood levels of haemoglobin A 2 (HbA 2 ) has been noted in sickle cell anaemia (SCA) patients. This study was designed to determine if there is a relationship between high HbA 2 levels and a reduction in the occurrence of vaso-occlusive crisis, complications and erythrocyte transfusion requirements in these patients. HbA 2 levels were determined in 330 patients (mean age was 23.69±10.94 years) who presented consecutively to the Haematology clinics of the University of Benin Teaching Hospital, Edo State Central Hospital and Sickle Cell Center in Benin City, Nigeria. Of these, 200 (81.3%) were SCA patients in steady state and 46 (18.7%) having vaso-occlusive crisis, and 84 age and sex matched subjects served as controls. An automated Coulter Counter was used to determine blood count values while the haemoglobin A 2 was estimated by HbS-free microcolumn chromatography. The mean HbA 2 in steady state, vaso-occlusive crisis and control were 4.52% ±1.16, 3.82% ±1.27 and 2.13% ±0.98 respectively. HbA 2 was significantly higher in steady state than during vaso-occlusive crisis (P 2 value (P=0.042 and P=0.038 respectively). High HbA 2 levels are associated with lower morbidity in sickle cell anaemia patients. Treatment strategies, which increase HbA 2 , may be beneficial in managing SCA. Keywords : Haemoglobin A 2 ; sickle cell anaemia; morbidity. International Journal of Biological and Chemical Sciences Vol. 1 (3) 2007: pp. 287-292

Increased Expression of the cGMP-Specific Phophodiesterase, PDE9A, in Sickle Cell Disease (SCD) Reticulocytes and Neutrophils, and Induction of Erythroid γ Globin Expression and Reduction of SCD Neutrophil Adhesion Following PDE9A Inhibition.

Modulation of the levels of the NO second messenger, cyclic guanosine monophosphate (cGMP), has been identified as a possible therapeutic approach for SCD treatment. Up-regulation of cGMP-dependent signaling may induce γ-globin production in erythrocytic lineage cells and diminish the adhesive properties of leukocytes, the adhesion of which contributes to vaso-occlusion. Since the phosphodiesterase (PDE) enzymes, which degrade intracellular cyclic nucleotides, display differing cellular expressions, their inhibition can provide tissue-specific induction of cGMP. We studied the gene expressions of 3 types of cGMP-specific PDE (−1A, −5A, −9A) in the reticulocytes (ret) and neutrophils (neu) of healthy controls (AA), steady-state SCD patients (SS) and SCD patients on hydroxyurea therapy (SSHU; 20–30 mg/kg/day). Ret and neu were separated from whole blood before extracting mRNA, synthesizing cDNA and quantifying PDE gene expression by Real-Time PCR relative to β-actin and GAPDH expression. PDE1 was not found expressed in ret of any of the groups, but was found expressed in the neu of all three groups (≤14.2 rel expression). Intermediate PDE5 expression was observed in ret of all groups (≤16.8 rel express); PDE5 expression in neu was, however, almost undetectable in all groups (<0.3 rel. express). In contrast, high PDE9A expressions were found in ret of all groups with significantly higher expression (P<0.05, unpaired t test) in the ret of SS than SSHU and AA (68.3±10.4; 181.8±50.0; 54.8±22.2 rel express for AA, SS, SSHU, resp., n=6). Extremely high PDE9A expressions were also found in the neu of all groups, although PDE9A was significantly higher in SCD neu than in control neu (101.7±24.0; 722.8±102.2; 873.9±221.7 rel. express for AA, SS, SSHU, resp., n≥4, P<0.05 for SS/SSHU comp. AA). Comparison of PDE9A expression in diverse cell types that included T98G glial cells, liver, colon, skin, spleen, lymphnode, mammary, ovary, uterus, testicle and K562 erythroleukemic cells demonstrated very low level or virtually undetectable gene PDE9A expression in all of these tissue types (<6.7 rel express in all tissues), with the exception of K562 cells (96.8 rel. express). Data indicate that PDE9A may be expressed at higher levels in hematopoietic lineage cells. In keeping with our results, co-culture of K562 cells in the presence of the PDE1 and PDE5 inhibitors, vinpocetine (1–50μM) and sildenafil (50–200μg/ml), respectively, did not significantly increase intracellular cGMP, nor γ-globin expression (data not shown, P>0.05), suggesting that PDE1 and PDE5 are not suitable targets for pharmacological inhibition in erythroid lineage cells. In contrast, co-culture of K562 cells in the presence of the PDE9A-specific inhibitor, BAY 73-6691, for 48 h significantly increased intracellular γ-globin expression by 97.0±7.8% (1.0μM, n=3, P<0.05). Furthermore, in vitro static adhesion assays showed that co-incubation of SS neu with BAY 73-6691 reversed augmented SS neu adhesion to immobilized fibronectin (20μg/ml) to control neu adhesion levels (AA neu adhesion: 8.7±1.3%; SS neu adhesion: 12.6±1.4%, reduced to 7.2±1.3% with 1.0 μM BAY 73-6691, n=4, P<0.01 comp.basal SS adhesion). The PDE9A enzyme may constitute a relatively hematopoietic cell-specific target for pharmacological inhibition with a potential to induce γ-globin production and to inhibit cell adhesion mechanisms, with beneficial consequences for individuals with SCD.

