John-John B. Schnog

Oxidative stress in sickle cell disease; pathophysiology and potential implications for disease management

Sickle cell disease (SCD) is a hemoglobinopathy characterized by hemolytic anemia, increased susceptibility to infections and vaso‐occlusion leading to a reduced quality of life and life expectancy. Oxidative stress is an important feature of SCD and plays a significant role in the pathophysiology of hemolysis, vaso‐occlusion and ensuing organ damage in sickle cell patients. Reactive oxygen species (ROS) and the (end‐)products of their oxidative reactions are potential markers of disease severity and could be targets for antioxidant therapies. This review will summarize the role of ROS in SCD and their potential implication for SCD management. Am. J. Hematol. 2011.

Evidence for a metabolic shift of arginine metabolism in sickle cell disease

Over the last few years, a pivotal role has been ascribed to reduced nitric oxide (NO) availability as a contributing factor to the vaso-occlusive process of sickle cell disease. We investigated whether arginine metabolism in sickle cell patients is different from healthy controls. Blood samples were drawn by venipuncture in the fasting state from 8 clinically asymptomatic HbSS patients and 14 race-matched HbAA controls. HbSS patients had decreased plasma arginine (p=0.001) and increased proline (p=0.015) levels as compared to controls. Ratios of arginine to ornithine (p<0.001), proline (p<0.001), glutamate (p=0.003), and citrulline (p=0.026) were lower in HbSS patients. There were significant correlations of ornithine (rs=−0.71, p=0.047), citrulline (rs=−0.79, p=0.021), arginine/ornithine (rs=0.93, p=0.001), and arginine/citrulline (rs=0.81, p=0.015) to hemoglobin and of arginine/proline (rs=−0.76, p=0.028) and citrulline (rs=0.71, p=0.048) to leukocyte counts. These data indicate that in clinically asymptomatic sickle cell patients increased arginine metabolism is shifted to the arginase pathway and that this seems to be more profound in patients with higher hemolytic rates and leukocyte counts.

Plasma levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, are elevated in sickle cell disease

In recent years an important role has been ascribed to a reduced nitric oxide (NO) availability in the pathophysiology of sickle cell disease (SCD). Endogenously produced inhibitors of NO synthase, in particular asymmetric dimethylarginine (ADMA), are currently considered of importance in various vascular disease states characterized by reduced NO availability. We determined ADMA levels in plasma of 12 adult sickle cell patients (eight HbSS and four HbSC), and compared these to plasma levels in race- and age-matched controls. Sickle cell patients were characterized by strongly elevated levels of ADMA [HbSS: median 0.63 μmol/l (interquartile range 0.54–0.85), HbSC: 0.43 μmol/l (0.40–0.46), HbAA: 0.33 μmol/l (0.32–0.35) p<0.001]. ADMA levels were highest in HbSS patients with lowest hemoglobin levels and highest leukocyte counts, and in HbSS patients ADMA levels were positively associated with serum levels of soluble vascular cell adhesion molecule-1. These results suggest an important role of ADMA in limiting NO availability in SCD, and its role in the pathophysiology of SCD should be further investigated.

Elevated homocysteine levels indicate suboptimal folate status in pediatric sickle cell patients

We investigated whether pediatric patients with sickle cell disease (SCD) (9 + 4 years; 27 homozygous SCD [HbSS]; 19 sickle‐C disease [HbSC]) have different folate status compared with age‐, sex‐, and race‐matched normal hemoglobin (HbAA) controls (n = 20), and whether their folate status can be improved by folate supplementation. The patients were supplemented with vitamins B6 and B12 during one week and with folate during the following week. Circulating folate, homocysteine, vitamin B6 and vitamin B12 levels were measured at baseline (patients and controls), after one week and after two weeks (patients). The patients had similar folate, vitamin B6, and vitamin B12, but higher homocysteine levels compared with HbAA controls (12.7 + 4.5 vs. 10.9 + 3.5 μmol/l; P = 0.04). Vitamin B6 and B12 supplementation did not change their homocysteine levels, but folate supplementation caused a 53% reduction (to 5.7 + 1.6). We conclude that patients with SCD have adequate vitamin B6 and B12 status, but suboptimal folate status, leading to elevated plasma homocysteine levels. They may therefore benefit from folate supplementation to reduce their high risk for endothelial damage. Am. J. Hematol. 59:192–198, 1998.

