Relationship of Omega-3 fatty acids DHA and EPA with the inflammatory biomarker hs-CRP in children with sickle cell anemia.

BACKGROUND: Inflammation and vaso-occlusion play key roles in Sickle Cell Disease (SCD) pathophysiology. Lipoxygenase products of the omega-3 fatty acids (O3FAs), docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, are potent anti-inflammatory mediators modulating pain. O3FAs decrease episodes of vaso-occlusion in SCD. METHODS: We assessed erythrocyte fatty acid composition in two major cell membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, in children with SCD HbSS-disease (n = 38) and age/race-matched HbAA-controls (n = 18). Ratio of pro-inflammatory arachidonic acid (AA) to anti-inflammatory DHA and EPA (FA-Ratio), and its relationship to hs-CRP were evaluated. RESULTS: FA-Ratios were increased in both phosphatidylcholine and phosphatidylethanolamine in HbSS compared to controls. Correlations were noted in HbSS subjects between hs-CRP and FA-Ratios (p = 0.011). FA-Ratios increased with age (p = 0.0007) due to an increase in pro-inflammatory AA with a concomitant decrease in anti-inflammatory DHA. CONCLUSIONS: Findings demonstrate relative deficiencies in HbSS of the anti-inflammatory precursor fatty acids DHA and EPA, which correlates positively with hs-CRP.

Tissue factor-positive monocytes in children with sickle cell disease: correlation with biomarkers of haemolysis.

Tissue Factor (TF) initiates thrombin generation, and whole blood TF (WBTF) is elevated in sickle cell disease (SCD). We sought to identify the presence of TF-positive monocytes in SCD and their relationship with the other coagulation markers including WBTF, microparticle-associated TF, thrombin-antithrombin (TAT) complexes and D-dimer. Whether major SCD-related pathobiological processes, including haemolysis, inflammation and endothelial activation, contribute to the coagulation abnormalities was also studied. The cohort comprised children with SCD (18 HbSS, 12 HbSC, mean age 3·6 years). We demonstrated elevated levels of TF-positive monocytes in HbSS, which correlated with WBTF, TAT and D-dimer (P = 0·02 to P = 0·0003). While TF-positive monocytes, WBTF, TAT and D-dimer correlated with several biomarkers of haemolysis, inflammation and endothelial activation in univariate analyses, in multiple regression models the haemolytic markers (reticulocytes and lactate dehydrogenase) contributed exclusively to the association with all four coagulant markers evaluated. The demonstration that haemolysis is the predominant operative pathology in the associated perturbations of coagulation in HbSS at a young age provides additional evidence for the early use of therapeutic agents, such as hydroxycarbamide to reduce the haemolytic component of this disease.

Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain.

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.

Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes.

Phosphatidylserine (PS)-positive erythrocytes adhere to endothelium and subendothelial matrix components. While thrombospondin mediates these inter-actions, it is unknown whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control HbAA erythrocytes, we demonstrate that PS-positive erythrocytes adhered to human lung microendothelial cells in the absence of plasma ligands, that this adhesion was enhanced following endothelial activation with IL-1alpha, TNF-alpha, LPS, hypoxia, and heme, and that this adhesive interaction was selective to erythrocyte PS. We next explored whether microendothelial cells express an adhesion receptor that recognizes cell surface-expressed PS (PSR) similar to that expressed on activated macrophages. We demonstrate constitutive expression of both PSR mRNA and protein that were up-regulated in a time-dependent manner following endothelial activation. While minimal PSR expression was noted on unstimulated cells, endothelial activation up-regulated PSR surface expression. In antibody-blocking studies, using PS-positive erythrocytes generated either artificially via ionophore treatment of control erythrocytes or from patients with sickle cell disease, we demonstrate that PSR was functional, supporting PS-mediated erythrocyte adhesion to activated endothelium. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the microendothelium that is up-regulated by such pathologically relevant agonists as hypoxia, cytokines, and heme.

Vaso-occlusion in children with sickle cell disease: clinical characteristics and biologic correlates.

Vaso-occlusive pain is a frequent manifestation of sickle cell disease, but most clinical studies have documented only those pain episodes for which patients seek acute care or require hospitalization. Based on limited previous studies, the authors suggest that pain episodes managed at home are more frequent then those resulting in acute care management but likely share a common pathophysiology. The authors determined the characteristics of vaso-occlusive pain managed at home in 30 subjects (ages 6-19 years) using a self-report diary daily for 6 months. A total of 175 pain episodes were reported in 4,885 days, with 51% lasting 1 day or less. Severe pain, rated as 7 to 10 on a 10-point scale, was reported on 12% of pain days, but most pain was of mild to moderate intensity. A combination of baseline hematologic parameters and biomarkers assessing erythrocyte/endothelial cell adhesion, including hematocrit, fetal hemoglobin, and adhesion ratio, were statistically significant predictors of pain frequency in statistical analyses. Given the overlap in clinical features and predictive hematologic parameters of home-managed and acute care-managed pain, both likely represent a continuum of frequency and severity rather than distinct clinical entities. The higher frequency of these home-managed episodes suggests their potential utility as additional outcome measures in studies of vaso-occlusive pain.

Hypoxaemia in sickle cell disease: biomarker modulation and relevance to pathophysiology.