Increased QT Dispersion in Sickle Cell Disease: Effect of Pulmonary Hypertension

Background: QT dispersion has been proposed to be a predictor of adverse outcomes in a variety of cardiac disease states. The objective of this study was to examine QT dispersion in patients with sickle cell disease (SCD) and to assess the effect of pulmonary hypertension (PHT) on QT dispersion. Methods: We performed Doppler echocardiographic assessments of pulmonary artery systolic pressure in 73 (mean age 18.5 ± 8.0 years) steady-state SCD patients and 25 (mean age 19.6 ± 7.2 years) healthy subjects. Resting 12-lead electrocardiogram was recorded and QT dispersion was calculated as the difference between maximum and minimum QT intervals. Bazett’s formula was used to obtain a rate-corrected value of the QT interval (QTc). Results: Maximum QTc, minimum QTc and QTc dispersion were significantly increased in SCD patients compared to the control subjects (p < 0.0001, p < 0.05, p < 0.0001, respectively). Among SCD patients, patients with PHT had higher maximum QTc and QTc dispersion than patients without PHT (p < 0.0001). However, minimum QTc showed no significant differences between the two patient groups. Conclusion: QTc dispersion is significantly increased in SCD patients, especially those with PHT indicating regional inhomogeneity of ventricular repolarization.

Cytokine profile of sickle cell disease in Oman

The aim of our study was to assess the cytokine profile of sickle cell disease (SCD) patients in steady state and in vaso-occlusive crisis (VOC). VOC has a complex nature, involving interactions between sickle red blood cells (RBC), the endothelium, and leucocytes. Endothelial damage due to recurrent adhesion of sickle RBCs may disrupt endothelial function, leading to altered cytokine release. It is therefore pertinent to study the cytokine profile of SCD patients in steady state and in crisis prior to exploring its contribution to vaso-occlusive manifestations, since it is believed that an altered balance of proinflammatory and anti-inflammatory cytokines plays an important role in painful crisis. Cytokines including IL-1beta, IL-2, IL-4, IL-6, IL-8, TNF-alpha, and IFN-gamma were measured by commercially available ELISA kits in SCD patients (n = 60); in steady state (n = 26) and in painful crisis (n = 34) and compared with nonanemic age- and sex-matched normal Omani controls (n = 20). SCD patients in crisis showed elevated levels of TNF-alpha (P < 0.092) and IL-6 (P < 0.024) when compared with steady state. It was also observed that SCD patients in steady state showed a significant elevation in IL-1beta (P < 0.04), IL-6 (P < 0.0001), and IFN-gamma (P < 0.02) as compared to normal subjects. It is thus evident that both type I and type II cytokines are significantly altered in SCD patients. In steady state, type II proinflammatory cytokines are elevated, whereas in crisis, an additional augmentation of type I cytokines occurs, with persistent elevation of type II cytokines, emphasizing the role of perturbed endothelium and activated monocytes in the pathophysiology of vaso-occlusion in sickle cell crisis.

Increased GM-CSF Levels in Sickle Cell Disease Are Associated with Increased Leukocyte Counts and Are Reversed by Hydroxyurea.