No association of the hypercoagulable state with sickle cell disease related pulmonary hypertension

Pulmonary hypertension (PHT) occurs in approximately 30% of adult patients with sickle cell disease (SCD) and is a risk factor for early death.[1][1] Hypercoagulability has been linked to PHT in general and pulmonary artery thrombosis contributes to PHT progression regardless of its cause.[2][2]

Steady-state sVCAM-1 serum levels in adults with sickle cell disease

Cytokines and adhesion molecules play an important role in the pathophysiology of vaso-occlusion in sickle cell disease (SCD), and their in vivo profiles are potential tools for assessing SCD severity. We compared steady-state soluble vascular cell adhesion molecule-1 (sVCAM-1) serum levels to clinical (painful crisis frequency, occurrence of acute chest syndrome, leg ulcers, and cerebrovascular disease) and related hematological parameters of SCD severity (such as HbF%, hemoglobin levels, and leukocyte counts) in 29 HbSS adults. Serum sVCAM-1 levels were not related to clinical severity, but an inverse correlation was demonstrated between sVCAM-1 and hemoglobin levels (r=−0.71, p<0.001) with a positive correlation to serum lactate dehydrogenase levels (r=0.59, p=0.008). Based upon these results, steady-state serum sVCAM-1 levels do not seem to reflect clinical disease severity. However, as VCAM-1 is involved in hematopoiesis, sVCAM-1 levels might reflect bone marrow activity in SCD. This underlies the pleiotropic nature of adhesion molecules in vivo and the need for further research in this area, especially since therapies targeting (cellular) adhesive interactions involving the endothelium are emerging for SCD.

Serum levels of angiogenic factors indicate a pro-angiogenic state in adults with sickle cell disease

Hypoxia‐induced angiogenesis may play an important role in the pathophysiology of sickle cell disease (SCD). Serum levels of angiopoietin (Ang)‐1, Ang‐2, vascular endothelial growth factor, placenta growth factor (PlGF), soluble tunica intima endothelial kinase 2 (sTIE2), erythropoietin (EPO) and endothelial activation markers (soluble vascular adhesion molecule‐1, soluble intercellular adhesion molecule‐1) were determined in controls, HbSS (n = 35) and HbSC (n = 23) patients. In the asymptomatic phase, serum Ang‐2 (P < 0·001), EPO (P < 0·001) and sTIE2 (P = 0·03) were elevated in patients. During painful crises, increased Ang‐2 (P < 0·001) and PlGF (P = 0·04) occurred in HbSS and Ang‐2 (P = 0·05) in HbSC patients. These results indicate a pro‐angiogenic state in SCD, mainly because of elevated Ang‐2 levels.

Sickle cell disease-related organ damage occurs irrespective of pain rate: implications for clinical practice

This study shows that clinically relevant forms of organ damage occur irrespective of the frequency of painful crises in adults with sickle cell disease. In daily clinical practice, the frequency of painful crises (pain rate) is an important parameter of sickle cell disease severity. We assessed the prevalence of sickle cell disease-related organ damage and complications and their relation to pain rate. Organ damage and history of vaso-occlusive complications were obtained via systematic screening of consecutive patients and by chart review. In 104 adult sickle cell patients pain rate was related to a history of acute chest syndromes, avascular osteonecrosis, iron overload, priapism and cholelithiasis. However, major disease-related complications, such as microalbuminuria and pulmonary hypertension, were detected in 23% and 24% respectively of patients without painful crises in the study period underlining the importance of systematic screening for developing organ damage in sickle cell patients irrespective of pain rate.

Nucleosomes and neutrophil activation in sickle cell disease painful crisis

Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ0-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ+-thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome.

Plasma asymmetric dimethylarginine concentrations in sickle cell disease are related to the hemolytic phenotype

Asymmetric dimethylarginine (ADMA) is associated with pulmonary hypertension (PHT) in sickle cell disease (SCD). We studied the relationship of ADMA to other SCD-related complications. Plasma ADMA and associated parameters were determined in 52 HbSS/HbSbeta(0)-thalassemia and 24 HbSC/HbSbeta(+)-thalassemia patients. As expected ADMA levels were higher in HbSS/HbSbeta(0)-thalassemia patients with PHT (p=0.018), but also in those with other hemolysis-associated complications such as leg ulcers (p=0.012), cholelithiasis (p=0.008) and priapism (p=0.02) compared with counterparts without these complications. ADMA levels did not differ between patients with and without other disease related complications such as retinopathy and avascular osteonecrosis. Higher ADMA concentrations therefore seem to be associated to the hemolytic phenotype of SCD.