BACKGROUND: Nocturnal oxyhaemoglobin desaturation might have a role in CNS complications related to sickle cell disease, and rates of painful crises. We attempted to examine the biological relations, and describe the haematological risk factors for oxyhaemoglobin desaturation. METHODS: The study population included children with sickle cell disease and controls. Cellular activation was assessed by measurement of soluble vascular cell adhesion molecule 1, P-selectin, L-selectin, and leukotriene B4. Erythrocyte-endothelial adhesion and routine haematological variables were assessed. Oxygen saturation (SaO2) was measured by pulse oximetry while children were awake and asleep. Children with a mean sleeping SaO2 of < or =93% were identified as hypoxaemic. Children were divided into four groups: controls (ten children), HbSC (nine, all normoxic), HbSS normoxic (13), and HbSS hypoxaemic (15). FINDINGS: Among haematological variables, sleeping SaO2 correlated only with packed-cell volume (r=0.7; p<0.0001). Inverse relations were noted between sleeping SaO2 and adhesion (-0.45; p<0.01), and markers of white-cell (-0.51; p<0.01), platelet (-0.61; p<0.001), and endothelial activation (-0.46; p<0.01). In the HbSS group who had sleeping hypoxaemia, waking SaO2 measurements showed continuing hypoxaemia, with similar correlation between SaO2 and cell activation markers. INTERPRETATION: Our adhesion-related findings suggest a potential mechanism for the increased occurrence of clinical vaso-occlusive crises in individuals with sickle cell disease who have oxyhaemoglobin desaturation. Release of cellular mediators in hypoxaemia, and the relation between anaemia and oxyhaemoglobin desaturation, suggest that risk factors for stroke, including anaemia, might have a role in CNS-vasculopathy through hypoxia-mediated pathways. Further more, hypoxaemia in the older child also occurs during the day; such mild untreated hypoxia could lead to an increased risk of vaso-occlusive episodes.

Eicosanoids in sickle cell disease: potential relevance of neutrophil leukotriene B4 to disease pathophysiology.

Neutrophil activation with the release of intracellular granule contents has been observed in sickle cell disease (SCD). Because leukotriene B(4) (LTB(4)), a 5-lipoxygenase metabolite of arachidonic acid in neutrophils, is a chemoattractant and enhances neutrophil adhesion to endothelium, we assessed plasma levels of this metabolite in controls (n = 9) and individuals with SCD, SS genotype, both in basal "steady state" (n = 37) and during episodes of vaso-occlusion (n = 10) and acute chest syndrome (n = 5). Thirteen patients with SCD, SC genotype, in steady state were also studied. Although no significant differences were noted between the control (136 +/- 32 fmol/mL) and SC genotype (177 +/- 83 fmol/mL, P >.15), LTB(4) levels were markedly increased in patients with SS genotype in basal steady state (207 +/- 64 fmol/mL, P <.003) compared with those in controls. Values were further increased during vaso-occlusion (264 +/- 94 fmol/mL) and acute chest syndrome (363 +/- 124 fmol/mL). These levels were significantly different from measurements taken during steady state (P <.04 and P <.0001, respectively). No correlation was noted between LTB(4) level and total white cell or neutrophil count. Additionally, the significant correlation noted in SCD between increased levels of plasma LTB(4) and soluble L-selectin (P <.03) reflects neutrophil activation. We also observed an effect of LTB(4) on red cell-endothelial adhesion at concentrations that appear clinically relevant (1-10 pmol/mL) with concomitant up-regulation of mRNA for the endothelial vitronectin receptor. These properties of LTB(4) are relevant to disease pathophysiology, providing further evidence of the contribution of the neutrophil to the proinflammatory and proadhesive phenotype in SCD.

Role of erythrocyte phosphatidylserine in sickle red cell-endothelial adhesion.

Phosphatidlyserine (PS) exposure on the erythrocyte surface endows the cell with the propensity of adhering to vascular endothelium. Because individuals with sickle cell disease (SCD) manifest loss of erythrocyte membrane asymmetry with PS exposure, we have assessed the contribution of this marker to the process of sickle erythrocyte-microendothelial adhesion. Assays for plasma-induced adhesion were conducted on unactivated endothelium, in the absence of immobilized ligands, such that PS was compared to the erythrocyte adhesion receptor CD36. Blocking studies with erythrocytes pretreated with annexin V (to cloak PS) or anti-CD36 or both revealed an inhibitory effect on adhesion of 36% +/- 10% and 23% +/- 8% with blocking of both sites suggestive of an additive effect. We next evaluated 87 blood samples from patients with SCD and grouped them into 4 categories based on adhesion marker (CD36 and PS) levels. Results revealed a striking correlation between erythrocyte PS positivity and adhesion. Analyses of the individual patient data demonstrated a positive correlation between PS and adhesion (R = 0.52, P <.000 001), whereas none was noted between adhesion and CD36 (R = 0.2, P >.07). The effect of PS on adhesion appears to be related to the quantitative differences in erythrocyte markers in SCD, with PS the predominant marker when compared to CD36 both in the total erythrocyte population, and when the adherence-prone erythrocyte, the CD71(+) stress reticulocyte, was evaluated. Our study signals the entrance of an important new contributor to the field of sickle erythrocyte-endothelial adhesion. The implications of erythrocyte PS exposure in relation to the vascular pathology of SCD need to be assessed.