The hematopoietic cytokine, GM-CSF, stimulates the proliferation and differentiation of progenitor cells to macrophages, eosinophils and megakaryocytes. Increased levels of GM-CSF have previously been reported in sickle cell disease (SCD) patients with low fetal hemoglobin (HbF) levels (Croizat, Br J Haematol. 87:592; 1994); however, a clearcut association between GM-CSF and HbF levels in SCD has yet to be established, since there is some evidence that levels may depend on other factors (Haider et al., Acta Haematol. 102:140; 1999). To further investigate the possible role of GM-CSF in SCD, plasma samples were collected from normal individuals (AA), steady-state sickle cell anemia patients (SS) and sickle cell patients on hydroxyurea therapy (SSHU; 20-30 mg/kg/day). Plasma GM-CSF levels were measured using a specific immunoenzymatic assay. HbF levels were quantified by ion-exchange HPLC and hematological measurements obtained using an Advia Hematology System. Plasma GM-CSF levels seem to be higher in SS patients than in normal controls (0.828 ± 0.215 pg/ml, n=39; 0.230 ± 0.081 pg/ml, n=9; respectively). In contrast, GM-CSF was significantly lower in SSHU patients than in SS without HU (0.288 ± 0.104 pg/ml; n=14; P = 0.045 compared to SS). A negative correlation between plasma GM-CSF and HbF levels was seen in patients (r = −0.332; P = 0.026; n=45; SS and SSHU groups) and, notably, a positive correlation between GM-CSF and white blood cell counts was observed (r = 0.302; P = 0.037; n= 48; SS and SSHU groups). Furthermore, GM-CSF (2.5 ng/ml) abolished the production of γ globin during erythropoietin-stimulated differentiation of TF-1 hematopoietic cells. These results provide further support to the hypothesis that plasma GM-CSF correlates negatively with HbF in sickle cell disease. Interestingly, in vitro data suggest that increased GM-CSF may, in fact, diminish HbF levels rather than reflect increased haematopoietic stress as a consequence of low HbF. Importantly, GM-CSF appears to correlate with leukocyte numbers in SCD, and levels were significantly decreased in patients taking HU, indicating that this cytokine may contribute to the leukocytosis seen in some SCD patients and may play a role in leukocyte count reduction by HU.

Increased levels of soluble ICAM-1 in the plasma of sickle cell patients are reversed by hydroxyurea.

Increased adhesive events between the blood vessel endothelium and red and white cells play a central role in the initiation of vasoocclusive crisis in sickle cell disease (SCD). Soluble VCAM-1 levels are increased in the plasma of sickle cell patients and may be reduced during hydroxyurea (HU) therapy. Reports regarding any changes in soluble ICAM-1 (sICAM-1) levels in sickle cell patients, however, are conflicting, and as yet no beneficial effect of HU upon levels has been observed. Thus, we sought to thoroughly investigate changes in sICAM-1 levels in SCD patients and the effect of HU therapy (20-30 mg/kg/day). Plasma sVCAM-1 levels were significantly higher in steady-state SCD patients than in normal controls (766 +/- 86 ng/mL vs. 325 +/- 38 ng/mL, respectively, P < 0.0001). sVCAM-1 levels were decreased in patients on HU therapy (543 +/- 69 ng/mL) compared to those not taking HU; however, this difference was not significant. Plasma sICAM-1 levels were significantly increased in steady-state SCD patients compared to normal individuals (285 +/- 20 ng/mL vs. 202 +/- 16 ng/mL, respectively, P = 0.002), and HU therapy significantly reduced sICAM-1 levels in patients (217 +/- 12, P = 0.027) to levels approaching those of healthy individuals. sVCAM-1 levels inversely correlated with fetal hemoglobin levels in SCD patients, while a nonsignificant inverse trend was observed between sICAM-1 levels and fetal hemoglobin. In conclusion, plasma sICAM-1 levels were significantly increased in SCD patients, and this increase was reversed by hydroxyurea therapy, possibly reflecting reduced endothelial activation in patients taking HU. Such an event may benefit patients by reducing adhesive interactions between white cells and the endothelium.

Differential Cell Count of Bone Marrow Aspirates in Steady-state Sickle Cell Anaemia Patients

Background: Megaloblastic arrest of the bone marrow is one of the causes of anaemic crises in patients with sickle cell anaemia. The diagnosis of this condition will require that the reference level of megaloblastosis during steady state be properly documented. Objectives:To document a reference differential cell count in the bone marrow aspirates of patients with sickle cell anaemia and determine the level of megaloblastosis that can be regarded as indicating megaloblastic crisis in such patients. Design: Systematic counting of haematopoietic cells in the bone marrow of children with confirmed Hb phenotype SS who are in steady state. Subjects and Methods: The subjects were 11 of 68 children with sickle cell anaemia attending the paediatric outpatient clinic at the Lagos University Teaching Hospital, Lagos, in respect of whom parental consent for bone marrow aspiration was obtained. About 4.5 ml of blood was obtained from the antecubital vein of each child, for full blood count. Bone marrow was aspirated from the posterior superior iliac spine. Slides were stained with MayGrünwald-Giemsa stain. Proportions of erythroid, myeloid, lymphoid and megakaryocytic cells out of 250 nucleated bone marrow cells were determined. Results: Steady state mean packed cell volume (PCV) was 0.2 ± 0.017 L/L. The mean reticulocyte count was 5.9 percent (95% CI, 5.3 – 7.0%) and the mean cell volume was 91.8 ± 2.7 fl. Erythroid precursors constituted 34.5 percent of the total nucleated bone marrow cells (NBMC). Of these, polychromatic and orthochromatic erythroid blasts predominated, constituting 49 and 36percent respectively, of total erythroid precursors. Polychromatic and orthochromatic megaloblasts constituted 16.5percent (95% CI, 7-25%) of total NBMC or 47.8 percent of erythroid precursors. The myeloid erythroid ratio was 1:1. The reference range of megaloblasts was 8-26 percent of the NBMC. Conclusion: Patients with sickle cell anaemia in steady-state may show megaloblastic bone marrow changes even with routine folate supplements. Megaloblastic crisis should not be diagnosed until megaloblasts are in excess of 26 percent of the total NBMC. Nigerian Journal of Paediatrics2003;30:54-59

Avascular Necrosis of the Femoral Head in Adult Kuwaiti Sickle Cell Disease Patients

While sickle cell disease (SCD) is generally mild in most Kuwaitis, because of their elevated fetal Hb levels, avascular necrosis of the femoral head (AVNFH) appears to be a common complication. It was recently documented in 26.7% of Kuwaiti children with SCD. There have, however, been no previous studies of adult patients. This is a 1-year study of consecutive, steady-state SCD patients seen in the hematology clinic of Mubarak Al-Kabeer Hospital. The patients’ charts were reviewed for frequency of hospitalizations, any documented complications and steady-state complete blood count (CBC). MRI was performed using T1- and T2-weighted FATSAT sequences in coronal and axial planes with 4-mm-thick slices on a 1.5-tesla GE super-conducting magnet. Thirty-five patients were studied, consisting of 25 SS and 10 Sβ⁰Thal patients aged between 17 and 44, with a mean age of 26.7 ± 9.3 years. Seventeen (48.6%) had varying degrees of AVNFH; among the 70 hips examined, 29 (41.1%) were affected. Of the 17 patients affected, 11 (64.7%) were SS, while 6 (35.3%) were Sβ⁰Thal. There were 14 (82.4%) males and 3 (17.6%) females (χ² = 8.6, p < 0.01). The mean age of those affected, 27.5 ± 10.7 years, was not significantly higher than that of the unaffected (26.3 ± 8.0 years). Eleven (64.7%) of those affected had a history of frequent vaso-occlusive crisis. No significant differences could be demonstrated in the mean CBC and Hb F values of the two groups; coexistent α-thal trait was not a factor in the SS group. Male gender was the only significant predisposing factor identified. While more patients with frequent vaso-occlusive crises were affected, the difference was not significant. AVNFH is, indeed, quite common among Kuwaiti SCD patients and there is a need for early institution of preventive and therapeutic protocols.

Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease.

To determine the effects of L-arginine (L-Arg) supplementation on nitric oxide metabolite (NOx) production, oral L-Arg was given to normal controls, sickle cell disease (SCD) patients at steady state and SCD patients hospitalized with a vaso-occlusive crisis (VOC). L-Arg (0.1 g/kg) increased NOx formation by 18.8 +/- 68% in normal controls, whereas steady-state SCD patients demonstrated a paradoxical decrease in NOx of -16.7 +/- 4% (P = 0.004). In contrast, patients with VOC demonstrated a dramatic increase in NOx production by +77.7 +/- 103%, a response that was dose dependent. L-Arg appears to be the rate-limiting step in NOx production during VOC. Oral arginine may therefore benefit SCD patients by inducing an increase in NO production during VOC.

Arginine therapy: a novel strategy to induce nitric oxide production in sickle cell disease

To determine the effects of l‐arginine (l‐Arg) supplementation on nitric oxide metabolite (NOx) production, oral l‐Arg was given to normal controls, sickle cell disease (SCD) patients at steady state and SCD patients hospitalized with a vaso‐occlusive crisis (VOC). l‐Arg (0·1 g/kg) increased NOx formation by 18·8 ± 68% in normal controls, whereas steady‐state SCD patients demonstrated a paradoxical decrease in NOx of −16·7 ± 4% (P = 0·004). In contrast, patients with VOC demonstrated a dramatic increase in NOx production by +77·7 ± 103%, a response that was dose dependent. l‐Arg appears to be the rate‐limiting step in NOx production during VOC. Oral arginine may therefore benefit SCD patients by inducing an increase in NO production during VOC.

Tumor necrosis factor-alpha is undetectable in the plasma of SS patients with elevated Hb F.

Steady-state sickle cell disease (SCD) patients may have increased plasma levels of acute phase reactants and pro-inflammatory cytokines because of subclinical inflammation. We have estimated TNF-alpha levels in the plasma and in supernatants following peripheral blood mononuclear cell (PBMC) activation with phytohemagglutinin (PHA) in a group of Kuwaiti SCD patients using ELISA. The group consisted of 28 SS, 8 Sbeta-thal, and 2 SD patients all in steady state; 5 SS patients were studied during 7 episodes of painful crisis. The subjects were aged 2 to 16 years, with a mean of 7.3 +/- 3.5 years. The beta(S)-globin gene cluster haplotype, alpha-tha1 status, and spleen function were determined in the SS group using standard techniques. Most (82%) were homozygous for the Saudi Arabia/India haplotype and had elevated Hb F levels ranging from 15% to 35%. There were 24 controls (Hb AA or AS), of whom 14 were healthy and 10 were acutely ill at the time of the study. None of the children with SCD (either in steady state or crisis) had detectable plasma TNF-alpha, but four controls (3 acutely ill and one healthy) had levels ranging from 61.7 to 249.8 pg/mL. Following PHA stimulation most subjects responded with high levels of TNF-alpha, with the median level among the steady-state SS patients being significantly higher than that in the controls (both the acutely ill or healthy). It therefore appears that because of the mild disease among our Arab SS children, TNF-alpha is not detectable in their plasma in steady state; these children, however, had a significantly higher response than controls following PBMC activation.

Pathophysiologic Role of Nitric Oxide (NO) in Sickle Cell Acute Chest Syndrome (ACS) • 796

NO, a potent cyto-protective signalling molecule, is produced by endothelial cells (ECs), and down-regulates EC expression of selective adhesion molecules. Recently, two case reports have suggested a role for NO inhalation in the treatment of ACS in patients with sickle cell disease (SCD). The data presented suggests a mechanism for the therapeutic use of NO in alleviating ACS. We have previously shown that hypoxia increased sickle red blood cell (RBC)-EC adhesion and that this effect was mediated via up-regulation of vascular cell adhesion molecule (VCAM)-1 on the EC surface. We now demonstrate that in ACS there is an increase in circulating soluble VCAM-1 (sVCAM-1) and a decrease in NO. We evaluated plasma levels of NO metabolites (assayed as nitrate and nitrite) and sVCAM-1 in 18 age-matched controls, 34 children with SCD in steady state, 13 patients in vaso-occlusive crisis (VOC) and 5 in ACS. Nitrate levels in controls, and patients with SCD in steady state, VOC and ACS were 22.4±1.6 (mean ± SE), 20.5±1.3, 14.3±0.4 and 11.1±1.4 μM, respectively. While there was no difference between the control group and the SCD patients in steady state, nitrate levels were significantly reduced in SCD patients in VOC and ACS with the greatest decrease in ACS (P<0.001). Circulatory levels of sVCAM-1 in controls, and patients with SCD in steady state, VOC and ACS were 491±90, 844±191, 856±64, and 1169±213 ng/ml, respectively. sVCAM-1 levels were significantly elevated in steady state, VOC and ACS (P<0.001) when compared to controls with the greatest increase occurring in ACS (significant increase over both steady state and VOC, P<0.02 to 0.001). Additionally, in in vitro experiments, the NO donor SIN-1 (n=6) inhibited hypoxia-induced sickle RBC adhesion to human lung micro-vascular ECs in a concentration-dependent manner with 57 and 73% inhibition noted at 10 and 100 μM, respectively. This effect was mediated via a concomitant down-regulation of hypoxia-induced VCAM-1 expression (66 and 85% inhibition). Our data shows that (1) hypoxia-induced VCAM-1 up-regulation on ECs occurs in vivo i.e. in ACS; (2) NO production is markedly decreased in ACS and (3) NO by attenuating hypoxia-induced RBC-EC adhesion may potentially modulate the clinical course of acute chest syndrome in patients with sickle cell disease.